Hormonal and metabolic regulation of source-sink relations under salinity and drought: From plant survival to crop yield stability Alfonso A. Albacete, Cristina Mart´ınez-And´ujar, Francisco P´erez-Alfocea PII: DOI: Reference:
S0734-9750(13)00182-1 doi: 10.1016/j.biotechadv.2013.10.005 JBA 6748
To appear in:
Biotechnology Advances
Received date: Revised date: Accepted date:
13 June 2013 17 October 2013 20 October 2013
Please cite this article as: Albacete Alfonso A., Mart´ınez-And´ ujar Cristina, P´erez-Alfocea Francisco, Hormonal and metabolic regulation of source-sink relations under salinity and drought: From plant survival to crop yield stability, Biotechnology Advances (2013), doi: 10.1016/j.biotechadv.2013.10.005
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ACCEPTED MANUSCRIPT Hormonal and metabolic regulation of source-sink relations under salinity and drought: from plant survival to crop yield stability
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Alfonso A. Albacete, Cristina Martínez-Andújar and Francisco Pérez-Alfocea*
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Departamento de Nutrición Vegetal, Centro de Edafología y Biología Aplicada del
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Segura (C.E.B.A.S.), Consejo Superior de Investigaciones Científicas (C.S.I.C.),
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Campus Universitario de Espinardo, P.O. Box 164, E-30100 Murcia, Spain
*Corresponding author:
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Francisco Pérez-Alfocea
Departamento de Nutrición Vegetal, Centro de Edafología y Biología Aplicada del
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Segura (C.E.B.A.S.), Consejo Superior de Investigaciones Científicas (C.S.I.C.),
Phone: +34 968 396342
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Fax: +34 968 396213
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Campus Universitario de Espinardo, 25, P.O. Box 164, E-30100 Murcia, Spain
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E-mail: [email protected]
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ACCEPTED MANUSCRIPT Abstract Securing food production for the growing population will require closing the gap between potential crop productivity under optimal conditions and the yield captured by
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farmers under a changing environment, which is termed agronomical stability. Drought
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and salinity are major environmental factors contributing to the yield gap ultimately by
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inducing premature senescence in the photosynthetic source tissues of the plant and by reducing the number and growth of the harvestable sink organs by affecting the transport and use of assimilates between and within them. However, the changes in
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source-sink relations induced by stress also include adaptive changes in the reallocation of photoassimilates that influence crop productivity, ranging from plant survival to yield stability. While the massive utilization of –omic technologies in model plants is
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discovering hundreds of genes with potential impacts in alleviating short-term applied drought and salinity stress (usually measured as plant survival), only in relatively few cases has an effect on crop yield stability been proven. However, achieving the former
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does not necessarily imply the latter. Plant survival only requires water status
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conservation and delayed leaf senescence (thus maintaining source activity) that is usually accompanied by growth inhibition. However, yield stability will additionally
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require the maintenance or increase in sink activity in the reproductive structures, thus contributing to the transport of assimilates from the source leaves and to delayed stressinduced leaf senescence. This review emphasizes the role of several metabolic and
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hormonal factors influencing not only the source strength, but especially the sink activity and their inter-relations, and their potential to improve yield stability under drought and salinity stresses. Keywords: assimilate transport, biomass partitioning, cytokinins, fruit/grain filling, gibberellins, hormonal signaling, invertases, leaf senescence, stay green, sucrose and starch metabolism.
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ACCEPTED MANUSCRIPT Contents 1. Introduction 2. Improving source strength
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2.1. Delaying leaf-senescence
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2.2. Stay-green genotypes
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2.3. Hormonal homeostasis and signalling 2.4. Carbon metabolism 3. Improving sink strength
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3.1. Invertases, pollen viability and grain/fruit filling
3.2. Hormonal signalling and regulation of sink activity
3.2.2. Cytokinins 3.2.3. Auxins
3.2.5. Jasmonates
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3.2.4. Abscisic acid
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3.2.1. Gibberellins
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3.2.6. Salicylic acid 3.2.7. Ethylene
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3.2.8. Strigolactones
3.3. Root-sink strength to improve stress adaptation 3.4. Increasing sink number
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4. Conclusions
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ACCEPTED MANUSCRIPT 1. Introduction About 70-90% more food is required to fulfil the food demands of a human population of 9 billion people expected in 2050. This includes a 50% increase in grain yield of
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major crop plants such as rice (Oryza sativa L.), wheat (Triticum aestivum L.) and
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maize (Zea mays L.) (Boyer, 1982; Godfray et al., 2010; Peleg et al., 2011). This
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increase must be achieved in a sustainable way without increasing resource demand (arable land, irrigation water, fertilizers) and overcoming the yield losses due to environmental stresses. According to the historical data on yield potential in rice, the
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most substantial progress (3-fold increase from 2.5 to 8 t·ha-1) was achieved with the delivery of semidwarf varieties (1965-1990). A subsequent 1.5-fold increase was achieved by introducing the F1 hybrids, while the next substantial improvement
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expected for 2010 with the introduction of C4 photosynthesis (Datta, 2004), has not been achieved so far. Despite considerable investment in cultivar improvement, about 20–70% of the potential crop yield in favourable ecosystems is not captured by farmers
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because of biotic and abiotic stresses (Cassman, 1999; Datta, 2004). In the case of major
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U.S. crops (corn, wheat, soybean, sorghum, oat, barley, potato, and sugar beet) the average production is only about 20% of the maximum yields attained under optimal
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conditions (Boyer, 1982; Godfray et al., 2010; Peleg et al., 2011). While biotic factors (diseases, pests, and weed competition) account for less than 10% of yield reductions, the remaining 70% is attributed to abiotic constraints such as drought and salinity, that
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are being exacerbated by climate change (Peleg et al., 2011). A similar situation occurs in other crops and regions worldwide, which is termed the ‘yield gap’ (Godfray et al., 2010). Although a considerable scope already exists for increasing the crop production limits by adopting new varieties, one major societal and scientific challenge is to fill the yield gap caused by abiotic factors. Abiotic stresses, such as salinity and drought, modify source-sink relations which influence plant growth as well as adaptation to stress and consequently affect crop productivity. Yield reduction is ultimately due to a decrease in the number and size of sink organs, since the source organs are unable to maintain the assimilate supply required to support the mutually competitive processes of vegetative growth, filling of reproductive structures and adaptation to stress. Only in extreme cases is the loss of productivity due to plant death. Therefore, yield stability under stress requires a 4
ACCEPTED MANUSCRIPT dynamic optimization of source-sink relations to maintain assimilate partitioning to reproductive structures while minimizing the requirements of other organs and processes to maintain physiological and morphological adaptive responses. Ideally, this
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optimization must be highly plastic and should be linked to the severity of the stress,
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ranging from plant survival (mostly by preserving source activity without growth) under severe stress conditions, to yield stability (both sink and source activities are required)
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under a wide range of productivity-compatible stress situations. However, this ideal scenario is far from being realised and increasing plant survival does not necessarily imply the most important trait of higher productivity or yield stability. Therefore,
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tolerance to abiotic stresses in functional studies in model plants should always be measured in terms of yield-related parameters rather than plant survival, because (i)
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both parameters are often inversely related (Ceccarelli, 1987), (ii) plant response to stress differs through the plant cycle, with the reproductive stage being one of the most sensitive (Parish et al., 2012), and (iii) other important factors affecting yield
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components, such as sink determination and growth can be missed, thus reducing the
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possibilities of applying the results to crop species in the field (Peleg et al., 2011). Drought and salt stress can initially reduce source photosynthetic activity by
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both stomatal and non-stomatal factors (Munns et al., 2006; Munns and Tester, 2008; Pérez-Alfocea et al., 2010). Regardless of the ionic component of salinity, both stresses can provoke premature senescence due to assimilate accumulation in source leaves as a
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result of decreased consumption in sink tissues. This assimilate accumulation provokes impaired consumption of NADPH by the Calvin Cycle, leading to the so called (feedback) photo-inhibition and photo-oxidation processes, with the subsequent transfer of photosynthetic electrons from over-reduced ferredoxin to oxygen (Mehler Reaction) producing toxic reactive oxygen species that damage cell structures (Balibrea et al., 2000; Keunen et al., 2013; Paul and Foyer, 2001; Stitt, 1991). Indeed, the processes causing photo-oxidative damage and premature leaf senescence are common to several abiotic root-zone stresses that induce water stress and inhibit growth of sink organs without a concomitant ionic component (Hare et al., 1997; He et al., 2001). There are strong indications from numerous studies that the regulation of senescence is particularly important for yield stability under stress conditions (Gregersen et al., 2013), and avoiding feedback inhibition of photosynthesis by coordinating assimilate transport between source and sink tissues may delay leaf senescence (Pérez-Alfocea et al., 2010). 5
ACCEPTED MANUSCRIPT However, this coordination requires the maintenance or increase of sink demand for assimilates from source tissues, since this process is primarily reduced by the stress in vegetative (Albacete et al., 2008; Balibrea et al., 2000; Munns, 1993) and reproductive
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tissues (Balibrea et al., 2003; Balibrea et al., 1999; Ghanem et al., 2009; Parish et al.,
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2012). Alternatively, reallocation of assimilates between source and sink tissues also allows the plant to adapt to biotic and abiotic stresses (Roitsch et al., 2003; Roitsch and
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González, 2004). One example is the increased root-to-shoot ratio due to growth maintenance in the root and the rapid inhibition of shoot growth (Albacete et al., 2008; Sharp, 2002), which can be considered an adaptive response to increase the water and
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nutrient acquisition from the root zone. This integrated growth plasticity involves longdistance communication between different organs, with hormones playing a major role
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(Ghanem et al., 2011a; Ghanem et al., 2011b; Pérez-Alfocea et al., 2011; Sachs, 2005). Growth regulation by drought and salinity is mediated primarily by the stress-related hormones abscisic acid (ABA) and ethylene (Albacete et al., 2008; Dodd and Davies,
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1996; Ghanem et al., 2008). However, other hormones, such as gibberellins (GAs),
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auxins and cytokinins (CKs) are also involved (Achard et al., 2006; Albacete et al., 2008; Ghanem et al., 2011a; Magome et al., 2004; 2008; Pérez-Alfocea et al., 2010;
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Rivero et al., 2007). Phytohormone balance can affect plant performance by influencing source-sink relations and metabolism, and thus, the stress tolerance and crop yield. The power of the hormone-related traits in improving yield has been already
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demonstrated. Indeed, the Green Revolution, that substantially improved worldwide food production in the 1960s and 1970s, was possible thanks to the breeding of crop varieties with improved vigour and harvest index. Those traits imply changes in sourcesink relations for producing more grain at the expense of the straw biomass. In the case of wheat and rice, those changes are explained by alterations in GAs homeostasis and signalling (Peng et al., 1999; Sasaki et al., 2002). In rice, the high-yielding semi-dwarf phenotype is due to a mutation in the gene encoding for the enzyme GA20 oxidase (GA20ox-2) that decreases GA synthesis in the vegetative sink tissues thus reducing plant height, while the isoform GA20ox-1 is expressed in reproductive organs allowing normal grain development (Sasaki et al., 2002). In wheat, the mutant dwarfing alleles encodes a transcription factor that reduces the sensitivity to GAs (GAI, Gibberellin Insensitive Gene) (Peng et al., 1999). Therefore, substantial increases in yield
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ACCEPTED MANUSCRIPT production have been due to changes in plant source-sink relations, induced by altering hormonal metabolism and signalling. However,
sustaining
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productivity under
unfavourable
conditions
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necessarily requires maintaining both assimilate production in sources tissues, and also
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transport to, and use within sink and harvestable tissues. While these processes may
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adjust to allow plant stress adaptation, their impairment for long periods causes a loss of productivity. This aspect is important since several abiotic stresses can co-occur in the field and the response to the combination can be different from the response to
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individual stresses. Hence, achieving tolerance to such combined stresses by exploiting general adaptive responses of the plant might lead to an important strategy for yield improvement. This review emphasizes that increased yield stability will require the
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increase or at least the maintenance of (a) source strength (photosynthetic efficiency and assimilate export) for longer (delayed stress-induced senescence) and/or (b) the sink strength (assimilate import) of the harvestable (fruits and seeds) and resource providing
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(new leaves and roots) organs (vigour maintenance). The homeostatic regulation of
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phytohormones and/or metabolic components could play significant roles in regulating source-sink activities and relations and their manipulation could provide new
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opportunities for increasing yield stability under abiotic stress conditions. However, it seems clear that a communication and interdependence between both sink and source organs are needed. For instance, increasing sink activity alone cannot be effective if the
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source is limited by stomatal factors or any other resource acquisition from the soil, while increasing the source efficiency is also ineffective if the sink activity is impaired by hormonal signals and/or limited water and nutrient availability. Hence, special attention should be paid to those genes/processes allowing sink activity (i.e. flower fertility and grain/fruit filling) under resource-limited conditions. Finally, although the major objective should be increasing yield stability of elite lines with high yield potential, a significant stress-induced yield reduction of high yielding lines may be preferable to a small stress-induced yield reduction of low yielding genotypes with agronomical stability. 2. Improving source strength 2.1. Delayed leaf-senescence
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ACCEPTED MANUSCRIPT Developmental leaf senescence is a programmed process influenced by several endogenous and exogenous factors, such as the plant developmental stage, leaf age, phytohormone levels, and light conditions. This process reduces local or systemic
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assimilate availability and therefore limits plant growth and productivity when it is
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prematurely induced by environmental factors such as drought and salinity (BuchananWollaston et al., 2003). Any genetic or physiological determinant delaying the
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progression of developmental or stress-induced senescence can promote the transfer of photosynthetic assimilates from sources (e.g. mature leaves) to harvestable sinks (e.g. developmental organs such as fruit and seeds and new leaves), thus potentially helping
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to close the yield gap under suboptimal conditions (Cassman, 1999; Godfray et al., 2010; Gregersen et al., 2013). Indeed, transcript profiling studies have revealed the
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occurrence of considerable cross-talk between stress responses and leaf senescence (Lim et al., 2007). For example, among 43 genes coding senescence-induced transcription factors (TFs), 28 were also induced by exposure to various stresses (Chen
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et al., 2002). Furthermore, the expression of many senescence-associated genes (SAGs),
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such as the Arabidopsis SEN1 gene, is commonly regulated by the initiation of natural leaf senescence and by exposure to stresses (Schenk et al., 2005). Importantly, the onset
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of stress-induced senescence is related to changes in endogenous hormones such as increases in ethylene and/or its precursor 1-aminocyclopropane-1-carboxylic acid (ACC) (Ghanem et al., 2008; Grbic and Bleecker, 1995), ABA (Zeevaart and Creelman, 1988), salicylic acid (SA) (Morris et al., 2000), and jasmonic acid (JA) (He et al., 2002),
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or decreased in CK levels (Gan and Amasino, 1995; Ghanem et al., 2008; Ghanem et al., 2011a). In addition, leaf senescence may also be induced by carbohydrate accumulation or by the availability of excess carbon relative to low levels of nitrogen (Wingler et al., 2006). Table 1 summarizes the hormonal and metabolic-related genes affecting source strength. 2.2. Stay-green genotypes The relationship between senescence and plant productivity is complex, but modulating the genetically determined senescence program can influence crop productivity under optimal and/or suboptimal conditions (reviewed in Gregersen et al. (2013)). Indeed, a positive correlation has been found between leaf area/photosynthetic rate duration and yield (grain or biomass) under water-limiting conditions in maize (Messmer et al., 8
ACCEPTED MANUSCRIPT 2011), sorghum (Borrell et al., 2000; Harris et al., 2007), wheat (Verma et al., 2004) and barley (Gonzalez et al., 2010; Vaezi et al., 2010). Hence, genetic determinants of functional stay-green genotypes are potentially valuable for conventional and
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biotechnological breeding approaches (Borrás et al., 2004; Gregersen et al., 2013). For
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example, the functional stay green wheat genotype CN17 exhibited significantly higher maximal photochemical efficiency for photosystem II (PSII) and higher efficiency of
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excitation capture by open PSII reaction centers (Fv/Fm) 21 days post-anthesis (Luo et al., 2013), thus extending the period of grain filling and presumably increasing yield (Mittler and Blumwald, 2010). In addition, chlorophyll degradation was delayed by
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approximately 14 days, and was not blocked as in ‘cosmetic’ stay-green phenotypes which retain chlorophyll but not photosynthetic activity. However, although functional
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stay-green varieties have been characterized physiologically and genetically in several plant species, the mechanism by which leaf greenness is coupled to effective photosynthetic competence remains unclear (Luo et al., 2013). Enhanced photosynthetic
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competence seems to be explained by a higher protection of PSII and granal stability, as
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well as increased levels of unsaturated fatty acids and antioxidative capacity, which provide more readiness to regenerate chloroplast ultrastructure by maintaining protein
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synthesis. In silico BLAST analysis indicated that four cell wall-associated hydrolases (i.e. cell wall invertases) were present in the CN17 stay-green genotype. The authors suggested that a mechanism similar to the C-repeat/dehydration-responsive element binding factor 2 (CBF2) gene described in Arabidopsis can be responsible for the
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regeneration of the photosynthetic apparatus (Luo et al., 2013). 2.3. Hormonal homeostasis and signalling The overexpression of the CBF2 gene in Arabidopsis delayed the onset of both natural (Sharabi-Schwager et al., 2010a) and artificially-induced leaf senescence by either darkness or phytohormones (ethylene, ABA, SA, and JA) in detached leaves (SharabiSchwager et al., 2010b). Affymetrix ATH1 genome array revealed significant changes in the expression of 286 genes in mature leaf tissue, 30 stress-related genes, 24 TFs, and 20 genes involved in protein metabolism, degradation, and post-translational modifications. Transcript profiling analysis of hormone metabolism and responsive genes revealed that overexpression of CBF2 induced expression of ABA biosynthesis genes (NCED, encoding the key enzyme in ABA biosynthesis, 9-cis-epoxycarotenoid 9
ACCEPTED MANUSCRIPT dioxygenase - (Thompson et al., 2000) and ABA-responsive genes, and suppressed SAand JA-related genes, including three lipoxygenases, allene oxide synthase and jasmonic acid carboxyl methyltransferase. Overexpression of CBF2 in Arabidopsis also
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suppressed leaf tissue responsiveness to ethylene (0.1-10 µl l-1) as compared to wild-
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type plants and significantly delayed senescence and chlorophyll degradation (SharabiSchwager et al., 2010a). Although only the expression of the ethylene receptor EIN4
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was significantly reduced, significant increases in 17 ABA biosynthetic and responsive genes occurred in CBF2-overexpressing plants. Thus, reduced leaf responsiveness to ethylene could be explained through interactions with other hormones such as ABA
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(Sharp, 2002). CBF2 seems to act directly on hormone metabolism and signalling, suppressing hormone-induced leaf senescence (Sharabi-Schwager et al., 2010b), and
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could potentially delay ethylene-mediated stress-induced senescence. Therefore, the manipulation of this kind of TF seems a possible strategy to increase yield under suboptimal conditions.
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However, the overexpression of the CBF1, CBF2, and CBF3 genes in
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Arabidopsis retarded growth causing a dwarf phenotype (Gilmour et al., 2004; Liu et al., 1998). Constitutive e CBF1 expression retarded growth by allowing the
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accumulation of DELLAs, a family of nuclear growth repressing proteins, whose degradation is stimulated by the growth-promoter hormones GAs (Achard et al., 2008). It is not clear if the observed delayed leaf senescence in CBF2-overexpressing plants is
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mediated by DELLA proteins as reported for the delayed flowering and dwarfism phenotypes (Achard et al., 2008), or rather is a DELLA-independent effect as noted for the increased sugar levels of CBF1-overexpressing plants (Wingler and Roitsch, 2008). Nevertheless, delayed senescence of growth-arrested phenotypes may be incompatible with yield promotion. Recent studies also suggest that growth impairment and adaptation to stress seems to occur through independent signalling processes and may differ depending on the stress condition (Dubois et al., 2013). However, even if activated by different processes, it seems that they occur simultaneously and can be induced by the same effector. Thus, the ethylene precursor ACC is implicated as an early signal related to plant growth arrest under both osmotic and salt stresses (Skirycz et al., 2011; Zhang et al., 2011) and the onset of salt-induced senescence (Ghanem et al., 2008). Unravelling 10
ACCEPTED MANUSCRIPT the molecular mechanisms and signalling processes involved could identify new targets for stress alleviation and crop improvement. Indeed, an analysis of the relatively abundant reports of transgenic Arabidopsis lines with enhanced survival reveals no
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improved growth under moderate drought conditions (Skirycz et al., 2011). However,
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the Arabidopsis TFs ETHYLENE RESPONSE FACTOR5 (ERF5) and ERF6 are rapidly induced under osmotic stress and seem to act as master regulators adapting leaf growth
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to environmental changes (Dubois et al., 2013; Skirycz et al., 2011). Enhanced ERF6 expression inhibits cell proliferation and leaf growth via GA degradation by gibberellin 2-oxidase6 (ga2-ox6) and subsequent DELLA-mediated inhibition of cyclin-dependent
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kinase A activity (Skirycz et al., 2011). As a result, ERF6 gain-of-function lines are dwarfed and hypersensitive to osmotic stress, while the growth of erf5erf6 loss-of-
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function double mutants is less affected by short- and long-term osmotic stress, probably due to the non-induction of ga2-ox6, thus avoiding the GA-DELLA cell-cycle inhibition. Surprisingly, the double erf5erf6 mutants were not more tolerant of moderate
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salinity (50 mM NaCl), suggesting that salinity-induced growth occurs independently of
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the ERF5/ERF6-growth regulatory pathway. This can be explained not only by a rapid accumulation of toxic ions (Dubois et al., 2013), but also by other hormone-related
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processes such as decreased levels of growth promoting hormones like CKs (Albacete et al., 2008; Ghanem et al., 2011a) or by interaction with ABA signalling (Pan et al., 2012). However, ERF6 also activates the expression of a plethora of osmotic stressresponsive genes (including the stress tolerance STZ, MYB51, and WRKY33) in a
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growth-independent manner (Dubois et al., 2013). Indeed, overexpression of SlERF5 in tomato plants conferred tolerance to drought (unwatered for 7 days) and salt (200 mM NaCl for 10 days) stresses by conserving chlorophyll concentrations and water status, but without additional effect on growth when compared to the WT (Pan et al., 2012). A similar response was found in transgenic rice plants overexpressing OsTZF1 (a member of the CCCH-type zinc finger gene family in rice) driven by a maize ubiquitin (Ubi) promoter. The Ubi::OsTZF1 plants exhibited not only delayed seed germination and growth retardation, but also delayed salt-, JA- and ABA-induced leaf senescence, indicating that delayed senescence might be due to a common factor like tolerance to oxidative stress (Jan et al., 2013). Again, the growth-retarded Ubi::OsTZF1 plants showed improved tolerance (plant survival) to high salinity (250 mM NaCl for 3 days) and drought stresses. The discovery of the missing link between plant survival and growth is of utmost importance. 11
ACCEPTED MANUSCRIPT The interesting finding that hormones target various members of protein families involved in growth, together with the identification of a small number of proteins that were co-regulated by multiple hormones, indicate synergistic action of metabolic
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pathways. Such pathway interactions probably occur through combinatorial regulation
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of common target proteins by various hormone-controlled kinases (Chen et al., 2010), with the common objective of adapting growth to the environmental conditions to
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improve plant survival. However, biotechnological manipulation of key proteins or hormones should focus not only on the ecological adaptation in terms of survival but also the agronomical stability for securing food production. For example, ABA induces
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the phosphorylation and activation of the AREBs transcription factors and the overexpression of the phosphorylated AREB1 induces the expression of many ABA-
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inducible genes (Choi et al., 2000; Furihata et al., 2006). Indeed, the overexpression of the SlAREB1 gene in tomato improved plant survival under drought and salinity stress through senescence-delaying and water-conservative mechanisms (Orellana et al.,
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2010a). Those ABA-mediated mechanisms are probably incompatible with growth
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maintenance, for which co-ordination with other growth-promoting hormones/proteins is required.
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Since CKs have an important role in delaying natural senescence (Gan and Amasino, 1995; 1997) and the concentration of the bioactive CKs decreases during the exposure to water (Havlová et al., 2008; Kudoyarova et al., 2007) and salt (Albacete et
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al., 2008; Ghanem et al., 2008; Ghanem et al., 2011a) stresses, it has been hypothesized that yield stability can be maintained by delaying stress-induced senescence and maintaining source activity through the stress-induced synthesis of cytokinins (reviewed in Gregersen et al. (2013)). About 80% of the studies published so far on transgenic crop plants with autoregulated (using essentially SAG or SARK promoters) isopentenyltransferase (IPT) gene expression reported delayed leaf senescence (stay green and chlorophyll content) and subsequently increased source photosynthetic activity over time. Interestingly, most of those studies reported increased biomass and/or yield-related parameters under drought stress in tobacco (Rivero et al., 2007), cassava (Zhang et al., 2010), creeping bent grass (Merewitz et al., 2010; 2011), peanut (Qin et al., 2011) and wheat (Peleg et al., 2011), and also in salinized cotton (Liu et al., 2012) and tomato (Ghanem et al., 2011a). Transient (2 hours) root specific IPT induction using a heat shock-specific promoter (HSP70::IPT) promoted plant growth in 12
ACCEPTED MANUSCRIPT salinized (100 mM NaCl) tomato plants by increasing leaf CK levels by 2-3 fold (Ghanem et al., 2011a). In this case, leaf ABA concentration was reduced by 20-40%. The attenuated growth inhibition of salinized CK overproducing plants may occur
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directly via the relative maintenance of CK-mediated cell division and cell wall
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extensibility and/or improved carbon status due to both delayed stomatal closure and leaf senescence through decreased ABA concentrations. Lower ABA concentrations in
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photosynthetically active leaves may also indirectly delay senescence, photoinhibition, and photooxidation during the osmotic phase of salinity (Ghanem et al., 2008; Ghanem
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et al., 2011a; Pérez-Alfocea et al., 2010).
Detailed physiological studies of the stress responses of CK-overproducing plants could identify new molecular targets to further increase crop productivity
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(Zalabák et al., 2013), not only in source but also in sink capacity. For example, water stressed transgenic rice plants expressing the IPT gene, driven by the stress- and maturation-induced promoter PSARK, exhibited improved water status because of lower
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water use, delayed drought symptoms by 4-7 days, and significant increases in plant
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biomass and grain yield (2-3 times), compared with WT plants (Peleg et al., 2011). During water-stress, PSARK::IPT plants displayed increased (i) expression of BR
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synthesis (DWF5: Delta14-sterol reductase and HYD1: C-8 sterol isomerase) and signaling (BRL3, BRI1, BRH1, BIM1, SERK1, BSK1, BIN4 and BAK1) genes and (ii) repression of JA-biosynthesis (OPR2) and signaling (MES3 and JAZ12) related genes.
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Moreover, the expression of genes encoding indole-3-acetic acid (IAA) transporters (OsPIN3a and OsPIN6) and IAA-responsive genes (OsIAA5, OsIAA21 and OsARF5) were down-regulated in the PSARK::IPT plants under drought conditions, while the levels of expression of GA-associated genes (GA receptor, OsGAI and chitin-inducible GAresponsive protein) were induced. In addition, ethylene responsive and signalling genes (flavonol synthase/flavanone 3 hydroxylase, ETR2, MTHFR2, RAP2.4 and EIN3) were up-regulated. Those changes in hormone homeostasis were associated with resource(s) mobilization during stress, thus modifying source-sink relationships and in stronger source and sink capacities in the aerial part of the PSARK::IPT plants during drought stress. As a result, the transgenic plants had higher grain yield with improved quality (nutrients and starch content, until 60-90% more than WT). The increased starch content of both flag leaves and grains correlated with the expression of the transcription factor OsMADS57 (a regulator of starch synthesis), under both control and stress conditions 13
ACCEPTED MANUSCRIPT (Peleg et al., 2011). The capacity to increase starch synthesis under water stress can be considered protective by avoiding feed-back inhibition of photosynthesis by soluble sugars when the assimilate export from source leaves is impaired, thus also influencing
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the timing of senescence (see section 2.4). Moreover, the mobilization of this starch
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during the night also contributes to increased source strength. The CK interaction with BRs (positive) and IAA (negative) signalling may explain the drought-resistant
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phenotype by decreasing stomatal density (Kim et al., 2012) and root development (Dello Ioio et al., 2008) in a constrained (pot) environment. An additional positive effect of CK-induced BR signalling is related to grain filling by stimulating the flow of
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assimilates from the source to the sink (Wu et al., 2008).
Although it has been widely reported that IPT gene expression in senescing
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leaves can enhance CK levels thus increasing vegetative biomass, the effect on crop yield is not always detectable. Many investigations with transgenic plants support the concept that delayed leaf senescence can have a positive impact on crop productivity
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but the positive effect depends on the plant species, the plant variable measured, and the
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environment, especially under drought, salinity and low nitrogen (Gregersen et al., 2013). Delayed senescence should therefore be maintained without growth reduction or
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at least with a growth recovery when the adaptive responses are induced. 2.4. Carbon metabolism
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Increasing radiation use efficiency (and thus source strength) of wheat by 50% could be achieved by modifying specificity, catalytic rate and regulation of Rubisco, upregulating Calvin cycle enzymes, introducing chloroplast CO2 concentrating mechanisms, optimizing light and N distribution of canopies while minimizing photoinhibition, and increasing spike photosynthesis (Reynolds et al., 2012). There is potentially to alter carbon metabolism to improve yield under environmental conditions, despite pleiotropic and deleterious effects observed in transgenic plants. These likely occur due to the alteration of basic developmental processes through sugar sensing and signalling, thus adequate (inducible) promoters are needed to avoid undesirable traits and any growth penalty in absence of stress (Peleg et al., 2011; Suárez et al., 2009). For example, constitutively (35S promoter) overexpressing the yeast trehalose-6-phosphate synthase (TPS1) gene in tomato provoked higher tolerance to salinity, drought and oxidative stress, but plants exhibited a plethora of pleiotropic changes such as thick 14
ACCEPTED MANUSCRIPT shoots, rigid dark-green leaves, erect branches, aberrant root development and higher chlorophyll and starch content compared to WT plants (Cortina and Culiáñez-Macià, 2005). Similarly, constitutive overexpression of a synthetic fusion of Escherichia coli
T
trehalose biosynthetic genes (otsA and otsB) in potato (Jang et al., 2003) and alfalfa
IP
(Suárez et al., 2009) caused a range of stunted plant growth phenotypes. However, those genes expressed under the light-regulated promoter of the Rubisco small subunit or an
SC R
ABA-inducible promoter produced from 3- to 10-fold more trehalose than WT plants, and increased tolerance to drought and salinity (100 mM NaCl) measured as vegetative growth. Although yield data are not available, those effects were correlated with a
NU
higher photosynthetic capacity under both control and stress conditions, 50% less in leaf Na+ accumulation, and delayed leaf senescence (Garg et al., 2002).
MA
Since the ability of the C4 photosynthetic pathway to suppress ribulose-1,5bisphosphate (RuBP) oxygenation and photorespiration represents the most efficient type of photosynthesis on Earth (Sage, 2004), engineering C4 photosynthetic pathway
D
into C3 crops has been considered a highly promising strategy to alleviate crop yield
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losses due to environmental factors (reviewed in Reguera et al. (2012)). Although the strategy of introducing C4 photosynthesis in C3 plants as rice seems extremely
in
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ambitious because of the coordinated relation between leaf morphology and metabolism C4 plants
(Hibberd et
al.,
2008),
the effects
of
overexpressing the
phosphoenolpyruvate carboxylase gene (PEPC) on photosynthesis in rice plants has
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shown controversial but encouraging results. Most studies on this topic have reported decreased photosynthesis with increasing activity of the key CO2-fixing enzyme PEPC in transgenic rice plants (Agarie et al., 2002; Fukayama et al., 2001). There are also reports (Bandyopadhyay et al., 2007; Ding et al., 2007; Jiao et al., 2002; Ku et al., 2000) of enhanced photosynthesis in transgenic rice plants but always under suboptimal (stress) conditions. However, yield results have been provided only recently. Rice plants transformed with the PEPC from millet (Seteria italica) under the control of light regulated Rubisco small subunit promoter had increased PEPC activity, net photosynthesis and grain yield (by 40-50%) under drought conditions (upland), while no differences were observed under optimal (wetland) conditions (Ding et al., 2013). These results suggest that the transgenic enzyme is photosynthetically effective only when the natural photosynthetic rate is limited, thus minimizing the switch between C3
15
ACCEPTED MANUSCRIPT and C4 metabolism described in the halophyte Mesembryanthemum crystallinum under osmotic stress (Scolombe et al., 2006). Senescence-regulated expression of the sucrolytic enzyme cell wall invertase
T
CIN1 (from Chenopodium rubrum L.) under control of the SAG12 promoter in leaves
IP
increased source strength and delayed natural senescence in tobacco, probably linked to
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a CK-mediated mechanism (Lara et al., 2004). This enzyme seems to activate metabolic carbohydrate flux that represses ethylene biosynthesis (Mayak and Borochov, 1984). It has been demonstrated that ethylene decreases cell wall invertase (cwInv) gene
NU
expression (Linden et al., 1996; Roitsch et al., 2003) and induces senescence (MunnéBosch and Alegre, 2004). Therefore, ethylene could mediate salt-induced senescence by inhibiting cwInv (Ghanem et al., 2008). ABA could also mediate stress-induced
MA
senescence by inducting the cwInv proteinaceous inhibitor in tomato and tobacco plants, thus changing the metabolic carbohydrate flux. Indeed, silencing the cwInv inhibitor delayed ABA-induced leaf senescence, and increased seed weight and fruit
D
hexose contents in tomato (Jin et al., 2009). GAs, BRs, auxins and CKs seem to
TE
upregulate cwInv and other sucrolytic enzyme genes, thus promoting sink strength and assimilate import in actively growing tissues (reviewed by Roitsch et al. (2003) and
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Roitsch and González (2004)). Assessing the role of hormones in regulating enzyme activities moderating source–sink relations (Roitsch et al., 2003; Roitsch and Ehness, 2000) will require measurement of these enzymes in hormone biosynthesis or sensitivity
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mutants.
Interestingly, tomato plants (Solanum lycopersicum cv. P-73) overexpressing the cwInv gene CIN1 from Chenopodium rubrum under the control of the putative fruit specific promoter from the vacuolar invertase gene InvLp6g from Solanum pimpinellifolium had significantly higher water use efficiency (WUE) and tolerance to water stress due to increased photosynthetic activity and lower water use (Albacete et al, unpublished results). One possible explanation for reduced transpiration is the increased CK/ABA ratio under stress conditions, since both hormones act antagonistically in stomatal regulation (Dodd, 2005). However, a metabolic effect of the sucrolytic enzymes in the leaves cannot be ruled out, since altered sucrose partitioning between storage and breakdown may affect stomatal function at the level of guard cells. Several studies have also revealed the importance of the apoplastic sucrose in regulating 16
ACCEPTED MANUSCRIPT stomatal aperture. High concentrations of apoplastic sucrose (reduced in the InvLp6g::CIN1 plants) can decrease stomatal aperture (Outlaw and De Vlieghere-He, 2001), while a high hexose/sucrose ratio and concentration of the organic acids malate
T
and citrate in the apoplast (increased in the InvLp6g::CIN1 plants) can influence
IP
stomatal aperture (Araújo et al., 2011) and fruit yield under water stress (Garchery et al., 2013). Potato (Solanum tuberosum) plants expressing an antisense construct targeted
SC R
against sucrose synthase 3 (SuSy3) had a lower stomatal conductance and slightly reduced CO2 fixation, while plants with increased guard cell acid invertase activity (by introducing the SUC2 gene from yeast) had increased stomatal conductance and CO2 14
CO2 feeding experiments
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fixation but decreased WUE (Antunes et al., 2012).
suggested that the changes in photosynthesis were mostly explained by stomatal
MA
regulation rather than altered mesophyll capacity for carbon fixation. Therefore, engineering sucrose metabolism in the apoplast or symplast of vegetative tissues also offers interesting opportunities to increase the source strength and reduce water use,
D
thus increasing stress tolerance and yield stability.
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Although photoassimilates are transported to heterotrophic sink organs where they are often again transiently or permanently stored as starch (Peterhansel and
CE P
Offermann, 2012), new data also indicate that a stronger accumulation of leaf starch used in plants to sustain metabolic functions during the night positively impacts both source strength and sink yield. Multivariate analysis revealed that starch integrates the
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overall metabolic response within a regulatory network that balances growth with the carbon supply (Peterhansel and Offermann, 2012; Sulpice et al., 2009). Constitutive overexpression of a starch synthase gene SSIV in Arabidopsis resulted in higher leaf starch contents at the end of the day and faster growth rates of transgenic plants suggesting greater photosynthetic efficiency (Gámez-Arjona et al., 2011). The physiological basis of these results was investigated in more detail in Arabidopsis and rice plants overexpressing the rate-limiting enzyme ADP glucose pyrophosphorylase (AGPase) (Gibson et al., 2011). In this study photosynthetic rates correlated with AGPase activity especially when plants were grown at high CO2 availability (1000 ppm). This was probably due to reduced feedback inhibition by photosynthetic products (Gibson et al., 2011) and could explain why additional starch was not synthesized at the expense of other biomass. Other studies have shown the cooperative action of sucrolytic enzymes with starch biosynthesis enzymes in controlling source-sink relations. Chopra 17
ACCEPTED MANUSCRIPT et al. (2005), reported the synergistic role of sucrose synthase, UGPase and AGPase for starch biosynthesis in mung bean, thus controlling the efficient partitioning of sucrose into ADP-glucose and finally controlling seed sink strength. Further, ultra-structural
T
analyses in leaves of tomato plants overexpressing a cell wall invertase gene CIN1
IP
revealed distinct and large starch grains than the WT under drought stress conditions (Albacete et al., unpublished results), suggesting that the cycle of starch synthesis and
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breakdown may modulate stress responses in transgenic plants (Sulpice et al., 2009) to maximize carbon uptake and growth.
NU
Taken together, transient or permanent sugar partitioning towards starch accumulation in both source and sink organs would maintain photoassimilate production (source strength), transport, and use (sink strength) that correlates with
MA
biomass production. Therefore, it would be expected that plants with high biomass production also show high starch accumulation during the day. This was recently tested by comparing growth and metabolite levels of 94 Arabidopsis accessions (Sulpice et al.,
D
2009). The most striking observation was that starch content at the end of the day was
TE
negatively correlated with above-ground biomass production. Thus, those plants that invested a large part of fixed carbon into transient starch for later use during the night,
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grew slower than those that used most fixed carbon for growth during the day (Sulpice et al., 2009; Sulpice et al., 2010). Instead, the percentage of total protein that was invested in enzymes of primary metabolism in the light correlated positively with
AC
biomass, supporting a link between metabolic activity during the day and final biomass. These data point to different strategies used in nature and in agricultural breeding systems to optimize biomass production and yield. Indeed, an active regulatory linkage between carbohydrates, sources and sinks has been suggested (Ainsworth and Bush, 2011), since there is a direct relation between assimilate loading and photosynthetic rate, since assimilate transport from leaves is required for continuous photosynthetic production (Nikinmaa et al., 2013; Turgeon, 2010). Nikinmaa et al. (2013) have postulated that assimilate transport in the phloem competes with transpiration for water, and an optimal level of stomatal opening should exist for a given conditions that maximizes phloem transport between source and sink organs, assuming that sugar loading is proportional to photosynthetic rate. Excess sugar loading, however, may block the assimilate transport because of viscosity build-up in 18
ACCEPTED MANUSCRIPT phloem sap, leading to starch accumulation and down-regulation of photosynthesis. Therefore, stomatal regulation seems to be directly connected with sink activity, plant structure and soil water availability as they all influence assimilate transport (Garchery
T
et al., 2013; Nikinmaa et al., 2013).
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It has been also recently postulated that reductions in stomata density and
SC R
stomatal aperture can reduce water loss by transpiration while maintaining sufficient CO2 uptake to sustain biomass and yield under water-deficit conditions (Yoo et al., 2009). Indeed, there are several examples where modifying a single gene decreased
NU
stomatal aperture and/or stomatal density and, consequently, increased WUE and resistance to water deficit-related stresses such as salinity and drought (reviewed in Yoo et al. (2009)). Molecular dissection of the genetic variability in WUE has only recently
MA
been initiated in the model Arabidopsis, where the ERECTA gene was found to be critical in altering transpiration efficiency by mechanisms including leaf epidermal and mesophyll differentiation (Masle et al., 2005).Therefore, an optimal relation between
D
transpiration and carbon assimilation and transport may be the reason for what reducing
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transpiration rates without affecting CO2 assimilation would result in increased WUE
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and may contribute to improve yield stability under stress conditions.
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3. Improving sink strength 3.1. Invertases, pollen viability and grain/fruit filling As already suggested, sink strength could act as a major driving force for maintaining assimilate transport, source activity and plant growth, and therefore, yield stability under adverse conditions. Indeed, an increasing body of direct or indirect evidence support this hypothesis. For instance, improving shoot Na+ exclusion capacity is considered a major target to increase salt tolerance in many crops, thus delaying the toxic effect and maintaining leaf function (Munns and Tester, 2008; Pérez-Alfocea et al., 2010). However, this capacity alone cannot always explain crop productivity under salinity. For example, wheat cv Tamaroi isogenic lines containing the Nax2 locus (encoding the HKT1 Na+-selective transporter) accumulated from 4 to 12-fold lesser Na+ in the flag leaf (according to the salinity level in the soil) than the cv Tamaroi in 19
ACCEPTED MANUSCRIPT field experiments (Munns et al., 2012). The improved yield (by 25%) of the Na+excluder line observed only under high salinity (169 mM NaCl) was explained by maintenance of the source strength (photosynthetic capacity) in the flag leaf during the
T
grain filling stage. Nevertheless, this yield improvement was difficult to explain only by
IP
decreased Na+ accumulation, since the excluder line was 3-times more sensitive to the leaf Na+ concentration than the cv Tamaroi (by plotting yield vs leaf Na+ concentration).
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Additionally, the yield increase was more likely due to a reduced flower abortion index because the effect was observed on a higher number of grains rather than on higher grain weight. Similarly, a QTL for salt tolerance (SKC1) that encodes a Na+ transporter
NU
and regulates K+/Na+ homeostasis has been identified in rice (Ren et al., 2005). Introducing this QTL has high potential to improve salt tolerance but, to our knowledge,
MA
no practical results in terms of improved yield under salinity have been reported so far. Those examples suggest that other traits regulating flower/grain sink activity could be more related to yield than leaf Na+ accumulation.
D
In this regard, many self-fertilizing crops are particularly sensitive to abiotic
TE
stress at the reproductive stage and pollen development is considered a limiting step in crop productivity (see Parish et al. (2012), for a review). In rice and wheat,
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developmental process for pollen reproduction implies a programmed cell death (PCD) at the tapetum (the feeding tissue of the microspore), and seems to be delayed or inhibited by various abiotic stresses, causing premature pollen and flower abortion and
AC
subsequent yield losses. According to Parish et al. (2012), two hypotheses can be formulated to explain the effect of abiotic stress on flower abortion-related processes at pollen development. On one hand, the ‘starvation hypothesis’ postulates that abiotic stress provokes a decrease of assimilates reaching the developing microspores as a consequence of the downregulation of a tapetal cwInv and hexose transporters, such as MST8 in rice, which is regulated by a MYB transcription factor (Zhang et al., 2010). On the other hand, the ‘PCD hypothesis’, that can be connected with the previous one through carbohydrate availability, suggests that stress delays or prevents developmental PCD at the tapetum, thus failing to provide materials required for microspore development. Indeed, a decrease in tapetal cwInv activity was correlated with pollen abortion and reductions in hexoses reaching the tapetum. The anther cwInv gene IVR1 is repressed at meiosis in drought-sensitive wheat lines resulting in sucrose accumulation, but not in tolerant ones (Koonjul et al., 2005). Similar inhibitory effects have been 20
ACCEPTED MANUSCRIPT observed in the rice cwInv OsINV4 and the monosaccharide transporter OsMST8 genes under cold stress (Oliver et al., 2005; Sheoran and Saini, 1996). Thus, tissue sink activity (capacity to use assimilates) is correlated with tolerance to cold and drought
T
stress in rice and wheat (Ji et al., 2010; Oliver et al., 2005). Table 2 summarizes the
IP
hormonal and metabolic-related genes affecting sink strength.
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A similar situation seems to occur in dicotyledonous species such as tomato (see Pérez-Alfocea et al. (2010) for a review). Decreased fruit number is a major factor reducing tomato yield at high salinity (>75–100 mM NaCl), in addition to decreased
NU
fruit weight (Cuartero and Fernández-Muñoz, 1998). Processes involved in fruit set and development must be responsible for yield reduction under salinity. A highly saline treatment (150 mM NaCl for 10 days) decreased pollen viability, inducing tomato
MA
flower abortion (Ghanem et al., 2009). However, low pollen viability was apparently not explained by local Na+ toxicity in either pollen grains or the tapetum. Instead, flower abortion was apparently explained by decreased carbohydrate availability in the and
pollen-producing
D
inflorescence
tissues,
despite
increased
carbohydrate
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concentrations within source leaves (Ghanem et al., 2009). This is probably due to reduced transport from source leaves and decreased sink activity, as suggested by
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dramatic reductions in sucrolytic activities of cwInv and sucrose synthase during floral development and maturation. Thus cwInv seems essential in maintaining sucrose import to sink tissues (Koch, 2004; Roitsch et al., 2003; Roitsch and Ehness, 2000) during
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pollen development (Roitsch and González, 2004), especially under potential sourcelimiting stress conditions. Assuming that the flower is fertilized, fruit or grain filling is also a susceptible process limiting yield. For example, tomato yield reduction by low to moderate salinity levels (25–75 mM NaCl) in irrigation water is due more to decreased fruit weight than number (Cuartero and Fernández-Muñoz, 1998). In general, salinity decreased both sink strength and sink activity measured as absolute and relative rates of dry matter accumulation during early fruit development (from 20 days after anthesis until start of ripening). Decreased sink activity was related to sucrose accumulation, and to decreased activity of the apoplastic and cytoplasmic sucrose cleaving enzymes; namely, cell wall invertase, neutral invertase and sucrose synthase. Genotypic differences in these enzyme
21
ACCEPTED MANUSCRIPT activities were correlated with the degree of salt tolerance of domestic and wild tomato species and hybrids (Balibrea et al., 2003; Balibrea et al., 1999; Koch, 2004). The importance of sucrose metabolism in grain filling has also been
T
demonstrated in rice. The GIF1 (Grain Incomplete Filling 1) gene encodes for cwInv
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required for carbon partitioning during early grain filling (Wang et al., 2008). Grains
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from the loss-of-function gif1 mutant show decreased glucose and fructose levels and sucrose accumulation, resulting in lower grain weight than the wild-type. The cultivated GIF1 allele showed a restricted expression pattern during grain filling compared with
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the wild rice allele because of accumulated mutations in the regulatory sequence of the gene by domestication. Ectopic expression of the cultivated GIF1 gene under the control of the 35S or rice Waxy promoters resulted in smaller grains, whereas
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overexpression of GIF1 driven by its native promoter increased grain production (Wang et al., 2008). It has been interpreted that the restricted expression of the GIF1 in the ovular vascular trace is key to increasing grain weight, suggesting that tissue-specific
TE
D
higher expression levels of GIF1 could provide a means to increase grain filling. Thus, tomato plants overexpressing a cwInv under the control of a fruit specific
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promoter (InvLp6g::CIN1) yielded up to 4-fold more than the WT and azygous lines under moderate salinity (75 mM NaCl) due to both decreased flower abortion (higher fruit number) and also due to a significant increase in the individual fruit weight.
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Increased sucrose partitioning from source leaves to the fruits in the salinized transgenic plants (compared to the WT and azygous lines) was explained by increased cwInv gene expression and activity, but curiously also by increased levels of trans-zeatin (t-Z) and reduced concentrations of the ethylene-precursor ACC (Albacete et al., unpublished results).Thus cwInv seems to function at the integration point of metabolic, hormonal and stress signals, providing a novel strategy to overcome drought- and salt-induced limitations to crop yield, without negatively affecting plant fitness under optimal growth conditions. An additional potential target to negative yield impacts of salt and drought conditions is the posttranslational relief of invertase from inhibition by a proteinaceous inhibitor (Ruan et al., 2010). Only recently, in vivo functionality and the physiological significance of these inhibitors on plant growth, development and stress responses have been demonstrated (Bonfig et al., 2010; Jin et al., 2009). Such post-translational
22
ACCEPTED MANUSCRIPT regulation is particularly relevant to cwInv because these proteins are intrinsically stable due to their glycosylation (Rausch and Greiner, 2004) Downregulation of some sucrolytic activities in response to drought or salinity
T
may be attenuated by ectopic regulation of these enzymes using promoters that are
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specific to sink tissues. However, these enzymes seem tightly regulated by
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phytohormones as part of an integrated and coordinated mechanism to control growth and development, and thus changes in phytohormone concentration provoked by the stress in vegetative tissues may decrease sucrolytic activities under salinity (Albacete et al., 2008; Pérez-Alfocea et al., 2011; Roitsch et al., 2003). Internal water status or the
NU
accumulation of toxic ions (Na+, Cl-) seem to be maintained in floral organs even following substantial leaf dehydration (i.e. droughted wheat, reviewed in Saini et al.
MA
(1999)) or Na+ accumulation (i.e. salinized tomatoes, Balibrea et al. (1999); Ghanem et al. (2009)). However, positive (i.e. CKs, GAs, auxins) and negative (i.e. ethylene, ABA) hormones coming from mature leaves or roots in response to stress may be good
D
candidates as signaling molecules, regulating sink activity in the reproductive organs at
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pre- or post-anthesis by influencing carbohydrate supply to the tapetum, microspores, and fertilized ovaries. Unravelling the regulation of those positive and negative signals
conditions.
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on sink activity could open new strategies to maintain growth and yield under stress
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3.2. Hormonal signalling and regulation of sink activity 3.2.1. Gibberellins
A large body of evidence supports the positive effects of exogenous gibberellic acid (GA3) application on plant growth and yield under stress conditions by inducing source and sink activities (reviewed in Iqbal et al. (2011)). GA3 increases source strength by improving photosynthetic efficiency via influencing some photosynthetic-related enzymes (i.e. Rubisco, fructose-1,6-biphosphatase and sucrose phosphate synthase), leaf area, light interception and phloem loading. Additionally, GA3 also induces sink strength by promoting cell division and growth and carbohydrate import through inducing the sucrolytic activities, namely the cwInv, a key enzyme in the regulation of phloem unloading (Roitsch and González, 2004). The induction of both source and sink strength increase the efficiency of assimilate production and transport, and thus source23
ACCEPTED MANUSCRIPT sink relations. GA signalling is necessary for normal tapetal function by influencing the availability of sugars to the tapetum (Plackett et al., 2011), but the effects of abiotic stress on GA level in developing anthers are not known (Parish et al., 2012).
T
Recently, it has been demonstrated in Arabidopsis and other model species that
IP
GA signalling is involved in metabolic adjustment for maintaining source–sink relations
SC R
of plants under limiting environmental conditions, and thus providing a plethora of potential molecular targets for biotechnological improvement of stress tolerance (Achard et al., 2006). Paradoxically, most of these studies indicate that GA signalling in
NU
response to stress seems to reduce growth to allow plant adaptation and survival, which cannot always be compatible with yield stability. Bioactive GA4 and GA1 levels decreased under high-salinity stress (100 mM NaCl) in Arabidopsis by more than 50%,
MA
which can be explained by the upregulation of six GA2oxidase genes, and thus reducing the levels of bioactive GAs (Achard et al., 2006; Magome et al., 2008). This response is mediated by the Dwarf and Delayed Flowering 1 (DDF1) gene, encoding an AP2
D
transcription factor of the DREB1/CBF subfamily that causes dwarfism by reducing
TE
bioactive GA concentrations in transgenic Arabidopsis (Magome et al., 2004). Further, the loss-of-function ga2ox7-2 mutant suppressed the dwarf genotype of DDF1–
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overexpressing plants and had similar shoot growth to the WT at 100-150 mM NaCl, although it showed increased root length at 50-100 mM NaCl (Magome et al., 2008). These results demonstrate that, under salinity stress, Arabidopsis plants actively reduce
AC
endogenous GA levels by inducing DDF1 and GA 2-oxidase, thus limiting growth for stress adaptation. Although other GA2oxs are upregulated by high-salinity stress in a DDF1-independent manner, DDF1 also directly upregulates the expression of several stress-responsive genes (such as RD29A and COR15A) that contribute to stress tolerance. A similar response occurred under osmotic stress (300 mM mannitol) and 100 μM ABA (Magome et al., 2008). Additionally, overexpression of the AtDREB1A in soybean (Glycine max L.) also produced typical GA-deficient dwarf phenotypes, due to the deactivation of bioactive GAs (Suo et al., 2012), with increased root length and tolerance to high salinity (300 mM NaCl) in tobacco (Cong et al., 2008), and drought resistance in tomato (SlDREB; Li et al. (2012)). These results suggest a key role in saltmediated sink growth impairment to the GA-DELLA signalling network (Achard et al., 2006).
24
ACCEPTED MANUSCRIPT Quadruple-DELLA mutants (lacking RGA, GAI, RGL1, and RGL2) had normal growth and lower salt tolerance under high (100-200 mM NaCl) salinity (Achard et al. (2006). Ectopic expression of the gain-of-function mutant Mhgai1 encoding a DELLA
T
protein (Malus hupehensis Redh. var. pingyiensis) in tomato reduced plant height and
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fruit set ratio, but it enhanced vegetative drought-tolerance (Wang et al., 2012). Moreover, a grafting experiment demonstrated the long-distance movement of Mhgai1
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mRNAs and its effect on dwarfing the scion genotype, which suggests root-targeted strategies to manipulate GA signalling in the plant and, consequently, source-sink relations and stress responses. Similar results were obtained by overexpressing the GA
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Methyl Transferase1 (AtGAMT1) gene that encodes for an enzyme that catalyzes the methylation of active GAs to generate inactive GA methyl esters (Varbanova et al.,
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2007). Tomato plants constitutively overexpressing the AtGAMT1 gene exhibited typical semi-dwarfed and greener GA-deficiency phenotypes and increased tolerance to drought stress (Nir et al., 2013). Transgenic plants maintained high leaf water status
D
under drought conditions, due to reduced stomatal conductance and whole-plant
TE
transpiration, thereby delaying desiccation and maintaining higher recovery rates. GA application to the transgenic plants restored normal growth and sensitivity to drought,
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supporting the role of GAs in coordinating growth and stress adaptation. Fruit growth in Arabidopsis also occurs via DELLA-dependent and DELLAindependent
(Gibberellin-Insensitive
DWARF1–mediated
GA
perception)
GA
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responses (Fuentes et al., 2012). Consequently, the growth of harvestable sinks is also regulated by GAs and increased endogenous GA concentrations could improve yield. Overexpression of the citrus gene CcGA20ox1, encoding the GA biosynthetic enzyme GA20-oxidase, in tomato (Solanum lycopersicum L. cv Micro-Tom) produced higher yield and number of fruits per plant with increased citric acid content (a component of ºBrix), and also elevated GA4 levels, thus mimicing GA treatments (García-Hurtado et al., 2012). Further studies of yield responses of GA-overproducing plants under adverse conditions are required to clarify the paradoxical relation between GAs, plant growth, plant survival and adaptation, and yield. 3.2.2. Cytokinins Similar to GAs, both increases and decreases in CK levels have been reported to have a positive impact on abiotic stress tolerance since CK homeostasis and signalling could 25
ACCEPTED MANUSCRIPT also play a key regulatory role between plant growth and plant survival (Ha et al., 2012; Zalabák et al., 2013). Consequently, CK impacts on source-sink relations and the subsequent morphological adjustment that optimizes growth and plant survival has a
T
different impact on crop productivity. While maintaining plant growth is a prerequisite
IP
for crop stability under stressful conditions, shoot growth inhibition and promotion of root growth (Sharp et al., 2004) has been regarded as an advantage in adverse
SC R
environments and is an integral part of plant stress tolerance to promote plant survival (Achard et al., 2006; Achard et al., 2008; Huang et al., 2009), as seems to occur in CKdeficient and CK receptor kinase mutants (Nishiyama et al., 2011). Hence, both
NU
constitutive overexpression of the CK-degrading enzyme cytokinin oxidase (CKX) or inhibition of the CK-biosynthetic IPT1, IPT3, IPT5, and IPT7 genes resulted in CK
MA
deficiency and enhanced drought- and salt stress-tolerant phenotypes. Enhanced drought tolerance of the CK-deficient plants was attributed to their capacity to maintain higher water content under stress, which was associated with their intact membrane structure,
D
as evidenced by lower electrolyte leakage (Nishiyama et al., 2011). Drought-tolerance
TE
was not explained by differences in stomatal opening or density since ABA response and concentration were similar to WT plants. Indeed, several studies have reported that
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plant growth responses to drought or salinity are not always a consequence of (or explained by) an alteration in stomata-related traits (Balibrea et al., 2000; Bartels and Sunkar, 2005; Fujita et al., 2006; Hirayama and Shinozaki, 2010). However, the tolerance was rather explained by a higher (i) ABA/CK ratio, (ii) sensitivity to ABA in
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the CK-deficient plants, (iii) subsequent higher expression of the ABA-related stress tolerance genes, such as structural protective proteins and/or intracellular solute concentrations(Verslues et al., 2006), (iv), shoot-to-root sink strength, as indicated above. This idea is also supported by the lower shoot sink activities due to both decreased cell number in leaf meristems of tobacco plants overexpressing the CKdegrading enzyme CKX (Werner et al., 2001) and sucrolytic activities (Werner et al., 2008). Similarly, growth inhibition of CK receptor- (Nishimura et al., 2004) and CK activating enzyme- (Kurakawa et al., 2007) defective mutants was associated with lower meristematic activity. However, CK-deficient and CK receptor His kinase mutants have enhanced root growth but retarded shoot growth, suggesting CKs are negative regulators in responses to water stress (Higuchi et al., 2004; Jeon et al., 2010; Miyawaki et al., 2006; Nishimura et al., 2004; Riefler et al., 2006; Tran et al., 2004; Werner et al., 2003), thus confirming a key role for a threshold CK concentration or sensitivity in 26
ACCEPTED MANUSCRIPT regulating plant growth and biomass partitioning, which seems critical for plant survival under abiotic stress. Nevertheless, increased CK levels under abiotic stress conditions were
T
proportional to the induced growth rate compared to the control plants. In tomato, root-
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specific IPT expression promoted leaf expansion up to 1.5-fold, shoot growth up to 1.9-
SC R
fold, and maximum efficiency of PSII (Fv/Fm) in salinized plants, compared to control plants (Ghanem et al., 2011a). Thus the average 2- to 2.5-fold increase in shoot biomass and relative growth rate was associated with a 2- to 2.6-fold increase in leaf CK
NU
concentration, while the 4.5-fold increase in biomass of droughted tobacco was associated with up to 5-fold increase in leaf CK concentration (PSARK::IPT; Rivero et al. (2007)). Moreover, grafting WT plants onto a constitutively (35S) expressing IPT
MA
rootstock increased fruit yield (by 30%) compared with salinized self-grafted WT/WT plants. This effect was probably due to increased shoot development and/or reduced flower abortion (as suggested by the 25% increase in fruit number), where increased t-Z
D
concentrations (from 1.5- to 2-fold) in the actively growing fruits promoted cell division
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and expansion, thus slightly increasing (by 5%) fruit weight (Ghanem et al., 2011a). In contrast, transiently elevated fruit CK concentrations (up to 9-fold) very early in tomato
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fruit development did not increase final fruit size or total fruit yield per plant in the absence of stress (AGPase::IPT; Luo et al. (2005)). However, IPT expression in senescing leaves increased individual tomato fruit weight by 20% (SAG12::IPT;
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Swartzberg et al. (2006)), probably due to increased sink assimilate import because of delayed source leaf senescence. Shoot growth maintenance (induced HSP70::IPT plants) and increased fruit production (WT/35S::IPT plants) under salinity may be result from not only increased source capacity to produce and/or export more assimilates due to both increased leaf area and delayed senescence, but also increased import and/or utilization of assimilates in sink organs achieved by an increased cell population attracting photoassimilates and/or an increased capacity to utilize imported sucrose metabolically through regulating sucrolytic enzymes (Lara et al., 2004; Roitsch and González, 2004). Depending on the promoter used and the growing conditions, it generally seems that the physiological impacts of overcoming decreased CK status (through transgenic IPT expression) were most pronounced in plants experiencing abiotic stress. However, 27
ACCEPTED MANUSCRIPT excessive overproduction of CKs above a threshold also caused stunted plant growth, abnormal tissue development, and sensitivity to drought (Havlová et al., 2008; Hewelt et al., 1994; Li et al., 1992; Synková et al., 1999; Wang et al., 1997). Consequently,
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appropriate manipulation of CK levels is necessary to increase leaf longevity and
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photosynthetic capacity (source strength) but also growth of sink organs (sink strength)
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under abiotic stress. 3.2.3. Auxins
Auxins can influence carbon partitioning in plants, and a number of studies have shown
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that both factors are interrelated. Thus auxins stimulate the mobilization of carbohydrates in leaves and the upper stem and increase the translocation of assimilates
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toward sink organs (Smith and Samach, 2013). Auxins may also influence carbohydrate utilization. Additionally, the interplay of auxin with other hormones may ultimately regulate carbohydrate levels and sugar signaling molecules (Hartig and Beck, 2006;
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Smith and Samach, 2013). Agulló-Antón et al. (2011) highlighted the interactions
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between auxin and carbohydrates and their regulatory role in sink establishment and growth in carnation cuttings. A transcriptomic study on hybrid embryos of Vicia faba
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has shown the importance of a set of genes related to auxin function and metabolism on the sink strength of hybrid seeds (Meitzel et al., 2011). These authors proposed a regulatory model on seed carbon allocation and growth where the auxin-related genes
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AUX/IAA, AXR1, and RBX1-like E3 gene play key roles. Further, it has been reported that auxins regulate the activity of the cwInv and thus sucrose allocation in sink organs (Albacete et al., 2008; Proels and Roitsch, 2009; Roitsch et al., 2003). To understand the molecular details of sugar–invertase-IAA relationships, genes from the two IAA biosynthetic pathways ZmYuc1 (tryptamine pathway) and ZmTar1 (indolpyruvic acid pathway) were studied in developing seeds of the invertase-deficient mutant miniature1 of maize (Chourey et al., 2010a). There were redundant trpdependent pathways of auxin biosynthesis in developing maize seeds, suggesting that homeostatic control of IAA in this important sink is highly complex and may be regulated by both sucrose metabolism and developmental signals, since cell division is fuelled by heterotrophic metabolism based on the supply of carbohydrates from source leaves. Furthermore, crosstalk between auxins, cytokinins and sugars has been reported in regulating the plant cell cycle (reviewed by Hartig and Beck (2006)). 28
ACCEPTED MANUSCRIPT 3.2.4. Abscisic acid ABA synthesis in the roots, and transport through the xylem to the shoot is one of the most rapid hormonal responses to drought and salinity, causing stomatal closure to
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reduce transpiration water loss and eventually restricting cellular growth. ABA can also
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be synthesized in leaf cells and transported through the plant (Dodd, 2005; Thompson et
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al., 2007), while guard-cell ABA production seems autonomous to regulate stomatal response (Bauer et al., 2013). ABA has been implicated in male sterility of tomato, rice and wheat (Morgan and King, 1984; Morgan, 1980; Westgate et al., 1996), with
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negative correlation between ABA levels and cold and drought tolerance (Ji et al., 2010; Oliver et al., 2007). Exogenous ABA application mimics the effect of stress provoking pollen sterility in barley by reducing sucrose import by inhibiting invertases and
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monosaccharide transporters, thus preventing microspores from PCD (Parish et al., 2012).
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Nevertheless, and contrarily to the reported negative effect of ABA on pollen
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viability some studies in cereals reported positive correlations between grain ABA content and efficient seed filling by optimizing faster remobilization events from stem
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reserves (nonstructural carbohydrates), a critical factor in sustaining grain filling and grain yield under drought stress (Yang et al., 2004; Yang et al., 2001). Indeed, overexpressing the LOS5 gene that encodes the key enzyme molybdenum-cofactor
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sulfurase in the last step of ABA biosynthesis, under the control of constitutive or drought-inducible promoters improved yield when water stress occurred during the initiation of panicle development by significantly increasing spikelet fertility. In contrast, the NCED2 gene, also involved in ABA biosynthesis, had no positive (or even negative) effects when compared to the WT (Xiao et al., 2009). Different responses in those transgenic plants may be due to the different hormone concentrations before and during the stress period. Indeed, the constitutive overexpression of LeNCED1 (droughtinducible and a rate-limiting enzyme for ABA biosynthesis) in tomato continuously elevated ABA accumulation, resulting in physiological and morphological changes in the transgenic plants. Under well-watered conditions, plants showed, leaf flooding, chlorosis and reduced assimilation rates occurred, but under water-deficit conditions these effects were insufficient to reduce biomass production, presumably because of counteracting positive effects on leaf expansion by improving water status and turgor, 29
ACCEPTED MANUSCRIPT and antagonising ethylene-induced epinastic growth (Thompson et al., 2007). Similarly, natural constitutively elevated ABA production in the citrus rootstock induced tolerance to water stress in sweet orange (Citrus sinensis) trees allowing a better regulation of
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gas-exchange parameters in the shoot, but reduced growth under control conditions
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(Allario et al., 2013). Interestingly, the drought tolerant (autotetraploid rootstock clone from Rangpur lime, Citrus limonia) graft combination showed decreased stomatal
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conductance, higher leaf and root ABA concentrations, and an increased expression of drought responsive genes, including CsNCED1 (key for ABA biosynthesis) than the same plant grafted onto the diploid rootstocks. These results highlight the importance of
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both constitutive (non-induced) expression of defence mechanisms and the longdistance ABA signalling in the adaptation to stress (Allario et al., 2013). The effect of
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pre- or over-elevated ABA on yield under optimal or suboptimal conditions needs to be addressed.
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3.2.5. Jasmonates
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The effect of jasmonates on sink activity may differ depending on the tissue and on the nature of the stress. Transgenic rice expressing PUbi1::AtJMT (JA carboxyl
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methyltransferase) displayed 6-fold increased JA levels and significantly lower grain yields (because of fewer spikelets per panicle, reduced grain filling rate and altered floral organ numbers), which was reproduced by applying exogenous methyl jasmonate
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(MeJA) (Kim et al., 2009). Interestingly, MeJA levels were increased by 19-fold in young WT panicles upon exposure to drought conditions, decreasing grain yield to a similar level to that observed in Ubi1::AtJMT plants. Leaf ABA concentrations only increased by 1.5-2-fold in non-droughted Ubi1::AtJMT and droughted WT plants, suggesting that biosynthesis was secondarily stimulated under drought conditions by the previous MeJA accumulation (Kim et al., 2009). Indeed, the OsSDR gene, an Arabidopsis ortholog which is essential for JA and ABA biosynthesis (Adie et al., 2007; González-Guzmán et al., 2002), was up-regulated in both Ubi1::AtJMT and droughttreated WT panicles, supporting its involvement in MeJA-dependent increases in ABA levels, and that ABA either precedes or cooperates with JA in signalling pathways under drought conditions (Kim et al., 2009). Similarly, Arabidopsis plants transformed with 35S::AtJMT produced 40% less total seed weight than WT controls due to fewer flowers (Cipollini, 2007), and the 30
ACCEPTED MANUSCRIPT application of exogenous MeJA on Pharbitis nil (Maciejewska and Kopcewicz, 2002) and Nicotiana sylvestris (Baldwin and Hamilton, 2000) dramatically reduced flowers number, which was explained by increased sterility (Zhu et al., 2004). In addition to the
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cost associated with constitutive overexpression of AtJMT and overproduction of MeJA,
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decreased energetic allocation towards reproductive structures in favour of activation of defence mechanisms cannot be ruled out (Cipollini, 2007; 2010). It is likely that
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increased levels of MeJA reduce spikelet number in Ubi1::AtJMT panicles by repressing cell cycle progression (Kim et al., 2009). This may be due to decreased assimilate transport to the reproductive structures due to changes in sink preferences under stress. 11
CO2 fixation and
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C-photosynthate
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In this regard, it was recently reported that both
export from the labelled source leaf increased rapidly (2 h) following MeJA treatment
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(simulating herbivore wounding action) relative to controls, with preferential allocation of radiolabel primarily to the roots and secondly to the young leaves, where label was incorporated into
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C-cinnamic acid and other defence-related phenolic compounds as
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anthocyanins (Ferrieri et al., 2013). The switch in relative sink strength correlated with
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increased cell wall and soluble invertase activities in both roots and young leaves and was suppressed in sucrose transporter mutant plants (suc2-1), indicating that this
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phenomenon may be controlled by phloem loading/unloading processes. It would be interesting if the same MeJA-mediated phenomenon could explain yield decrease under abiotic stress conditions.
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Since adventitious root formation requires an adequate supply of carbohydrates to the region of root regeneration, it has been used as model system to study the molecular and biochemical processes involved in the interaction between JA and primary metabolism. In a study with Petunia hybrida cuttings, increased transcripts coding for cwInv and the apoplastic localized monosaccharide transporter gen STP4 were related to transient JA accumulation (Ahkami et al., 2009). Similarly, Henkes et al. (2008) showed that JA treatment to a part of barley roots caused signal transduction from the treated roots to the shoot, leading to an increase in carbon allocation from the leaves, simulating a wounding effect. Therefore, bioactive jasmonates as JA and MeJA could be involved in redirecting resources from growth to defence mechanisms (Chen et al., 2011). This
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ACCEPTED MANUSCRIPT differential metabolic response may be responsible for the decrease in yield-related parameters in both droughted and jasmonate overproducing plants.
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3.2.6. Salicylic acid
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It has been considered that SA plays a crucial role in the plant energy status,
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translocation and storage of assimilates in response to biotic and abiotic factors. Carbohydrate metabolism in both source and sink tissue was regulated by SA activating plant defence response against pathogens and other biotic stresses through increasing invertase activity or vice versa (Ehness and Roitsch, 1997; LeClere et al., 2008). Foliar
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SA (10-6-10-4 M) sprays decreased hexose concentrations in the leaf but increased them in fruits of salinized pepper plants, with concomitant increases in sink strength and fruit
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growth (Elwan and El-Hamahmy, 2009). Increased sink strength by exogenous application of SA was also found in wheat (Arfan et al., 2007) and maize (Gunes et al., 2007). Further, SA can interact with other hormones in regulating source-sink
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relationships under stress conditions (Iqbal et al., 2011). For example, constitutive
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overexpression of a GA-responsive gene FSGASA4 in Arabidopsis plants subjected to salt stress increased not only the endogenous levels of SA, but also the expression of
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ICS1 and NPR1 genes involved in SA biosynthesis and action, respectively (AlonsoRamírez et al., 2009a; b). This hypothesis was supported by the finding that sid2 mutants, impaired in SA biosynthesis, were more sensitive to salt stress than the wild
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type without any apparent effect of exogenous application of GAs (Alonso-Ramírez et al., 2009b).
3.2.7. Ethylene
Although ethylene can stimulate the growth of certain plant organs under specific circumstances (i.e. internode extension of deep-water rice), it is often regarded as a growth inhibitor (Pierik et al., 2007). Exogenous application of the ethylene-releasing compound ethephon decreased both root and fruit sink strength in salinized tomato plants by decreasing invertase activity (Albacete et al, unpublished results). Thus, researchers have pursued chemical, genetic, and even microbiological ways of decreasing crop ethylene production. In this sense, some rhizosphere bacteria use ACC, the immediate precursor of ethylene in higher plants, by hydrolysing it through the enzyme ACC deaminase into α-ketobutyrate and ammonia, and in this way promote 32
ACCEPTED MANUSCRIPT sink strength and root growth by lowering endogenous ethylene levels in the microrhizo environment (Hayat et al., 2010). Different biotechnological strategies have been also use to reduce ethylene biosynthesis and perception (reviewed by Stearns and Glick
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(2003)), mainly focused on the ACC deaminase gene, and have been shown in many
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cases to regulate carbon metabolism and sink strength. Additionally, attempts were made to manipulate the growth and development of the pollen grains by exogenous
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application of chemicals regulating formation/action of ethylene. For example, application of ethylene action (AgNO3) and synthesis inhibitor (Co(NO3)2, paclobutrazol and uniconazole) improved grain setting in rice (Naik and Mohapatra,
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1999), related to enhanced activities of acid invertases and sucrose synthase.
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3.2.8. Strigolactones
The novel class of plant hormones strigolactones (SLs) has a clear impact in source-sink relations, since they modulate shoot branching and height and could have an important
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implication in crop productivity in diverse species (de Saint Germain et al., 2013). For
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example, the reduction in SL production in rice mutants affected in carotenoid-derived MAX/RMS/D (more axillary branching) pathway resulted in increased tillers and
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reduced plant height and spikelet fertility (Lin et al., 2009; Liu et al., 2013) Although the effect of SL on stem elongation seems to be independent of GAs (de Saint Germain et al., 2013), strong evidence supporting the role of SLs in the regulation of growth
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redistribution in the shoot by rapidly modulating auxin transport (Shinohara et al., 2013) has been provided. This SL-mediated regulatory effect is important to coordinate biomass reallocation in response to stress, since auxin has been proposed to mediate the systemic growth coordination by sensing the environmental cues. In particular, a positive feedback mechanism that establishes auxin flow from active shoot meristems (auxin sources) to the roots (auxin sinks) has been proposed to mediate competition between shoot meristems and to balance shoot and root growth (Shinohara et al., 2013). Indeed, this regulatory mechanism might be responsible for the plant response to nutrient starvation, water and saline stresses, since an induction in SL production would minimize shoot branching, increase the root/shoot ratio and promote root colonization by AM fungi, thus alleviating the stress impact (Aroca et al., 2013; Xie and Yoneyama, 2010). Further research is required to elucidate the potential of SL manipulation in order
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ACCEPTED MANUSCRIPT to improve crop productivity by altering biomass allocation under abiotic stress conditions.
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3.3. Root-sink strength to improve stress adaptation
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Many genes having a positive effect on abiotic stress tolerance seem to have a direct or
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indirect effect on source-sink relations, including the roots. Although enhanced root growth could be initially considered as a waste of assimilates to the detriment of shoot growth and yield, it has been hypothesized that modified sucrose allocation to the roots in plants growing under saline or drought stress may be an adaptive response to recover
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the functional equilibrium of the plant under the new conditions (Albacete et al., 2008; Balibrea et al., 2000; Sharp et al., 2004; Siahpoosh et al., 2012). Indeed, root-specific
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traits can be exploited to improve resource (water and nutrients) capture and plant development under resource-limited conditions (Ghanem et al., 2011b). The reduction of root sucrose levels in sensitive rice cultivars after exposure to high-salt
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concentrations was associated with a complex metabolic depletion syndrome
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(Siahpoosh et al., 2012). Because sucrose transporters were previously implied to be part of the rice salt acclimation process (Shinozaki and Yamaguchi-Shinozaki, 2007),
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the increased supply of assimilates to the root organ (i.e. manipulation of the sucrose transporters and/or invertases) should be considered in breeding programs for increasing salt tolerance, which would help to maintain root functioning (i.e. toxic ions exclusion)
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and adaptation to stress (Ghanem et al., 2011a; Ghanem et al., 2011b; Pérez-Alfocea et al., 2010; Siahpoosh et al., 2012). Increased assimilate partitioning to the roots of salinized tomato plants can be explained by changes in cwInv activity in both roots and leaves (Albacete et al., 2008), as reported in CK-deficient tobacco plants (Werner et al., 2008). This enzyme regulates sucrose transport by controlling the apoplastic unloading step from phloem (Roitsch et al., 2003). However, although CKs are major hormones establishing sink activity through the co-ordinated induction of cwInv and hexose transporters in order to enhance phloem carbohydrate supply to actively growing tissues (Roitsch et al., 2000), this response seems to depend on the tissue, and CKs are unlikely to be directly responsible for the cwInv induction in the roots. This idea is supported by the induction or maintenance of this enzyme activity reported in roots of CKXoverexpressing tobacco plants with lowered total and bioactive CKs (Werner et al., 2001). 34
ACCEPTED MANUSCRIPT However, IAA seems to induce both sink (sucrose allocation) and cwInv activities in both young leaves and roots (Albacete et al., 2008; Roitsch et al., 2003), supporting its role in the relative increase in root sink strength under drought and
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salinity. Indeed, lateral root initiation is suppressed by ABA signals while auxin is a
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crucial phytohormone for the initiation of lateral roots by promoting lateral root formation under water stress (Seo et al., 2009). Several MYB genes have been identified
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constitutive
expression
of
the
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for their important roles in auxin-induced lateral root formation (Dubois et al., 2013). R2R3-MYB
transcription
factor
gene
35S::MdSIMYB1 from apple (Malus × domestica) in tobacco and apple resulted in
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ABA- and NaCl- insensitive germination and enhanced tolerance to high salinity, drought and cold by up-regulating the stress-responsive genes NtDREB1A, NtERD10B
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and NtERD10C (Wang et al., 2013). Transgenic tobacco plants also exhibited increased root sink strength (measured as biomass allocation) because of enhanced expression of the auxin-responsive genes NtIAA4.2, NtIAA4.1, and NtIAA2.5 under stress conditions.
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Gene transcripts were abundant in the leaves, flowers, and fruits compared to other
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organs, and were induced by abiotic stresses and plant hormones. Dexamethasone-induced expression of the Arabidopsis HARDY (HRD) gene,
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encoding for AP2/ethylene response factor (ERF)-like transcription factor that belongs to the BREB/CRB family in rice, improves WUE by enhancing photosynthetic assimilation (40-55% more than WT under control and drought conditions, respectively)
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and reducing transpiration, thus increasing both intrinsic (photosynthetic rate/stomatal conductance) and agronomical (biomass/water use) WUE (Karaba et al., 2007). Although no yield data were provided, these drought-tolerant low-water-consuming rice plants exhibit increased shoot biomass under well watered conditions and an adaptive increase in root biomass under drought stress (soil at 70% of field capacity) without growth penalty under control conditions. The HRD gene was identified by a gain-offunction Arabidopsis mutant hrd-D having roots with enhanced strength, branching and cortical cells, which exhibited drought resistance and salt (300 mM NaCl) tolerance accompanied by enhanced expression of abiotic stress-associated genes. HRD overexpression in Arabidopsis produced thicker leaves with more chloroplast-bearing mesophyll cells and, in rice, leaf biomass and bundle sheath cells were increased which probably contributed to the enhanced photosynthetic assimilation and efficiency. The mechanism(s) involved in regulating these responses remains unknown, but gene 35
ACCEPTED MANUSCRIPT clusters repressed under drought are up-regulated by HRD, suggesting a protective influence on essential processes, such as protein biosynthesis and carbohydrate metabolism (Karaba et al., 2007). Increased photosynthesizing area and carbon
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assimilation (better mesophyll efficiency) would significantly improve canopy
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photosynthesis, enhancing the capacity of both source and sink tissues and resulting in high biomass. Therefore, the increase in root-sink activity is an example not only of
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adaptation to drought and salinity, but also of source activity maintenance, as indicated by the unaltered maximum quantum yield of PSII (Fv/Fm), with a potential positive
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impact on yield stability. 3.4. Increasing sink number
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Modifying plant architecture, which implies alterations in source-sink balance, can potentially increase crop yield also under suboptimal conditions, as stated above. Since grain yield is controlled by QTLs derived from natural variations in many crop plants,
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fruit/grain number and weight are the most important traits in determining crop
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productivity. Three promising recent examples alter the plant architecture through modifying shoot meristematic sink activity. Grain number 1a (Gn1a) is a major QTL of
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grain number in rice that encodes the CK-degrading enzyme OsCKX2. This gene causes CK accumulation in the inflorescence meristem and increases the number of reproductive organs, increasing grain number per panicle (45%) and per plant (34%) in
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the near isogenic line (NIL)-Gn1a with respect to the Koshihikari genetic background (Ashikari et al., 2005). Similarly, the gain-of-function mutation allele of DEP1 (Dense and Erect Panicle1), coding for a truncated phosphatidylethanolamine-binding proteinlike domain protein, enhances meristematic activity and reduces length of the inflorescence internode resulting in three dominant pleiotropic phenotypes: dense and erect panicle, increased number of grains per panicle and a consequent increase in grain yield (40%), although they also have decreased grain weight (Huang et al., 2009). More recently, a point mutation in the OsSPL14 (Souamosa Promoter Binding Protein-Like 14 gene), located in the IPA1 (ideal plant architecture) QTL, perturbs the microRNA OsmiR156-directed regulation of OsSPL14, generating an ideal rice plant with a reduced tiller number, increased lodging resistance and more and bigger grains per panicle (Jiao et al., 2010). It was demonstrated that introducting the OsSPL14 ipa1 allele into the common japonica rice variety Xiushui 11 cultivated in the South of China increased the 36
ACCEPTED MANUSCRIPT grain yield by more than 10% in the test plot (Jiao et al., 2010). The yield stability of those high yielding lines under changing environments needs to be addressed given the critical role of CKs and microRNAs in both plant development and response to abiotic
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stresses (see Lima et al. (2011), for a review).
4. Conclusions
Figures 1 and 2 summarize the different hormonal and metabolic factors that regulate
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source-sink relations with demonstrated or potential impact on plant performance under abiotic stresses that have been addressed in this review. Since salinity and drought
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decrease crop productivity by reducing the activity of the source leaves, and/or the number and size of the harvestable sink tissues, maintaining source and sink activities is the best strategy to preserve crop productivity. However, the reallocation of assimilates
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between different sink organs also represents an adaptive response to stress in terms of
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plant survival and/or growth (and yield) stability. Delayed senescence with concomitant growth penalty (plant survival) could help to withstand transient stress but will not
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guarantee food security under permanent environmental constraints. Nevertheless, since the physiological plant adaptation has an energetic cost, yield stability will probably require an increase in either photosynthetic and/or adaptability efficiencies. Photosynthetic efficiency (source activity) can be optimized in an integrated process
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with stomatal opening, assimilate transport and sink activity (optimization of sourcesink relations), probably requiring a modelling approach. However, Omics technologies have revealed hundreds of genes involved in adaptation and maintenance of source activity by delaying senescence, but in detriment of growth (sink activity). Ideally, maintaining or increasing sink activity in the plant (individual or collectively) will help to avoid initial photoinhibition and premature stress-induced senescence, thus maintaining assimilate production and transport in/from the source tissues. For this reason it is essential to gain insights into the factors regulating sink activity (i.e. at inflorescence setting, flower fertilization and fruit/grain filling). Hence, the discovery of any novel gene involved in stress adaptation in model species should include an investigation on sink activity and, ultimately, on yield performance in crop plants. Increasing sink activity in the reproductive organs by either (i) the overexpression of sucrolytic activities, for example under the control of a fruit/grain specific promoter, or 37
ACCEPTED MANUSCRIPT (ii) the positive (i.e. CKs, GAs) or negative (i.e. ethylene) modulation of hormonal factors regulating the sink number and activity, could be useful strategies for improving yield under suboptimal abiotic stress conditions. Other specific metabolic alterations
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(i.e. carbon metabolism) can also improve the photosynthesis and WUE under resource
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limiting conditions. Finally, some of those factors seem to be able to simultaneously increase or maintain both source and sink activities (i.e. CK signalling; cell wall
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invertase) under stress, thus minimizing the negative impact on yield. Root-targeted strategies could be used to optimize source-sink relations through manipulating root-to-
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shoot hormonal and genetic (ie. miRNA) communication.
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Acknowledgements
AA is very grateful to CSIC (Spain) for a postdoc research grant (I3P program). The
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authors thank Dr Ian C Dodd for revising the English version of the manuscript and for
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constructive criticism, the European Commission (ROOTOPOWER Contract # 289365), the EU COST office (action FA1204 on vegetable grafting), the Fundación
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Séneca de la Región de Murcia, Spain (project 08712/PI/08), and the Spanish MINECO-FEDER (AGL2011-27996/AGR) for support of source-sink relations and
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hormonal signalling research.
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ACCEPTED MANUSCRIPT Legend to figures
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Figure 1. Metabolic (right) and hormonal (left) factors regulating source-sink relations that could influence plant growth, adaptation and yield under drought and salinity. See main text and Tables for explanation. Arrow or bar heads indicate positive and negative regulation, respectively. Abbreviations: ACCd, ACC deaminase; AGPs, ADP-glucose pyrophosphorylase; AREB/DREB, abscisic acid/dehydration responsive element binding transcription factors; CBF2, C-repeat/dehydration-responsive element binding factor; CCD, carotenoid cleavage deoxygenase; CIN1, cell wall invertase (cvInv); CKX, cytokinin oxidase; CSA, MYB domain protein; CYP, sterol C-22 hydroxylase; DDF, Dwarf and delayed flowering gene; ERF, ethylene response factor; G1-1/2, genes involved in sink activation; GAMT, gibberellin methyl transferase; GAox, gibberellin oxidase; GIF1, Grain Incomplete Filling 1, cell wall invertase; GPT, glucose-6phosphate/translocator; HRD, AP2/ethylene response factor-like transcription factor; IAA, indole 3-acetic acid responsive genes; IPT, isopentenyltransferase; IVR1, anther cell wall invertase; JMT, jasmonic acid carboxyl methyltransferase; LOS5,molybdenum-cofactor sulfurase; MADS, regulator of starch synthesis; MAX, more axillary growth; MYB, transcription factor; NCED, 9-cis-epoxycarotenoid dioxygenase; NTT1, adenilate translocator; ORF, rolB-like part gene PEPC; phosphoenolpyruvate carboxylase; PIN, indole 3-acetic acid transporter; SPS, sucrose phosphate synthase; SSIV, starch synthase; SUC2, phloem-localized sucrose/H+ symporter; SUS, sucrose synthase; SUT, sucrose transporter; TDY1, phloem-expressed transmembrane protein; TZF, CCCH-type zinc finger transcription factor.
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Figure 2. Interactions between hormones and carbon metabolism in the regulation of source and sink activities, phloem assimilate (un)loading and transport, and their relative influence on plant survival (only source activity is required) and on growth/yield (sink activity and transport from the source are also required) related processes under drought and salinity conditions. See main text and Tables for explanation, and legend to Figure 1 for abbreviation list. Arrow or bar heads indicate positive and negative regulation, respectively.
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ACCEPTED MANUSCRIPT Table 1 List of genes related to source strength, and their effect on growth, plant survival and/or yield Effects on growth/survival/yield
CBF2
Overexpression of CBF2 in Arabidopsis (Luo et al., 2013; Sharabi-Schwager et al., 2010a)
Delay of senescence by interaction with ABA-, JA-, SA- and ethylene-related genes
CBF1, CBF2, CBF3
Overexpression of CBF1, CBF2, and CBF3 in Arabidopsis (Gilmour et al., 2004; Liu et al., 1998)
Growth retardation and dwarf phenotype by accumulation DELLAs, incompatible with yield promotion
ERF6
ERF6 overexpression in Arabidopsis (Skirycz et al., 2011)
Hypersensitive to osmotic stress, and inhibition of cell proliferation and growth via GA degradation by GA2-oxidase6
ERF5, ERF6
erf5erf6 loss-of-function double mutants (Dubois et al., 2013)
Arabidopsis
Less affected by salinity, but no clear effect on growth
SlERF5
SlERF5 overexpression in tomato under drought and salinity stress (Pan et al., 2012)
Increased tolerance to drought and salinity, increasing plant survival but without additional effect on growth
OsTZF1
Overexpression of OsTZF1in rice (Jan et al., 2013)
Delay of salt-, JA-, and ABA-induced senescence, and improved tolerance (plant survival) to high salinity and drought
SlAREB1
Overexpression in tomato of the AREB1 transcription factor implicated in the expression of ABA-related genes (Orellana et al., 2010b)
Increased plant survival under drought and salinity stress through senescence-delaying and water-conservative mechanisms
SAG12::IPT
Overexpression of the IPT gene under the control of senescence promoter SAG12 in tobacco plants (Cowan et al., 2005)
Increased CK content in SAG12::IPT plants impacts source-sink relations
PSARK::IPT
Overexpression of the IPT gene in rice (Peleg et al., 2011)
SAG39::IPT
Identification and application of a rice senescenceassociated promoter (Liu et al., 2010)
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ZmCYP, AtCYP, OsCYP
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Gene Description of the study Hormone-related genes
Overexpression of genes encoding sterol C-22 hydroxylases that control brassinosteroid levels in rice (Wu et al., 2008)
Source-sink modifications and strong sink capacity of PSARK::IPT plants during water stress SAG39::IPT plants show increased yield and rapid sugar reutilization at the onset of leaf senescence Brassinosteroids stimulates the flow of assimilates from the source to the sink, increasing grain yield
Carbon metabolism-related genes
ADP-Glucose pyrophosphorylase mutation (agpsm1) in maize (Schlosser et al., 2012)
Leaf maize starch mutation diminished field growth and productivity
C-metabolism and other genes
Transcriptome profiling in Vitis vinifera berries (Pastore et al., 2011)
Increasing source-sink ratio induces extensive transcriptome reprograming
AGPase, SUS, SUT1, SUT2, SPS
Sucrose metabolism genes under sink limitation in Citrus (Nebauer et al., 2011)
Inhibition of sucrose synthesis and transport genes, and increase in AGPase gene
SSIV
Overexpression of starch synthase SSIV in Arabidopsis and potato (Gámez-Arjona et al., 2011)
SSIV overexpression lead to accumulation of starch in source and sink organs, increasing growth rate and yield
OsSUT2
Study of ossut2 rice mutants (Eom et al., 2011)
Essential role of SUT2 in sugar export from source leaves to sink organs
AtSUC2
Characterization of a phloem-localized sucrose/H+ symporter AtSUC2 in Arabidopsis (Srivastava et al., 2009)
AtSUC2 overexpression in mutants restores growth and carbon partitioning
SUT1
Function of sucrose transporter1 gene in maize using sut1 mutants (Slewinski et al., 2009)
Sut1 mutants have altered biomass partitioning and reduced phloem loading of sucrose
AtSUC2
Functional
sucrose/H+
Overexpression of AtSUC2 supports a role in retrieval but not efflux along the phloem
AC
AGPS
characterization
of
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ACCEPTED MANUSCRIPT transport
Array 128 genes related to photosynthesis and Cmetabolism
Changes in expression of photosynthesis and Cmetabolism related genes (McCormick et al., 2008)
Sink demand limits source activity through a kinase-mediated sugar signaling mechanism
SPS1, RSUS1
Changes in sucrose phosphate synthase (SPS1) and sucrose synthase (RSUS1) genes under elevated CO2 in rice (Li et al., 2008)
Important role of SPS in regulating leaf sink to source transition under elevated CO2 conditions
SPS
Overexpression of SPS in potato under field conditions (Ishimaru et al., 2008)
SPS overexpression improves supply of photosynthate from source (leaves) to sink (tubers), increasing yield
TPS1
Constitutive overexpression of TPS1 in tomato (Cortina and Culiáñez-Macià, 2005)
Improved plant survival under salinity, drought, and oxidative stress, but abnormal growth (pleiotropic changes)
ostA, ostB
Constitutive overexpression of a synthetic fusion ostA and ostB in potato (Jang et al., 2003) and alfalfa (Suárez et al., 2009)
Stunted plant growth.
ostA, ostB
Light regulated or ABA-inducible expression of ostA and ostB fusion (Garg et al., 2002)
Increased tolerance to drought and salinity in terms of vegetative growth, with higher photosynthetic capacity
PEPC
Overexpression of PEPC in rice (Ding et al., 2013)
Effective when photosynthesis is limited. Higher photosynthesis and final grain yield under drought stress
CIN1
CIN1 overexpression under the control of senescence-associated promoter SAG12 (Lara et al., 2004)
Increased source senescence.
CIN1
CIN1 overexpression under the control of a putative fruit specific promoter (Albacete et. al, unpublished)
Increased WUE and tolerance to drought due to increased photosynthetic capacity and lower water use
Ascorbate oxidase RNAi in tomato (Garchery et al., 2013)
Diminution of ascorbate oxidase affects Callocation and increases tomato yield under drought stress
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AO
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Other genes
TDY1
T
symporter in Arabidopsis (Srivastava et al., 2008)
strength
and
delayed
Identification and characterization of a collection of phloem-expressed transmembrane (tdy1) maize mutants (Ma et al., 2009)
TDY1 functions in carbon partitioning by promoting phloem loading
rolB-like part of ORF8 gene
Overexpression of ORF8 in tobacco (Umber et al., 2002)
Transgenic plants reduces sucrose export from source leaves to sink organs
SAGs, SEN1
Expression of different senescence associated genes in Arabidopsis (Schenk et al., 2005)
Regulatory expression of SAGs by senescence and stress with reduction of photosynthetic activity
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Effects on growth/survival/yield
DDF1
ga2ox7-2 Arabidopsis mutant in DDF1overexpressing background (Magome et al., 2008)
ga2ox7-2 mutant suppress dwarf genotype of DDF1-overexpressing plants under salt stress, indicating key role in salt-mediated growth impairment
DREB1A
Overexpression of AtDREB1A in tobacco (Cong et al., 2008) and SlDREB in tomato (Li et al., 2012)
GA-deficient dwarf genotypes, with increased root sink growth and tolerance to drought and salinity
AtGAMT1A
Overexpression of AtGAMT1A implicated in the degradation of GAs in tomato (Nir et al., 2013)
Semi-dwarfed genotype and increased tolerance to drought stress
CKX
CKX overexpression in tobacco (Werner et al., 2008)
Decreased shoot sink activity due to reduced cell number in meristems and sucrolytic activities
35S::IPT
35S::IPT tomato rootstocks (Ghanem et al., 2011a)
Grafting WT plants onto 35S::IPT rootstocks increased fruit yield under salinity
SAG12::IPT
Overexpression of SAG12::IPT (Swartzberg et al., 2006)
tomato
Higher fruit weight due to increased sink assimilate import and delayed leaf senescence
IPT
IPT overexpression in axillary buds of tobacco (Guivarc'h et al., 2002)
Cytokinins play a role in tuberization and sink formation
AUX/IAA, AXR1, RBX1like E3
Transcriptomic study on hybrid embryos of Vicia faba (Meitzel et al., 2011)
Regulatory model on seed carbon allocation and growth of auxin-related genes
LOS5
Overexpression of the LOS5 gene encoding a key enzyme of ABA biosynthesis (Xiao et al., 2009)
Increased spikelet fertility and subsequent improved yield under water stress
LeNCED1
LeNCED1 overexpression in tomato (Thompson et al., 2007)
Growth maintenance under drought stress, but growth penalty under control conditions
Ubi1::AtJMT
AtJMT overexpression in rice, encoding JA carboxyl methyltransferase (Kim et al., 2009)
Lower spikelet number and yield under drought stress
35S::AtJMT
Constitutive overexpression Arabidopsis (Cipollini, 2010)
in
Reduced yield due to a reduction in seed weight
Overexpression of genes encoding sterol C-22 hydroxylases that control brassinosteroid levels in rice (Wu et al., 2008)
Brassinosteroids stimulates the flow of assimilates from the source to the sink, increasing grain yield
SC R
IP
T
Gene Description of the study Hormone-related genes
NU
Table 2 List of genes related to sink strength, and their effect on growth, plant survival and/or yield.
CE P
TE
D
MA
in
ZmCYP, AtCYP, OsCYP
of
AtJMT
Carbon metabolism-related genes
Regulatory expression of the anther cell wall invertase gene IVR1 in wheat (Koonjul et al., 2005)
Higher expression of IVR1 in anthers, increased sink strength and cell wall invertase in tolerant wheat cultivars to drought stress
OsINV4, OsMST8
Expression of cell wall invertase and sugar transporter genes in rice (Oliver et al., 2005)
Increased expression in sink organs, existing a correlation between tissue sink activity and tolerance to drought and cold stress
GIF1
Cell wall invertase gene GIF1 overexpression driven by its native promoter in rice (Wang et al., 2008)
Increased sink strength during grain filling and final yield
CIN1
Overexpression of CIN1 gene under the control of a putative fruit-specific promoter in tomato (Albacete et al., unpublished)
Specific increase in fruit sink activity, reduced flower abortion, and higher fruit yield under salinity
GPT, NTT1
Simultaneous overexpression of glucose-6phosphate/translocator (GPT) and adenilate translocator (NTT1) genes in potato (Zhang et al., 2008)
Overriding co-limiting import of carbon and energy into tubers increases starch content and yield
ISI1
Study of Arabidopsis mutants of the impaired sucrose induction1 gene (Rook et al., 2006)
Isi1 mutants do not utilize available sugars efficiently due to a lack of control of sugarresponsive gene expression
Wheat isogenic lines containing the Nax2 locus encoding the HKT1 Na+ transporter (Munns et al., 2012)
Reduced Na+ in flag leaf and flower abortion, and increased photosynthetic activity during grain filling and yield.
AC
IVR1
Other genes HKT1
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ACCEPTED MANUSCRIPT QTL for salt tolerance SKC1 in rice, encoding a Na+ transporter (Ren et al., 2005)
Higher salt tolerance but no practical results in terms of improved yield
MdSIMYB1
Overexpression of the MdSIMYB1 transcription factor in tobacco and apple (Wang et al., 2013)
Increased resistance to salt, drought and cold stress, and biomass partitioning to the root
HRD
Overexpression of the HRD transcription factor in Arabidopsis and rice (Karaba et al., 2007)
Increased root sink strength and biomass under drought stress, but no yield data.
Gn1a
QTL of grain number in rice that encodes OsCKX2 (Ashikari et al., 2005)
Enhanced grain number per panicle and per plant, increasing final yield
DEP1
Gain-of-function mutation of allele DEP1 (Huang et al., 2009)
Dense panicle, increased number of grains and consequent increase in grain yield
OsSPL14
Point mutation in OsSPL14 in rice (Jiao et al., 2010)
Ideal rice with reduced tiller number, more and bigger grains per panicle, and increased yield
TDY1
Identification and characterization of a collection of phloem-expressed transmembrane (tdy1) maize mutants (Ma et al., 2009)
TDY1 functions in carbon partitioning by promoting phloem loading
rolB-like part of ORF8 gene
Overexpression of ORF8 in tobacco (Umber et al., 2002)
Transgenic plants reduces sucrose export from source leaves to sink organs
SAGs, SEN1
Expression of different senescence associated genes in Arabidopsis (Schenk et al., 2005)
Regulatory expression of SAGs by senescence and stress with reduction of photosynthetic activity
R2R3 MYB transcription factor, MST8
Characterization of carbon starved anther (csa) rice mutant encoding a MYB Domain Protein (Zhang et al., 2010)
CSA is a key transcriptional regulator for sugar partitioning in rice during male reproductive development
G1-1, LeG1-1, LeG1-2
Changes in G1-1 expression in potato tubers and LeG1-1 and LeG1-2 expression in tomato seeds (Agrimonti et al., 2007)
G1-1 and its homologs in tomato are involved in maintenance of sink function of potato tubers and tomato seeds
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SKC1
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Werner T, Motyka V, Laucou V, Smets R, Van Onckelen H, Schmülling T. Cytokinin-deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite functions of cytokinins in the regulation of shoot and root meristem activity. Plant Cell. 2003;15:2532-50. Werner T, Motyka V, Strnad M, Schmülling T. Regulation of plant growth by cytokinin. Proc Natl Acad Sci USA. 2001;98:10487-92. Westgate ME, Passioura JB, Munns R. Water status and ABA content of floral organs in drought-stressed wheat. Aust J Plant Physiol. 1996;23:763-72. Wingler A, Purdy S, MacLean JA, Pourtau N. The role of sugars in integrating environmental signals during the regulation of leaf senescence. J Exp Bot. 2006;57:391-9. Wingler A, Roitsch T. Metabolic regulation of leaf senescence: Interactions of sugar signalling with biotic and abiotic stress responses. Plant Biol. 2008;10:50-62. Wu CY, Trieu A, Radhakrishnan P, Kwok SF, Harris S, Zhang K, et al. Brassinosteroids regulate grain filling in rice. Plant Cell. 2008;20:2130-45. Xiao BZ, Chen X, Xiang CB, Tang N, Zhang QF, Xiong LZ. Evaluation of seven functionknown candidate genes for their effects on improving drought resistance of transgenic rice under field conditions. Mol Plant. 2009;2:73-83. Xie X, Yoneyama K. The strigolactone story. 2010. p. 93-117. Yang J, Zhang J, Wang Z, Xu G, Zhu Q. Activities of key enzymes in sucrose-to-starch conversion in wheat grains subjected to water deficit during grain filling. Plant Physiol. 2004;135:1621-9. Yang J, Zhang J, Wang Z, Zhu Q, Liu L. Water deficit–induced senescence and its relationship to the remobilization of pre-stored carbon in wheat during grain filling. Agron J. 2001;93:196-206. Yoo CY, Pence HE, Hasegawa PM, Mickelbart MV. Regulation of transpiration to improve crop water use. Crit Rev Plant Sci. 2009;28:410-31. Zalabák D, Pospíšilová H, Šmehilová M, Mrízová K, Frébort I, Galuszka P. Genetic engineering of cytokinin metabolism: Prospective way to improve agricultural traits of crop plants. Biotechnol Adv. 2013;31:97-117. Zeevaart J, Creelman R. Metabolism and physiology of abscisic acid. Annu Rev Plant Physiol Plant Mol Biol. 1988;39:439-73. Zhang H, Liang W, Yang X, Luo X, Jiang N, Ma H, et al. Carbon Starved Anther encodes a MYB domain protein that regulates sugar partitioning required for rice pollen development. Plant Cell. 2010;22:672-89. Zhang H, Wu X, Li G, Qin P. Interactions between arbuscular mycorrhizal fungi and phosphatesolubilizing fungus (Mortierella sp.) and their effects on Kostelelzkya virginica growth and enzyme activities of rhizosphere and bulk soils at different salinities. Biol Fertil Soils. 2011:1-12. Zhang L, Häusler RE, Greiten C, Hajirezaei MR, Haferkamp I, Neuhaus HE, et al. Overriding the co-limiting import of carbon and energy into tuber amyloplasts increases the starch content and yield of transgenic potato plants. Plant Biotechnol J. 2008;6:453-64. Zhu Q-H, Ramm K, Shivakkumar R, Dennis ES, Upadhyaya NM. The ANTHER INDEHISCENCE1 gene encoding a single MYB domain protein is involved in anther development in rice. Plant Physiol. 2004;135:1514-25.
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S0734-9750(13)00182-1 doi: 10.1016/j.biotechadv.2013.10.005 JBA 6748
To appear in:
Biotechnology Advances
Received date: Revised date: Accepted date:
13 June 2013 17 October 2013 20 October 2013
Please cite this article as: Albacete Alfonso A., Mart´ınez-And´ ujar Cristina, P´erez-Alfocea Francisco, Hormonal and metabolic regulation of source-sink relations under salinity and drought: From plant survival to crop yield stability, Biotechnology Advances (2013), doi: 10.1016/j.biotechadv.2013.10.005
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ACCEPTED MANUSCRIPT Hormonal and metabolic regulation of source-sink relations under salinity and drought: from plant survival to crop yield stability
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Alfonso A. Albacete, Cristina Martínez-Andújar and Francisco Pérez-Alfocea*
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Departamento de Nutrición Vegetal, Centro de Edafología y Biología Aplicada del
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Segura (C.E.B.A.S.), Consejo Superior de Investigaciones Científicas (C.S.I.C.),
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Campus Universitario de Espinardo, P.O. Box 164, E-30100 Murcia, Spain
*Corresponding author:
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Francisco Pérez-Alfocea
Departamento de Nutrición Vegetal, Centro de Edafología y Biología Aplicada del
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Segura (C.E.B.A.S.), Consejo Superior de Investigaciones Científicas (C.S.I.C.),
Phone: +34 968 396342
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Fax: +34 968 396213
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Campus Universitario de Espinardo, 25, P.O. Box 164, E-30100 Murcia, Spain
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E-mail: [email protected]
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ACCEPTED MANUSCRIPT Abstract Securing food production for the growing population will require closing the gap between potential crop productivity under optimal conditions and the yield captured by
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farmers under a changing environment, which is termed agronomical stability. Drought
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and salinity are major environmental factors contributing to the yield gap ultimately by
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inducing premature senescence in the photosynthetic source tissues of the plant and by reducing the number and growth of the harvestable sink organs by affecting the transport and use of assimilates between and within them. However, the changes in
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source-sink relations induced by stress also include adaptive changes in the reallocation of photoassimilates that influence crop productivity, ranging from plant survival to yield stability. While the massive utilization of –omic technologies in model plants is
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discovering hundreds of genes with potential impacts in alleviating short-term applied drought and salinity stress (usually measured as plant survival), only in relatively few cases has an effect on crop yield stability been proven. However, achieving the former
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does not necessarily imply the latter. Plant survival only requires water status
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conservation and delayed leaf senescence (thus maintaining source activity) that is usually accompanied by growth inhibition. However, yield stability will additionally
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require the maintenance or increase in sink activity in the reproductive structures, thus contributing to the transport of assimilates from the source leaves and to delayed stressinduced leaf senescence. This review emphasizes the role of several metabolic and
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hormonal factors influencing not only the source strength, but especially the sink activity and their inter-relations, and their potential to improve yield stability under drought and salinity stresses. Keywords: assimilate transport, biomass partitioning, cytokinins, fruit/grain filling, gibberellins, hormonal signaling, invertases, leaf senescence, stay green, sucrose and starch metabolism.
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ACCEPTED MANUSCRIPT Contents 1. Introduction 2. Improving source strength
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2.1. Delaying leaf-senescence
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2.2. Stay-green genotypes
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2.3. Hormonal homeostasis and signalling 2.4. Carbon metabolism 3. Improving sink strength
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3.1. Invertases, pollen viability and grain/fruit filling
3.2. Hormonal signalling and regulation of sink activity
3.2.2. Cytokinins 3.2.3. Auxins
3.2.5. Jasmonates
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3.2.4. Abscisic acid
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3.2.1. Gibberellins
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3.2.6. Salicylic acid 3.2.7. Ethylene
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3.2.8. Strigolactones
3.3. Root-sink strength to improve stress adaptation 3.4. Increasing sink number
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4. Conclusions
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ACCEPTED MANUSCRIPT 1. Introduction About 70-90% more food is required to fulfil the food demands of a human population of 9 billion people expected in 2050. This includes a 50% increase in grain yield of
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major crop plants such as rice (Oryza sativa L.), wheat (Triticum aestivum L.) and
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maize (Zea mays L.) (Boyer, 1982; Godfray et al., 2010; Peleg et al., 2011). This
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increase must be achieved in a sustainable way without increasing resource demand (arable land, irrigation water, fertilizers) and overcoming the yield losses due to environmental stresses. According to the historical data on yield potential in rice, the
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most substantial progress (3-fold increase from 2.5 to 8 t·ha-1) was achieved with the delivery of semidwarf varieties (1965-1990). A subsequent 1.5-fold increase was achieved by introducing the F1 hybrids, while the next substantial improvement
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expected for 2010 with the introduction of C4 photosynthesis (Datta, 2004), has not been achieved so far. Despite considerable investment in cultivar improvement, about 20–70% of the potential crop yield in favourable ecosystems is not captured by farmers
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because of biotic and abiotic stresses (Cassman, 1999; Datta, 2004). In the case of major
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U.S. crops (corn, wheat, soybean, sorghum, oat, barley, potato, and sugar beet) the average production is only about 20% of the maximum yields attained under optimal
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conditions (Boyer, 1982; Godfray et al., 2010; Peleg et al., 2011). While biotic factors (diseases, pests, and weed competition) account for less than 10% of yield reductions, the remaining 70% is attributed to abiotic constraints such as drought and salinity, that
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are being exacerbated by climate change (Peleg et al., 2011). A similar situation occurs in other crops and regions worldwide, which is termed the ‘yield gap’ (Godfray et al., 2010). Although a considerable scope already exists for increasing the crop production limits by adopting new varieties, one major societal and scientific challenge is to fill the yield gap caused by abiotic factors. Abiotic stresses, such as salinity and drought, modify source-sink relations which influence plant growth as well as adaptation to stress and consequently affect crop productivity. Yield reduction is ultimately due to a decrease in the number and size of sink organs, since the source organs are unable to maintain the assimilate supply required to support the mutually competitive processes of vegetative growth, filling of reproductive structures and adaptation to stress. Only in extreme cases is the loss of productivity due to plant death. Therefore, yield stability under stress requires a 4
ACCEPTED MANUSCRIPT dynamic optimization of source-sink relations to maintain assimilate partitioning to reproductive structures while minimizing the requirements of other organs and processes to maintain physiological and morphological adaptive responses. Ideally, this
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optimization must be highly plastic and should be linked to the severity of the stress,
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ranging from plant survival (mostly by preserving source activity without growth) under severe stress conditions, to yield stability (both sink and source activities are required)
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under a wide range of productivity-compatible stress situations. However, this ideal scenario is far from being realised and increasing plant survival does not necessarily imply the most important trait of higher productivity or yield stability. Therefore,
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tolerance to abiotic stresses in functional studies in model plants should always be measured in terms of yield-related parameters rather than plant survival, because (i)
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both parameters are often inversely related (Ceccarelli, 1987), (ii) plant response to stress differs through the plant cycle, with the reproductive stage being one of the most sensitive (Parish et al., 2012), and (iii) other important factors affecting yield
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components, such as sink determination and growth can be missed, thus reducing the
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possibilities of applying the results to crop species in the field (Peleg et al., 2011). Drought and salt stress can initially reduce source photosynthetic activity by
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both stomatal and non-stomatal factors (Munns et al., 2006; Munns and Tester, 2008; Pérez-Alfocea et al., 2010). Regardless of the ionic component of salinity, both stresses can provoke premature senescence due to assimilate accumulation in source leaves as a
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result of decreased consumption in sink tissues. This assimilate accumulation provokes impaired consumption of NADPH by the Calvin Cycle, leading to the so called (feedback) photo-inhibition and photo-oxidation processes, with the subsequent transfer of photosynthetic electrons from over-reduced ferredoxin to oxygen (Mehler Reaction) producing toxic reactive oxygen species that damage cell structures (Balibrea et al., 2000; Keunen et al., 2013; Paul and Foyer, 2001; Stitt, 1991). Indeed, the processes causing photo-oxidative damage and premature leaf senescence are common to several abiotic root-zone stresses that induce water stress and inhibit growth of sink organs without a concomitant ionic component (Hare et al., 1997; He et al., 2001). There are strong indications from numerous studies that the regulation of senescence is particularly important for yield stability under stress conditions (Gregersen et al., 2013), and avoiding feedback inhibition of photosynthesis by coordinating assimilate transport between source and sink tissues may delay leaf senescence (Pérez-Alfocea et al., 2010). 5
ACCEPTED MANUSCRIPT However, this coordination requires the maintenance or increase of sink demand for assimilates from source tissues, since this process is primarily reduced by the stress in vegetative (Albacete et al., 2008; Balibrea et al., 2000; Munns, 1993) and reproductive
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tissues (Balibrea et al., 2003; Balibrea et al., 1999; Ghanem et al., 2009; Parish et al.,
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2012). Alternatively, reallocation of assimilates between source and sink tissues also allows the plant to adapt to biotic and abiotic stresses (Roitsch et al., 2003; Roitsch and
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González, 2004). One example is the increased root-to-shoot ratio due to growth maintenance in the root and the rapid inhibition of shoot growth (Albacete et al., 2008; Sharp, 2002), which can be considered an adaptive response to increase the water and
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nutrient acquisition from the root zone. This integrated growth plasticity involves longdistance communication between different organs, with hormones playing a major role
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(Ghanem et al., 2011a; Ghanem et al., 2011b; Pérez-Alfocea et al., 2011; Sachs, 2005). Growth regulation by drought and salinity is mediated primarily by the stress-related hormones abscisic acid (ABA) and ethylene (Albacete et al., 2008; Dodd and Davies,
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1996; Ghanem et al., 2008). However, other hormones, such as gibberellins (GAs),
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auxins and cytokinins (CKs) are also involved (Achard et al., 2006; Albacete et al., 2008; Ghanem et al., 2011a; Magome et al., 2004; 2008; Pérez-Alfocea et al., 2010;
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Rivero et al., 2007). Phytohormone balance can affect plant performance by influencing source-sink relations and metabolism, and thus, the stress tolerance and crop yield. The power of the hormone-related traits in improving yield has been already
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demonstrated. Indeed, the Green Revolution, that substantially improved worldwide food production in the 1960s and 1970s, was possible thanks to the breeding of crop varieties with improved vigour and harvest index. Those traits imply changes in sourcesink relations for producing more grain at the expense of the straw biomass. In the case of wheat and rice, those changes are explained by alterations in GAs homeostasis and signalling (Peng et al., 1999; Sasaki et al., 2002). In rice, the high-yielding semi-dwarf phenotype is due to a mutation in the gene encoding for the enzyme GA20 oxidase (GA20ox-2) that decreases GA synthesis in the vegetative sink tissues thus reducing plant height, while the isoform GA20ox-1 is expressed in reproductive organs allowing normal grain development (Sasaki et al., 2002). In wheat, the mutant dwarfing alleles encodes a transcription factor that reduces the sensitivity to GAs (GAI, Gibberellin Insensitive Gene) (Peng et al., 1999). Therefore, substantial increases in yield
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ACCEPTED MANUSCRIPT production have been due to changes in plant source-sink relations, induced by altering hormonal metabolism and signalling. However,
sustaining
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productivity under
unfavourable
conditions
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necessarily requires maintaining both assimilate production in sources tissues, and also
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transport to, and use within sink and harvestable tissues. While these processes may
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adjust to allow plant stress adaptation, their impairment for long periods causes a loss of productivity. This aspect is important since several abiotic stresses can co-occur in the field and the response to the combination can be different from the response to
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individual stresses. Hence, achieving tolerance to such combined stresses by exploiting general adaptive responses of the plant might lead to an important strategy for yield improvement. This review emphasizes that increased yield stability will require the
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increase or at least the maintenance of (a) source strength (photosynthetic efficiency and assimilate export) for longer (delayed stress-induced senescence) and/or (b) the sink strength (assimilate import) of the harvestable (fruits and seeds) and resource providing
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(new leaves and roots) organs (vigour maintenance). The homeostatic regulation of
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phytohormones and/or metabolic components could play significant roles in regulating source-sink activities and relations and their manipulation could provide new
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opportunities for increasing yield stability under abiotic stress conditions. However, it seems clear that a communication and interdependence between both sink and source organs are needed. For instance, increasing sink activity alone cannot be effective if the
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source is limited by stomatal factors or any other resource acquisition from the soil, while increasing the source efficiency is also ineffective if the sink activity is impaired by hormonal signals and/or limited water and nutrient availability. Hence, special attention should be paid to those genes/processes allowing sink activity (i.e. flower fertility and grain/fruit filling) under resource-limited conditions. Finally, although the major objective should be increasing yield stability of elite lines with high yield potential, a significant stress-induced yield reduction of high yielding lines may be preferable to a small stress-induced yield reduction of low yielding genotypes with agronomical stability. 2. Improving source strength 2.1. Delayed leaf-senescence
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ACCEPTED MANUSCRIPT Developmental leaf senescence is a programmed process influenced by several endogenous and exogenous factors, such as the plant developmental stage, leaf age, phytohormone levels, and light conditions. This process reduces local or systemic
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assimilate availability and therefore limits plant growth and productivity when it is
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prematurely induced by environmental factors such as drought and salinity (BuchananWollaston et al., 2003). Any genetic or physiological determinant delaying the
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progression of developmental or stress-induced senescence can promote the transfer of photosynthetic assimilates from sources (e.g. mature leaves) to harvestable sinks (e.g. developmental organs such as fruit and seeds and new leaves), thus potentially helping
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to close the yield gap under suboptimal conditions (Cassman, 1999; Godfray et al., 2010; Gregersen et al., 2013). Indeed, transcript profiling studies have revealed the
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occurrence of considerable cross-talk between stress responses and leaf senescence (Lim et al., 2007). For example, among 43 genes coding senescence-induced transcription factors (TFs), 28 were also induced by exposure to various stresses (Chen
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et al., 2002). Furthermore, the expression of many senescence-associated genes (SAGs),
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such as the Arabidopsis SEN1 gene, is commonly regulated by the initiation of natural leaf senescence and by exposure to stresses (Schenk et al., 2005). Importantly, the onset
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of stress-induced senescence is related to changes in endogenous hormones such as increases in ethylene and/or its precursor 1-aminocyclopropane-1-carboxylic acid (ACC) (Ghanem et al., 2008; Grbic and Bleecker, 1995), ABA (Zeevaart and Creelman, 1988), salicylic acid (SA) (Morris et al., 2000), and jasmonic acid (JA) (He et al., 2002),
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or decreased in CK levels (Gan and Amasino, 1995; Ghanem et al., 2008; Ghanem et al., 2011a). In addition, leaf senescence may also be induced by carbohydrate accumulation or by the availability of excess carbon relative to low levels of nitrogen (Wingler et al., 2006). Table 1 summarizes the hormonal and metabolic-related genes affecting source strength. 2.2. Stay-green genotypes The relationship between senescence and plant productivity is complex, but modulating the genetically determined senescence program can influence crop productivity under optimal and/or suboptimal conditions (reviewed in Gregersen et al. (2013)). Indeed, a positive correlation has been found between leaf area/photosynthetic rate duration and yield (grain or biomass) under water-limiting conditions in maize (Messmer et al., 8
ACCEPTED MANUSCRIPT 2011), sorghum (Borrell et al., 2000; Harris et al., 2007), wheat (Verma et al., 2004) and barley (Gonzalez et al., 2010; Vaezi et al., 2010). Hence, genetic determinants of functional stay-green genotypes are potentially valuable for conventional and
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biotechnological breeding approaches (Borrás et al., 2004; Gregersen et al., 2013). For
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example, the functional stay green wheat genotype CN17 exhibited significantly higher maximal photochemical efficiency for photosystem II (PSII) and higher efficiency of
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excitation capture by open PSII reaction centers (Fv/Fm) 21 days post-anthesis (Luo et al., 2013), thus extending the period of grain filling and presumably increasing yield (Mittler and Blumwald, 2010). In addition, chlorophyll degradation was delayed by
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approximately 14 days, and was not blocked as in ‘cosmetic’ stay-green phenotypes which retain chlorophyll but not photosynthetic activity. However, although functional
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stay-green varieties have been characterized physiologically and genetically in several plant species, the mechanism by which leaf greenness is coupled to effective photosynthetic competence remains unclear (Luo et al., 2013). Enhanced photosynthetic
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competence seems to be explained by a higher protection of PSII and granal stability, as
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well as increased levels of unsaturated fatty acids and antioxidative capacity, which provide more readiness to regenerate chloroplast ultrastructure by maintaining protein
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synthesis. In silico BLAST analysis indicated that four cell wall-associated hydrolases (i.e. cell wall invertases) were present in the CN17 stay-green genotype. The authors suggested that a mechanism similar to the C-repeat/dehydration-responsive element binding factor 2 (CBF2) gene described in Arabidopsis can be responsible for the
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regeneration of the photosynthetic apparatus (Luo et al., 2013). 2.3. Hormonal homeostasis and signalling The overexpression of the CBF2 gene in Arabidopsis delayed the onset of both natural (Sharabi-Schwager et al., 2010a) and artificially-induced leaf senescence by either darkness or phytohormones (ethylene, ABA, SA, and JA) in detached leaves (SharabiSchwager et al., 2010b). Affymetrix ATH1 genome array revealed significant changes in the expression of 286 genes in mature leaf tissue, 30 stress-related genes, 24 TFs, and 20 genes involved in protein metabolism, degradation, and post-translational modifications. Transcript profiling analysis of hormone metabolism and responsive genes revealed that overexpression of CBF2 induced expression of ABA biosynthesis genes (NCED, encoding the key enzyme in ABA biosynthesis, 9-cis-epoxycarotenoid 9
ACCEPTED MANUSCRIPT dioxygenase - (Thompson et al., 2000) and ABA-responsive genes, and suppressed SAand JA-related genes, including three lipoxygenases, allene oxide synthase and jasmonic acid carboxyl methyltransferase. Overexpression of CBF2 in Arabidopsis also
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suppressed leaf tissue responsiveness to ethylene (0.1-10 µl l-1) as compared to wild-
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type plants and significantly delayed senescence and chlorophyll degradation (SharabiSchwager et al., 2010a). Although only the expression of the ethylene receptor EIN4
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was significantly reduced, significant increases in 17 ABA biosynthetic and responsive genes occurred in CBF2-overexpressing plants. Thus, reduced leaf responsiveness to ethylene could be explained through interactions with other hormones such as ABA
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(Sharp, 2002). CBF2 seems to act directly on hormone metabolism and signalling, suppressing hormone-induced leaf senescence (Sharabi-Schwager et al., 2010b), and
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could potentially delay ethylene-mediated stress-induced senescence. Therefore, the manipulation of this kind of TF seems a possible strategy to increase yield under suboptimal conditions.
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However, the overexpression of the CBF1, CBF2, and CBF3 genes in
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Arabidopsis retarded growth causing a dwarf phenotype (Gilmour et al., 2004; Liu et al., 1998). Constitutive e CBF1 expression retarded growth by allowing the
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accumulation of DELLAs, a family of nuclear growth repressing proteins, whose degradation is stimulated by the growth-promoter hormones GAs (Achard et al., 2008). It is not clear if the observed delayed leaf senescence in CBF2-overexpressing plants is
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mediated by DELLA proteins as reported for the delayed flowering and dwarfism phenotypes (Achard et al., 2008), or rather is a DELLA-independent effect as noted for the increased sugar levels of CBF1-overexpressing plants (Wingler and Roitsch, 2008). Nevertheless, delayed senescence of growth-arrested phenotypes may be incompatible with yield promotion. Recent studies also suggest that growth impairment and adaptation to stress seems to occur through independent signalling processes and may differ depending on the stress condition (Dubois et al., 2013). However, even if activated by different processes, it seems that they occur simultaneously and can be induced by the same effector. Thus, the ethylene precursor ACC is implicated as an early signal related to plant growth arrest under both osmotic and salt stresses (Skirycz et al., 2011; Zhang et al., 2011) and the onset of salt-induced senescence (Ghanem et al., 2008). Unravelling 10
ACCEPTED MANUSCRIPT the molecular mechanisms and signalling processes involved could identify new targets for stress alleviation and crop improvement. Indeed, an analysis of the relatively abundant reports of transgenic Arabidopsis lines with enhanced survival reveals no
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improved growth under moderate drought conditions (Skirycz et al., 2011). However,
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the Arabidopsis TFs ETHYLENE RESPONSE FACTOR5 (ERF5) and ERF6 are rapidly induced under osmotic stress and seem to act as master regulators adapting leaf growth
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to environmental changes (Dubois et al., 2013; Skirycz et al., 2011). Enhanced ERF6 expression inhibits cell proliferation and leaf growth via GA degradation by gibberellin 2-oxidase6 (ga2-ox6) and subsequent DELLA-mediated inhibition of cyclin-dependent
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kinase A activity (Skirycz et al., 2011). As a result, ERF6 gain-of-function lines are dwarfed and hypersensitive to osmotic stress, while the growth of erf5erf6 loss-of-
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function double mutants is less affected by short- and long-term osmotic stress, probably due to the non-induction of ga2-ox6, thus avoiding the GA-DELLA cell-cycle inhibition. Surprisingly, the double erf5erf6 mutants were not more tolerant of moderate
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salinity (50 mM NaCl), suggesting that salinity-induced growth occurs independently of
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the ERF5/ERF6-growth regulatory pathway. This can be explained not only by a rapid accumulation of toxic ions (Dubois et al., 2013), but also by other hormone-related
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processes such as decreased levels of growth promoting hormones like CKs (Albacete et al., 2008; Ghanem et al., 2011a) or by interaction with ABA signalling (Pan et al., 2012). However, ERF6 also activates the expression of a plethora of osmotic stressresponsive genes (including the stress tolerance STZ, MYB51, and WRKY33) in a
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growth-independent manner (Dubois et al., 2013). Indeed, overexpression of SlERF5 in tomato plants conferred tolerance to drought (unwatered for 7 days) and salt (200 mM NaCl for 10 days) stresses by conserving chlorophyll concentrations and water status, but without additional effect on growth when compared to the WT (Pan et al., 2012). A similar response was found in transgenic rice plants overexpressing OsTZF1 (a member of the CCCH-type zinc finger gene family in rice) driven by a maize ubiquitin (Ubi) promoter. The Ubi::OsTZF1 plants exhibited not only delayed seed germination and growth retardation, but also delayed salt-, JA- and ABA-induced leaf senescence, indicating that delayed senescence might be due to a common factor like tolerance to oxidative stress (Jan et al., 2013). Again, the growth-retarded Ubi::OsTZF1 plants showed improved tolerance (plant survival) to high salinity (250 mM NaCl for 3 days) and drought stresses. The discovery of the missing link between plant survival and growth is of utmost importance. 11
ACCEPTED MANUSCRIPT The interesting finding that hormones target various members of protein families involved in growth, together with the identification of a small number of proteins that were co-regulated by multiple hormones, indicate synergistic action of metabolic
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pathways. Such pathway interactions probably occur through combinatorial regulation
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of common target proteins by various hormone-controlled kinases (Chen et al., 2010), with the common objective of adapting growth to the environmental conditions to
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improve plant survival. However, biotechnological manipulation of key proteins or hormones should focus not only on the ecological adaptation in terms of survival but also the agronomical stability for securing food production. For example, ABA induces
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the phosphorylation and activation of the AREBs transcription factors and the overexpression of the phosphorylated AREB1 induces the expression of many ABA-
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inducible genes (Choi et al., 2000; Furihata et al., 2006). Indeed, the overexpression of the SlAREB1 gene in tomato improved plant survival under drought and salinity stress through senescence-delaying and water-conservative mechanisms (Orellana et al.,
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2010a). Those ABA-mediated mechanisms are probably incompatible with growth
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maintenance, for which co-ordination with other growth-promoting hormones/proteins is required.
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Since CKs have an important role in delaying natural senescence (Gan and Amasino, 1995; 1997) and the concentration of the bioactive CKs decreases during the exposure to water (Havlová et al., 2008; Kudoyarova et al., 2007) and salt (Albacete et
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al., 2008; Ghanem et al., 2008; Ghanem et al., 2011a) stresses, it has been hypothesized that yield stability can be maintained by delaying stress-induced senescence and maintaining source activity through the stress-induced synthesis of cytokinins (reviewed in Gregersen et al. (2013)). About 80% of the studies published so far on transgenic crop plants with autoregulated (using essentially SAG or SARK promoters) isopentenyltransferase (IPT) gene expression reported delayed leaf senescence (stay green and chlorophyll content) and subsequently increased source photosynthetic activity over time. Interestingly, most of those studies reported increased biomass and/or yield-related parameters under drought stress in tobacco (Rivero et al., 2007), cassava (Zhang et al., 2010), creeping bent grass (Merewitz et al., 2010; 2011), peanut (Qin et al., 2011) and wheat (Peleg et al., 2011), and also in salinized cotton (Liu et al., 2012) and tomato (Ghanem et al., 2011a). Transient (2 hours) root specific IPT induction using a heat shock-specific promoter (HSP70::IPT) promoted plant growth in 12
ACCEPTED MANUSCRIPT salinized (100 mM NaCl) tomato plants by increasing leaf CK levels by 2-3 fold (Ghanem et al., 2011a). In this case, leaf ABA concentration was reduced by 20-40%. The attenuated growth inhibition of salinized CK overproducing plants may occur
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directly via the relative maintenance of CK-mediated cell division and cell wall
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extensibility and/or improved carbon status due to both delayed stomatal closure and leaf senescence through decreased ABA concentrations. Lower ABA concentrations in
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photosynthetically active leaves may also indirectly delay senescence, photoinhibition, and photooxidation during the osmotic phase of salinity (Ghanem et al., 2008; Ghanem
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et al., 2011a; Pérez-Alfocea et al., 2010).
Detailed physiological studies of the stress responses of CK-overproducing plants could identify new molecular targets to further increase crop productivity
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(Zalabák et al., 2013), not only in source but also in sink capacity. For example, water stressed transgenic rice plants expressing the IPT gene, driven by the stress- and maturation-induced promoter PSARK, exhibited improved water status because of lower
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water use, delayed drought symptoms by 4-7 days, and significant increases in plant
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biomass and grain yield (2-3 times), compared with WT plants (Peleg et al., 2011). During water-stress, PSARK::IPT plants displayed increased (i) expression of BR
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synthesis (DWF5: Delta14-sterol reductase and HYD1: C-8 sterol isomerase) and signaling (BRL3, BRI1, BRH1, BIM1, SERK1, BSK1, BIN4 and BAK1) genes and (ii) repression of JA-biosynthesis (OPR2) and signaling (MES3 and JAZ12) related genes.
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Moreover, the expression of genes encoding indole-3-acetic acid (IAA) transporters (OsPIN3a and OsPIN6) and IAA-responsive genes (OsIAA5, OsIAA21 and OsARF5) were down-regulated in the PSARK::IPT plants under drought conditions, while the levels of expression of GA-associated genes (GA receptor, OsGAI and chitin-inducible GAresponsive protein) were induced. In addition, ethylene responsive and signalling genes (flavonol synthase/flavanone 3 hydroxylase, ETR2, MTHFR2, RAP2.4 and EIN3) were up-regulated. Those changes in hormone homeostasis were associated with resource(s) mobilization during stress, thus modifying source-sink relationships and in stronger source and sink capacities in the aerial part of the PSARK::IPT plants during drought stress. As a result, the transgenic plants had higher grain yield with improved quality (nutrients and starch content, until 60-90% more than WT). The increased starch content of both flag leaves and grains correlated with the expression of the transcription factor OsMADS57 (a regulator of starch synthesis), under both control and stress conditions 13
ACCEPTED MANUSCRIPT (Peleg et al., 2011). The capacity to increase starch synthesis under water stress can be considered protective by avoiding feed-back inhibition of photosynthesis by soluble sugars when the assimilate export from source leaves is impaired, thus also influencing
T
the timing of senescence (see section 2.4). Moreover, the mobilization of this starch
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during the night also contributes to increased source strength. The CK interaction with BRs (positive) and IAA (negative) signalling may explain the drought-resistant
SC R
phenotype by decreasing stomatal density (Kim et al., 2012) and root development (Dello Ioio et al., 2008) in a constrained (pot) environment. An additional positive effect of CK-induced BR signalling is related to grain filling by stimulating the flow of
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assimilates from the source to the sink (Wu et al., 2008).
Although it has been widely reported that IPT gene expression in senescing
MA
leaves can enhance CK levels thus increasing vegetative biomass, the effect on crop yield is not always detectable. Many investigations with transgenic plants support the concept that delayed leaf senescence can have a positive impact on crop productivity
D
but the positive effect depends on the plant species, the plant variable measured, and the
TE
environment, especially under drought, salinity and low nitrogen (Gregersen et al., 2013). Delayed senescence should therefore be maintained without growth reduction or
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at least with a growth recovery when the adaptive responses are induced. 2.4. Carbon metabolism
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Increasing radiation use efficiency (and thus source strength) of wheat by 50% could be achieved by modifying specificity, catalytic rate and regulation of Rubisco, upregulating Calvin cycle enzymes, introducing chloroplast CO2 concentrating mechanisms, optimizing light and N distribution of canopies while minimizing photoinhibition, and increasing spike photosynthesis (Reynolds et al., 2012). There is potentially to alter carbon metabolism to improve yield under environmental conditions, despite pleiotropic and deleterious effects observed in transgenic plants. These likely occur due to the alteration of basic developmental processes through sugar sensing and signalling, thus adequate (inducible) promoters are needed to avoid undesirable traits and any growth penalty in absence of stress (Peleg et al., 2011; Suárez et al., 2009). For example, constitutively (35S promoter) overexpressing the yeast trehalose-6-phosphate synthase (TPS1) gene in tomato provoked higher tolerance to salinity, drought and oxidative stress, but plants exhibited a plethora of pleiotropic changes such as thick 14
ACCEPTED MANUSCRIPT shoots, rigid dark-green leaves, erect branches, aberrant root development and higher chlorophyll and starch content compared to WT plants (Cortina and Culiáñez-Macià, 2005). Similarly, constitutive overexpression of a synthetic fusion of Escherichia coli
T
trehalose biosynthetic genes (otsA and otsB) in potato (Jang et al., 2003) and alfalfa
IP
(Suárez et al., 2009) caused a range of stunted plant growth phenotypes. However, those genes expressed under the light-regulated promoter of the Rubisco small subunit or an
SC R
ABA-inducible promoter produced from 3- to 10-fold more trehalose than WT plants, and increased tolerance to drought and salinity (100 mM NaCl) measured as vegetative growth. Although yield data are not available, those effects were correlated with a
NU
higher photosynthetic capacity under both control and stress conditions, 50% less in leaf Na+ accumulation, and delayed leaf senescence (Garg et al., 2002).
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Since the ability of the C4 photosynthetic pathway to suppress ribulose-1,5bisphosphate (RuBP) oxygenation and photorespiration represents the most efficient type of photosynthesis on Earth (Sage, 2004), engineering C4 photosynthetic pathway
D
into C3 crops has been considered a highly promising strategy to alleviate crop yield
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losses due to environmental factors (reviewed in Reguera et al. (2012)). Although the strategy of introducing C4 photosynthesis in C3 plants as rice seems extremely
in
CE P
ambitious because of the coordinated relation between leaf morphology and metabolism C4 plants
(Hibberd et
al.,
2008),
the effects
of
overexpressing the
phosphoenolpyruvate carboxylase gene (PEPC) on photosynthesis in rice plants has
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shown controversial but encouraging results. Most studies on this topic have reported decreased photosynthesis with increasing activity of the key CO2-fixing enzyme PEPC in transgenic rice plants (Agarie et al., 2002; Fukayama et al., 2001). There are also reports (Bandyopadhyay et al., 2007; Ding et al., 2007; Jiao et al., 2002; Ku et al., 2000) of enhanced photosynthesis in transgenic rice plants but always under suboptimal (stress) conditions. However, yield results have been provided only recently. Rice plants transformed with the PEPC from millet (Seteria italica) under the control of light regulated Rubisco small subunit promoter had increased PEPC activity, net photosynthesis and grain yield (by 40-50%) under drought conditions (upland), while no differences were observed under optimal (wetland) conditions (Ding et al., 2013). These results suggest that the transgenic enzyme is photosynthetically effective only when the natural photosynthetic rate is limited, thus minimizing the switch between C3
15
ACCEPTED MANUSCRIPT and C4 metabolism described in the halophyte Mesembryanthemum crystallinum under osmotic stress (Scolombe et al., 2006). Senescence-regulated expression of the sucrolytic enzyme cell wall invertase
T
CIN1 (from Chenopodium rubrum L.) under control of the SAG12 promoter in leaves
IP
increased source strength and delayed natural senescence in tobacco, probably linked to
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a CK-mediated mechanism (Lara et al., 2004). This enzyme seems to activate metabolic carbohydrate flux that represses ethylene biosynthesis (Mayak and Borochov, 1984). It has been demonstrated that ethylene decreases cell wall invertase (cwInv) gene
NU
expression (Linden et al., 1996; Roitsch et al., 2003) and induces senescence (MunnéBosch and Alegre, 2004). Therefore, ethylene could mediate salt-induced senescence by inhibiting cwInv (Ghanem et al., 2008). ABA could also mediate stress-induced
MA
senescence by inducting the cwInv proteinaceous inhibitor in tomato and tobacco plants, thus changing the metabolic carbohydrate flux. Indeed, silencing the cwInv inhibitor delayed ABA-induced leaf senescence, and increased seed weight and fruit
D
hexose contents in tomato (Jin et al., 2009). GAs, BRs, auxins and CKs seem to
TE
upregulate cwInv and other sucrolytic enzyme genes, thus promoting sink strength and assimilate import in actively growing tissues (reviewed by Roitsch et al. (2003) and
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Roitsch and González (2004)). Assessing the role of hormones in regulating enzyme activities moderating source–sink relations (Roitsch et al., 2003; Roitsch and Ehness, 2000) will require measurement of these enzymes in hormone biosynthesis or sensitivity
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mutants.
Interestingly, tomato plants (Solanum lycopersicum cv. P-73) overexpressing the cwInv gene CIN1 from Chenopodium rubrum under the control of the putative fruit specific promoter from the vacuolar invertase gene InvLp6g from Solanum pimpinellifolium had significantly higher water use efficiency (WUE) and tolerance to water stress due to increased photosynthetic activity and lower water use (Albacete et al, unpublished results). One possible explanation for reduced transpiration is the increased CK/ABA ratio under stress conditions, since both hormones act antagonistically in stomatal regulation (Dodd, 2005). However, a metabolic effect of the sucrolytic enzymes in the leaves cannot be ruled out, since altered sucrose partitioning between storage and breakdown may affect stomatal function at the level of guard cells. Several studies have also revealed the importance of the apoplastic sucrose in regulating 16
ACCEPTED MANUSCRIPT stomatal aperture. High concentrations of apoplastic sucrose (reduced in the InvLp6g::CIN1 plants) can decrease stomatal aperture (Outlaw and De Vlieghere-He, 2001), while a high hexose/sucrose ratio and concentration of the organic acids malate
T
and citrate in the apoplast (increased in the InvLp6g::CIN1 plants) can influence
IP
stomatal aperture (Araújo et al., 2011) and fruit yield under water stress (Garchery et al., 2013). Potato (Solanum tuberosum) plants expressing an antisense construct targeted
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against sucrose synthase 3 (SuSy3) had a lower stomatal conductance and slightly reduced CO2 fixation, while plants with increased guard cell acid invertase activity (by introducing the SUC2 gene from yeast) had increased stomatal conductance and CO2 14
CO2 feeding experiments
NU
fixation but decreased WUE (Antunes et al., 2012).
suggested that the changes in photosynthesis were mostly explained by stomatal
MA
regulation rather than altered mesophyll capacity for carbon fixation. Therefore, engineering sucrose metabolism in the apoplast or symplast of vegetative tissues also offers interesting opportunities to increase the source strength and reduce water use,
D
thus increasing stress tolerance and yield stability.
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Although photoassimilates are transported to heterotrophic sink organs where they are often again transiently or permanently stored as starch (Peterhansel and
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Offermann, 2012), new data also indicate that a stronger accumulation of leaf starch used in plants to sustain metabolic functions during the night positively impacts both source strength and sink yield. Multivariate analysis revealed that starch integrates the
AC
overall metabolic response within a regulatory network that balances growth with the carbon supply (Peterhansel and Offermann, 2012; Sulpice et al., 2009). Constitutive overexpression of a starch synthase gene SSIV in Arabidopsis resulted in higher leaf starch contents at the end of the day and faster growth rates of transgenic plants suggesting greater photosynthetic efficiency (Gámez-Arjona et al., 2011). The physiological basis of these results was investigated in more detail in Arabidopsis and rice plants overexpressing the rate-limiting enzyme ADP glucose pyrophosphorylase (AGPase) (Gibson et al., 2011). In this study photosynthetic rates correlated with AGPase activity especially when plants were grown at high CO2 availability (1000 ppm). This was probably due to reduced feedback inhibition by photosynthetic products (Gibson et al., 2011) and could explain why additional starch was not synthesized at the expense of other biomass. Other studies have shown the cooperative action of sucrolytic enzymes with starch biosynthesis enzymes in controlling source-sink relations. Chopra 17
ACCEPTED MANUSCRIPT et al. (2005), reported the synergistic role of sucrose synthase, UGPase and AGPase for starch biosynthesis in mung bean, thus controlling the efficient partitioning of sucrose into ADP-glucose and finally controlling seed sink strength. Further, ultra-structural
T
analyses in leaves of tomato plants overexpressing a cell wall invertase gene CIN1
IP
revealed distinct and large starch grains than the WT under drought stress conditions (Albacete et al., unpublished results), suggesting that the cycle of starch synthesis and
SC R
breakdown may modulate stress responses in transgenic plants (Sulpice et al., 2009) to maximize carbon uptake and growth.
NU
Taken together, transient or permanent sugar partitioning towards starch accumulation in both source and sink organs would maintain photoassimilate production (source strength), transport, and use (sink strength) that correlates with
MA
biomass production. Therefore, it would be expected that plants with high biomass production also show high starch accumulation during the day. This was recently tested by comparing growth and metabolite levels of 94 Arabidopsis accessions (Sulpice et al.,
D
2009). The most striking observation was that starch content at the end of the day was
TE
negatively correlated with above-ground biomass production. Thus, those plants that invested a large part of fixed carbon into transient starch for later use during the night,
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grew slower than those that used most fixed carbon for growth during the day (Sulpice et al., 2009; Sulpice et al., 2010). Instead, the percentage of total protein that was invested in enzymes of primary metabolism in the light correlated positively with
AC
biomass, supporting a link between metabolic activity during the day and final biomass. These data point to different strategies used in nature and in agricultural breeding systems to optimize biomass production and yield. Indeed, an active regulatory linkage between carbohydrates, sources and sinks has been suggested (Ainsworth and Bush, 2011), since there is a direct relation between assimilate loading and photosynthetic rate, since assimilate transport from leaves is required for continuous photosynthetic production (Nikinmaa et al., 2013; Turgeon, 2010). Nikinmaa et al. (2013) have postulated that assimilate transport in the phloem competes with transpiration for water, and an optimal level of stomatal opening should exist for a given conditions that maximizes phloem transport between source and sink organs, assuming that sugar loading is proportional to photosynthetic rate. Excess sugar loading, however, may block the assimilate transport because of viscosity build-up in 18
ACCEPTED MANUSCRIPT phloem sap, leading to starch accumulation and down-regulation of photosynthesis. Therefore, stomatal regulation seems to be directly connected with sink activity, plant structure and soil water availability as they all influence assimilate transport (Garchery
T
et al., 2013; Nikinmaa et al., 2013).
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It has been also recently postulated that reductions in stomata density and
SC R
stomatal aperture can reduce water loss by transpiration while maintaining sufficient CO2 uptake to sustain biomass and yield under water-deficit conditions (Yoo et al., 2009). Indeed, there are several examples where modifying a single gene decreased
NU
stomatal aperture and/or stomatal density and, consequently, increased WUE and resistance to water deficit-related stresses such as salinity and drought (reviewed in Yoo et al. (2009)). Molecular dissection of the genetic variability in WUE has only recently
MA
been initiated in the model Arabidopsis, where the ERECTA gene was found to be critical in altering transpiration efficiency by mechanisms including leaf epidermal and mesophyll differentiation (Masle et al., 2005).Therefore, an optimal relation between
D
transpiration and carbon assimilation and transport may be the reason for what reducing
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transpiration rates without affecting CO2 assimilation would result in increased WUE
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and may contribute to improve yield stability under stress conditions.
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3. Improving sink strength 3.1. Invertases, pollen viability and grain/fruit filling As already suggested, sink strength could act as a major driving force for maintaining assimilate transport, source activity and plant growth, and therefore, yield stability under adverse conditions. Indeed, an increasing body of direct or indirect evidence support this hypothesis. For instance, improving shoot Na+ exclusion capacity is considered a major target to increase salt tolerance in many crops, thus delaying the toxic effect and maintaining leaf function (Munns and Tester, 2008; Pérez-Alfocea et al., 2010). However, this capacity alone cannot always explain crop productivity under salinity. For example, wheat cv Tamaroi isogenic lines containing the Nax2 locus (encoding the HKT1 Na+-selective transporter) accumulated from 4 to 12-fold lesser Na+ in the flag leaf (according to the salinity level in the soil) than the cv Tamaroi in 19
ACCEPTED MANUSCRIPT field experiments (Munns et al., 2012). The improved yield (by 25%) of the Na+excluder line observed only under high salinity (169 mM NaCl) was explained by maintenance of the source strength (photosynthetic capacity) in the flag leaf during the
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grain filling stage. Nevertheless, this yield improvement was difficult to explain only by
IP
decreased Na+ accumulation, since the excluder line was 3-times more sensitive to the leaf Na+ concentration than the cv Tamaroi (by plotting yield vs leaf Na+ concentration).
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Additionally, the yield increase was more likely due to a reduced flower abortion index because the effect was observed on a higher number of grains rather than on higher grain weight. Similarly, a QTL for salt tolerance (SKC1) that encodes a Na+ transporter
NU
and regulates K+/Na+ homeostasis has been identified in rice (Ren et al., 2005). Introducing this QTL has high potential to improve salt tolerance but, to our knowledge,
MA
no practical results in terms of improved yield under salinity have been reported so far. Those examples suggest that other traits regulating flower/grain sink activity could be more related to yield than leaf Na+ accumulation.
D
In this regard, many self-fertilizing crops are particularly sensitive to abiotic
TE
stress at the reproductive stage and pollen development is considered a limiting step in crop productivity (see Parish et al. (2012), for a review). In rice and wheat,
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developmental process for pollen reproduction implies a programmed cell death (PCD) at the tapetum (the feeding tissue of the microspore), and seems to be delayed or inhibited by various abiotic stresses, causing premature pollen and flower abortion and
AC
subsequent yield losses. According to Parish et al. (2012), two hypotheses can be formulated to explain the effect of abiotic stress on flower abortion-related processes at pollen development. On one hand, the ‘starvation hypothesis’ postulates that abiotic stress provokes a decrease of assimilates reaching the developing microspores as a consequence of the downregulation of a tapetal cwInv and hexose transporters, such as MST8 in rice, which is regulated by a MYB transcription factor (Zhang et al., 2010). On the other hand, the ‘PCD hypothesis’, that can be connected with the previous one through carbohydrate availability, suggests that stress delays or prevents developmental PCD at the tapetum, thus failing to provide materials required for microspore development. Indeed, a decrease in tapetal cwInv activity was correlated with pollen abortion and reductions in hexoses reaching the tapetum. The anther cwInv gene IVR1 is repressed at meiosis in drought-sensitive wheat lines resulting in sucrose accumulation, but not in tolerant ones (Koonjul et al., 2005). Similar inhibitory effects have been 20
ACCEPTED MANUSCRIPT observed in the rice cwInv OsINV4 and the monosaccharide transporter OsMST8 genes under cold stress (Oliver et al., 2005; Sheoran and Saini, 1996). Thus, tissue sink activity (capacity to use assimilates) is correlated with tolerance to cold and drought
T
stress in rice and wheat (Ji et al., 2010; Oliver et al., 2005). Table 2 summarizes the
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hormonal and metabolic-related genes affecting sink strength.
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A similar situation seems to occur in dicotyledonous species such as tomato (see Pérez-Alfocea et al. (2010) for a review). Decreased fruit number is a major factor reducing tomato yield at high salinity (>75–100 mM NaCl), in addition to decreased
NU
fruit weight (Cuartero and Fernández-Muñoz, 1998). Processes involved in fruit set and development must be responsible for yield reduction under salinity. A highly saline treatment (150 mM NaCl for 10 days) decreased pollen viability, inducing tomato
MA
flower abortion (Ghanem et al., 2009). However, low pollen viability was apparently not explained by local Na+ toxicity in either pollen grains or the tapetum. Instead, flower abortion was apparently explained by decreased carbohydrate availability in the and
pollen-producing
D
inflorescence
tissues,
despite
increased
carbohydrate
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concentrations within source leaves (Ghanem et al., 2009). This is probably due to reduced transport from source leaves and decreased sink activity, as suggested by
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dramatic reductions in sucrolytic activities of cwInv and sucrose synthase during floral development and maturation. Thus cwInv seems essential in maintaining sucrose import to sink tissues (Koch, 2004; Roitsch et al., 2003; Roitsch and Ehness, 2000) during
AC
pollen development (Roitsch and González, 2004), especially under potential sourcelimiting stress conditions. Assuming that the flower is fertilized, fruit or grain filling is also a susceptible process limiting yield. For example, tomato yield reduction by low to moderate salinity levels (25–75 mM NaCl) in irrigation water is due more to decreased fruit weight than number (Cuartero and Fernández-Muñoz, 1998). In general, salinity decreased both sink strength and sink activity measured as absolute and relative rates of dry matter accumulation during early fruit development (from 20 days after anthesis until start of ripening). Decreased sink activity was related to sucrose accumulation, and to decreased activity of the apoplastic and cytoplasmic sucrose cleaving enzymes; namely, cell wall invertase, neutral invertase and sucrose synthase. Genotypic differences in these enzyme
21
ACCEPTED MANUSCRIPT activities were correlated with the degree of salt tolerance of domestic and wild tomato species and hybrids (Balibrea et al., 2003; Balibrea et al., 1999; Koch, 2004). The importance of sucrose metabolism in grain filling has also been
T
demonstrated in rice. The GIF1 (Grain Incomplete Filling 1) gene encodes for cwInv
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required for carbon partitioning during early grain filling (Wang et al., 2008). Grains
SC R
from the loss-of-function gif1 mutant show decreased glucose and fructose levels and sucrose accumulation, resulting in lower grain weight than the wild-type. The cultivated GIF1 allele showed a restricted expression pattern during grain filling compared with
NU
the wild rice allele because of accumulated mutations in the regulatory sequence of the gene by domestication. Ectopic expression of the cultivated GIF1 gene under the control of the 35S or rice Waxy promoters resulted in smaller grains, whereas
MA
overexpression of GIF1 driven by its native promoter increased grain production (Wang et al., 2008). It has been interpreted that the restricted expression of the GIF1 in the ovular vascular trace is key to increasing grain weight, suggesting that tissue-specific
TE
D
higher expression levels of GIF1 could provide a means to increase grain filling. Thus, tomato plants overexpressing a cwInv under the control of a fruit specific
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promoter (InvLp6g::CIN1) yielded up to 4-fold more than the WT and azygous lines under moderate salinity (75 mM NaCl) due to both decreased flower abortion (higher fruit number) and also due to a significant increase in the individual fruit weight.
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Increased sucrose partitioning from source leaves to the fruits in the salinized transgenic plants (compared to the WT and azygous lines) was explained by increased cwInv gene expression and activity, but curiously also by increased levels of trans-zeatin (t-Z) and reduced concentrations of the ethylene-precursor ACC (Albacete et al., unpublished results).Thus cwInv seems to function at the integration point of metabolic, hormonal and stress signals, providing a novel strategy to overcome drought- and salt-induced limitations to crop yield, without negatively affecting plant fitness under optimal growth conditions. An additional potential target to negative yield impacts of salt and drought conditions is the posttranslational relief of invertase from inhibition by a proteinaceous inhibitor (Ruan et al., 2010). Only recently, in vivo functionality and the physiological significance of these inhibitors on plant growth, development and stress responses have been demonstrated (Bonfig et al., 2010; Jin et al., 2009). Such post-translational
22
ACCEPTED MANUSCRIPT regulation is particularly relevant to cwInv because these proteins are intrinsically stable due to their glycosylation (Rausch and Greiner, 2004) Downregulation of some sucrolytic activities in response to drought or salinity
T
may be attenuated by ectopic regulation of these enzymes using promoters that are
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specific to sink tissues. However, these enzymes seem tightly regulated by
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phytohormones as part of an integrated and coordinated mechanism to control growth and development, and thus changes in phytohormone concentration provoked by the stress in vegetative tissues may decrease sucrolytic activities under salinity (Albacete et al., 2008; Pérez-Alfocea et al., 2011; Roitsch et al., 2003). Internal water status or the
NU
accumulation of toxic ions (Na+, Cl-) seem to be maintained in floral organs even following substantial leaf dehydration (i.e. droughted wheat, reviewed in Saini et al.
MA
(1999)) or Na+ accumulation (i.e. salinized tomatoes, Balibrea et al. (1999); Ghanem et al. (2009)). However, positive (i.e. CKs, GAs, auxins) and negative (i.e. ethylene, ABA) hormones coming from mature leaves or roots in response to stress may be good
D
candidates as signaling molecules, regulating sink activity in the reproductive organs at
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pre- or post-anthesis by influencing carbohydrate supply to the tapetum, microspores, and fertilized ovaries. Unravelling the regulation of those positive and negative signals
conditions.
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on sink activity could open new strategies to maintain growth and yield under stress
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3.2. Hormonal signalling and regulation of sink activity 3.2.1. Gibberellins
A large body of evidence supports the positive effects of exogenous gibberellic acid (GA3) application on plant growth and yield under stress conditions by inducing source and sink activities (reviewed in Iqbal et al. (2011)). GA3 increases source strength by improving photosynthetic efficiency via influencing some photosynthetic-related enzymes (i.e. Rubisco, fructose-1,6-biphosphatase and sucrose phosphate synthase), leaf area, light interception and phloem loading. Additionally, GA3 also induces sink strength by promoting cell division and growth and carbohydrate import through inducing the sucrolytic activities, namely the cwInv, a key enzyme in the regulation of phloem unloading (Roitsch and González, 2004). The induction of both source and sink strength increase the efficiency of assimilate production and transport, and thus source23
ACCEPTED MANUSCRIPT sink relations. GA signalling is necessary for normal tapetal function by influencing the availability of sugars to the tapetum (Plackett et al., 2011), but the effects of abiotic stress on GA level in developing anthers are not known (Parish et al., 2012).
T
Recently, it has been demonstrated in Arabidopsis and other model species that
IP
GA signalling is involved in metabolic adjustment for maintaining source–sink relations
SC R
of plants under limiting environmental conditions, and thus providing a plethora of potential molecular targets for biotechnological improvement of stress tolerance (Achard et al., 2006). Paradoxically, most of these studies indicate that GA signalling in
NU
response to stress seems to reduce growth to allow plant adaptation and survival, which cannot always be compatible with yield stability. Bioactive GA4 and GA1 levels decreased under high-salinity stress (100 mM NaCl) in Arabidopsis by more than 50%,
MA
which can be explained by the upregulation of six GA2oxidase genes, and thus reducing the levels of bioactive GAs (Achard et al., 2006; Magome et al., 2008). This response is mediated by the Dwarf and Delayed Flowering 1 (DDF1) gene, encoding an AP2
D
transcription factor of the DREB1/CBF subfamily that causes dwarfism by reducing
TE
bioactive GA concentrations in transgenic Arabidopsis (Magome et al., 2004). Further, the loss-of-function ga2ox7-2 mutant suppressed the dwarf genotype of DDF1–
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overexpressing plants and had similar shoot growth to the WT at 100-150 mM NaCl, although it showed increased root length at 50-100 mM NaCl (Magome et al., 2008). These results demonstrate that, under salinity stress, Arabidopsis plants actively reduce
AC
endogenous GA levels by inducing DDF1 and GA 2-oxidase, thus limiting growth for stress adaptation. Although other GA2oxs are upregulated by high-salinity stress in a DDF1-independent manner, DDF1 also directly upregulates the expression of several stress-responsive genes (such as RD29A and COR15A) that contribute to stress tolerance. A similar response occurred under osmotic stress (300 mM mannitol) and 100 μM ABA (Magome et al., 2008). Additionally, overexpression of the AtDREB1A in soybean (Glycine max L.) also produced typical GA-deficient dwarf phenotypes, due to the deactivation of bioactive GAs (Suo et al., 2012), with increased root length and tolerance to high salinity (300 mM NaCl) in tobacco (Cong et al., 2008), and drought resistance in tomato (SlDREB; Li et al. (2012)). These results suggest a key role in saltmediated sink growth impairment to the GA-DELLA signalling network (Achard et al., 2006).
24
ACCEPTED MANUSCRIPT Quadruple-DELLA mutants (lacking RGA, GAI, RGL1, and RGL2) had normal growth and lower salt tolerance under high (100-200 mM NaCl) salinity (Achard et al. (2006). Ectopic expression of the gain-of-function mutant Mhgai1 encoding a DELLA
T
protein (Malus hupehensis Redh. var. pingyiensis) in tomato reduced plant height and
IP
fruit set ratio, but it enhanced vegetative drought-tolerance (Wang et al., 2012). Moreover, a grafting experiment demonstrated the long-distance movement of Mhgai1
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mRNAs and its effect on dwarfing the scion genotype, which suggests root-targeted strategies to manipulate GA signalling in the plant and, consequently, source-sink relations and stress responses. Similar results were obtained by overexpressing the GA
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Methyl Transferase1 (AtGAMT1) gene that encodes for an enzyme that catalyzes the methylation of active GAs to generate inactive GA methyl esters (Varbanova et al.,
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2007). Tomato plants constitutively overexpressing the AtGAMT1 gene exhibited typical semi-dwarfed and greener GA-deficiency phenotypes and increased tolerance to drought stress (Nir et al., 2013). Transgenic plants maintained high leaf water status
D
under drought conditions, due to reduced stomatal conductance and whole-plant
TE
transpiration, thereby delaying desiccation and maintaining higher recovery rates. GA application to the transgenic plants restored normal growth and sensitivity to drought,
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supporting the role of GAs in coordinating growth and stress adaptation. Fruit growth in Arabidopsis also occurs via DELLA-dependent and DELLAindependent
(Gibberellin-Insensitive
DWARF1–mediated
GA
perception)
GA
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responses (Fuentes et al., 2012). Consequently, the growth of harvestable sinks is also regulated by GAs and increased endogenous GA concentrations could improve yield. Overexpression of the citrus gene CcGA20ox1, encoding the GA biosynthetic enzyme GA20-oxidase, in tomato (Solanum lycopersicum L. cv Micro-Tom) produced higher yield and number of fruits per plant with increased citric acid content (a component of ºBrix), and also elevated GA4 levels, thus mimicing GA treatments (García-Hurtado et al., 2012). Further studies of yield responses of GA-overproducing plants under adverse conditions are required to clarify the paradoxical relation between GAs, plant growth, plant survival and adaptation, and yield. 3.2.2. Cytokinins Similar to GAs, both increases and decreases in CK levels have been reported to have a positive impact on abiotic stress tolerance since CK homeostasis and signalling could 25
ACCEPTED MANUSCRIPT also play a key regulatory role between plant growth and plant survival (Ha et al., 2012; Zalabák et al., 2013). Consequently, CK impacts on source-sink relations and the subsequent morphological adjustment that optimizes growth and plant survival has a
T
different impact on crop productivity. While maintaining plant growth is a prerequisite
IP
for crop stability under stressful conditions, shoot growth inhibition and promotion of root growth (Sharp et al., 2004) has been regarded as an advantage in adverse
SC R
environments and is an integral part of plant stress tolerance to promote plant survival (Achard et al., 2006; Achard et al., 2008; Huang et al., 2009), as seems to occur in CKdeficient and CK receptor kinase mutants (Nishiyama et al., 2011). Hence, both
NU
constitutive overexpression of the CK-degrading enzyme cytokinin oxidase (CKX) or inhibition of the CK-biosynthetic IPT1, IPT3, IPT5, and IPT7 genes resulted in CK
MA
deficiency and enhanced drought- and salt stress-tolerant phenotypes. Enhanced drought tolerance of the CK-deficient plants was attributed to their capacity to maintain higher water content under stress, which was associated with their intact membrane structure,
D
as evidenced by lower electrolyte leakage (Nishiyama et al., 2011). Drought-tolerance
TE
was not explained by differences in stomatal opening or density since ABA response and concentration were similar to WT plants. Indeed, several studies have reported that
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plant growth responses to drought or salinity are not always a consequence of (or explained by) an alteration in stomata-related traits (Balibrea et al., 2000; Bartels and Sunkar, 2005; Fujita et al., 2006; Hirayama and Shinozaki, 2010). However, the tolerance was rather explained by a higher (i) ABA/CK ratio, (ii) sensitivity to ABA in
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the CK-deficient plants, (iii) subsequent higher expression of the ABA-related stress tolerance genes, such as structural protective proteins and/or intracellular solute concentrations(Verslues et al., 2006), (iv), shoot-to-root sink strength, as indicated above. This idea is also supported by the lower shoot sink activities due to both decreased cell number in leaf meristems of tobacco plants overexpressing the CKdegrading enzyme CKX (Werner et al., 2001) and sucrolytic activities (Werner et al., 2008). Similarly, growth inhibition of CK receptor- (Nishimura et al., 2004) and CK activating enzyme- (Kurakawa et al., 2007) defective mutants was associated with lower meristematic activity. However, CK-deficient and CK receptor His kinase mutants have enhanced root growth but retarded shoot growth, suggesting CKs are negative regulators in responses to water stress (Higuchi et al., 2004; Jeon et al., 2010; Miyawaki et al., 2006; Nishimura et al., 2004; Riefler et al., 2006; Tran et al., 2004; Werner et al., 2003), thus confirming a key role for a threshold CK concentration or sensitivity in 26
ACCEPTED MANUSCRIPT regulating plant growth and biomass partitioning, which seems critical for plant survival under abiotic stress. Nevertheless, increased CK levels under abiotic stress conditions were
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proportional to the induced growth rate compared to the control plants. In tomato, root-
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specific IPT expression promoted leaf expansion up to 1.5-fold, shoot growth up to 1.9-
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fold, and maximum efficiency of PSII (Fv/Fm) in salinized plants, compared to control plants (Ghanem et al., 2011a). Thus the average 2- to 2.5-fold increase in shoot biomass and relative growth rate was associated with a 2- to 2.6-fold increase in leaf CK
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concentration, while the 4.5-fold increase in biomass of droughted tobacco was associated with up to 5-fold increase in leaf CK concentration (PSARK::IPT; Rivero et al. (2007)). Moreover, grafting WT plants onto a constitutively (35S) expressing IPT
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rootstock increased fruit yield (by 30%) compared with salinized self-grafted WT/WT plants. This effect was probably due to increased shoot development and/or reduced flower abortion (as suggested by the 25% increase in fruit number), where increased t-Z
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concentrations (from 1.5- to 2-fold) in the actively growing fruits promoted cell division
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and expansion, thus slightly increasing (by 5%) fruit weight (Ghanem et al., 2011a). In contrast, transiently elevated fruit CK concentrations (up to 9-fold) very early in tomato
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fruit development did not increase final fruit size or total fruit yield per plant in the absence of stress (AGPase::IPT; Luo et al. (2005)). However, IPT expression in senescing leaves increased individual tomato fruit weight by 20% (SAG12::IPT;
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Swartzberg et al. (2006)), probably due to increased sink assimilate import because of delayed source leaf senescence. Shoot growth maintenance (induced HSP70::IPT plants) and increased fruit production (WT/35S::IPT plants) under salinity may be result from not only increased source capacity to produce and/or export more assimilates due to both increased leaf area and delayed senescence, but also increased import and/or utilization of assimilates in sink organs achieved by an increased cell population attracting photoassimilates and/or an increased capacity to utilize imported sucrose metabolically through regulating sucrolytic enzymes (Lara et al., 2004; Roitsch and González, 2004). Depending on the promoter used and the growing conditions, it generally seems that the physiological impacts of overcoming decreased CK status (through transgenic IPT expression) were most pronounced in plants experiencing abiotic stress. However, 27
ACCEPTED MANUSCRIPT excessive overproduction of CKs above a threshold also caused stunted plant growth, abnormal tissue development, and sensitivity to drought (Havlová et al., 2008; Hewelt et al., 1994; Li et al., 1992; Synková et al., 1999; Wang et al., 1997). Consequently,
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appropriate manipulation of CK levels is necessary to increase leaf longevity and
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photosynthetic capacity (source strength) but also growth of sink organs (sink strength)
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under abiotic stress. 3.2.3. Auxins
Auxins can influence carbon partitioning in plants, and a number of studies have shown
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that both factors are interrelated. Thus auxins stimulate the mobilization of carbohydrates in leaves and the upper stem and increase the translocation of assimilates
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toward sink organs (Smith and Samach, 2013). Auxins may also influence carbohydrate utilization. Additionally, the interplay of auxin with other hormones may ultimately regulate carbohydrate levels and sugar signaling molecules (Hartig and Beck, 2006;
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Smith and Samach, 2013). Agulló-Antón et al. (2011) highlighted the interactions
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between auxin and carbohydrates and their regulatory role in sink establishment and growth in carnation cuttings. A transcriptomic study on hybrid embryos of Vicia faba
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has shown the importance of a set of genes related to auxin function and metabolism on the sink strength of hybrid seeds (Meitzel et al., 2011). These authors proposed a regulatory model on seed carbon allocation and growth where the auxin-related genes
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AUX/IAA, AXR1, and RBX1-like E3 gene play key roles. Further, it has been reported that auxins regulate the activity of the cwInv and thus sucrose allocation in sink organs (Albacete et al., 2008; Proels and Roitsch, 2009; Roitsch et al., 2003). To understand the molecular details of sugar–invertase-IAA relationships, genes from the two IAA biosynthetic pathways ZmYuc1 (tryptamine pathway) and ZmTar1 (indolpyruvic acid pathway) were studied in developing seeds of the invertase-deficient mutant miniature1 of maize (Chourey et al., 2010a). There were redundant trpdependent pathways of auxin biosynthesis in developing maize seeds, suggesting that homeostatic control of IAA in this important sink is highly complex and may be regulated by both sucrose metabolism and developmental signals, since cell division is fuelled by heterotrophic metabolism based on the supply of carbohydrates from source leaves. Furthermore, crosstalk between auxins, cytokinins and sugars has been reported in regulating the plant cell cycle (reviewed by Hartig and Beck (2006)). 28
ACCEPTED MANUSCRIPT 3.2.4. Abscisic acid ABA synthesis in the roots, and transport through the xylem to the shoot is one of the most rapid hormonal responses to drought and salinity, causing stomatal closure to
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reduce transpiration water loss and eventually restricting cellular growth. ABA can also
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be synthesized in leaf cells and transported through the plant (Dodd, 2005; Thompson et
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al., 2007), while guard-cell ABA production seems autonomous to regulate stomatal response (Bauer et al., 2013). ABA has been implicated in male sterility of tomato, rice and wheat (Morgan and King, 1984; Morgan, 1980; Westgate et al., 1996), with
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negative correlation between ABA levels and cold and drought tolerance (Ji et al., 2010; Oliver et al., 2007). Exogenous ABA application mimics the effect of stress provoking pollen sterility in barley by reducing sucrose import by inhibiting invertases and
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monosaccharide transporters, thus preventing microspores from PCD (Parish et al., 2012).
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Nevertheless, and contrarily to the reported negative effect of ABA on pollen
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viability some studies in cereals reported positive correlations between grain ABA content and efficient seed filling by optimizing faster remobilization events from stem
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reserves (nonstructural carbohydrates), a critical factor in sustaining grain filling and grain yield under drought stress (Yang et al., 2004; Yang et al., 2001). Indeed, overexpressing the LOS5 gene that encodes the key enzyme molybdenum-cofactor
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sulfurase in the last step of ABA biosynthesis, under the control of constitutive or drought-inducible promoters improved yield when water stress occurred during the initiation of panicle development by significantly increasing spikelet fertility. In contrast, the NCED2 gene, also involved in ABA biosynthesis, had no positive (or even negative) effects when compared to the WT (Xiao et al., 2009). Different responses in those transgenic plants may be due to the different hormone concentrations before and during the stress period. Indeed, the constitutive overexpression of LeNCED1 (droughtinducible and a rate-limiting enzyme for ABA biosynthesis) in tomato continuously elevated ABA accumulation, resulting in physiological and morphological changes in the transgenic plants. Under well-watered conditions, plants showed, leaf flooding, chlorosis and reduced assimilation rates occurred, but under water-deficit conditions these effects were insufficient to reduce biomass production, presumably because of counteracting positive effects on leaf expansion by improving water status and turgor, 29
ACCEPTED MANUSCRIPT and antagonising ethylene-induced epinastic growth (Thompson et al., 2007). Similarly, natural constitutively elevated ABA production in the citrus rootstock induced tolerance to water stress in sweet orange (Citrus sinensis) trees allowing a better regulation of
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gas-exchange parameters in the shoot, but reduced growth under control conditions
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(Allario et al., 2013). Interestingly, the drought tolerant (autotetraploid rootstock clone from Rangpur lime, Citrus limonia) graft combination showed decreased stomatal
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conductance, higher leaf and root ABA concentrations, and an increased expression of drought responsive genes, including CsNCED1 (key for ABA biosynthesis) than the same plant grafted onto the diploid rootstocks. These results highlight the importance of
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both constitutive (non-induced) expression of defence mechanisms and the longdistance ABA signalling in the adaptation to stress (Allario et al., 2013). The effect of
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pre- or over-elevated ABA on yield under optimal or suboptimal conditions needs to be addressed.
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3.2.5. Jasmonates
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The effect of jasmonates on sink activity may differ depending on the tissue and on the nature of the stress. Transgenic rice expressing PUbi1::AtJMT (JA carboxyl
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methyltransferase) displayed 6-fold increased JA levels and significantly lower grain yields (because of fewer spikelets per panicle, reduced grain filling rate and altered floral organ numbers), which was reproduced by applying exogenous methyl jasmonate
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(MeJA) (Kim et al., 2009). Interestingly, MeJA levels were increased by 19-fold in young WT panicles upon exposure to drought conditions, decreasing grain yield to a similar level to that observed in Ubi1::AtJMT plants. Leaf ABA concentrations only increased by 1.5-2-fold in non-droughted Ubi1::AtJMT and droughted WT plants, suggesting that biosynthesis was secondarily stimulated under drought conditions by the previous MeJA accumulation (Kim et al., 2009). Indeed, the OsSDR gene, an Arabidopsis ortholog which is essential for JA and ABA biosynthesis (Adie et al., 2007; González-Guzmán et al., 2002), was up-regulated in both Ubi1::AtJMT and droughttreated WT panicles, supporting its involvement in MeJA-dependent increases in ABA levels, and that ABA either precedes or cooperates with JA in signalling pathways under drought conditions (Kim et al., 2009). Similarly, Arabidopsis plants transformed with 35S::AtJMT produced 40% less total seed weight than WT controls due to fewer flowers (Cipollini, 2007), and the 30
ACCEPTED MANUSCRIPT application of exogenous MeJA on Pharbitis nil (Maciejewska and Kopcewicz, 2002) and Nicotiana sylvestris (Baldwin and Hamilton, 2000) dramatically reduced flowers number, which was explained by increased sterility (Zhu et al., 2004). In addition to the
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cost associated with constitutive overexpression of AtJMT and overproduction of MeJA,
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decreased energetic allocation towards reproductive structures in favour of activation of defence mechanisms cannot be ruled out (Cipollini, 2007; 2010). It is likely that
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increased levels of MeJA reduce spikelet number in Ubi1::AtJMT panicles by repressing cell cycle progression (Kim et al., 2009). This may be due to decreased assimilate transport to the reproductive structures due to changes in sink preferences under stress. 11
CO2 fixation and
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C-photosynthate
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In this regard, it was recently reported that both
export from the labelled source leaf increased rapidly (2 h) following MeJA treatment
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(simulating herbivore wounding action) relative to controls, with preferential allocation of radiolabel primarily to the roots and secondly to the young leaves, where label was incorporated into
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C-cinnamic acid and other defence-related phenolic compounds as
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anthocyanins (Ferrieri et al., 2013). The switch in relative sink strength correlated with
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increased cell wall and soluble invertase activities in both roots and young leaves and was suppressed in sucrose transporter mutant plants (suc2-1), indicating that this
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phenomenon may be controlled by phloem loading/unloading processes. It would be interesting if the same MeJA-mediated phenomenon could explain yield decrease under abiotic stress conditions.
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Since adventitious root formation requires an adequate supply of carbohydrates to the region of root regeneration, it has been used as model system to study the molecular and biochemical processes involved in the interaction between JA and primary metabolism. In a study with Petunia hybrida cuttings, increased transcripts coding for cwInv and the apoplastic localized monosaccharide transporter gen STP4 were related to transient JA accumulation (Ahkami et al., 2009). Similarly, Henkes et al. (2008) showed that JA treatment to a part of barley roots caused signal transduction from the treated roots to the shoot, leading to an increase in carbon allocation from the leaves, simulating a wounding effect. Therefore, bioactive jasmonates as JA and MeJA could be involved in redirecting resources from growth to defence mechanisms (Chen et al., 2011). This
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ACCEPTED MANUSCRIPT differential metabolic response may be responsible for the decrease in yield-related parameters in both droughted and jasmonate overproducing plants.
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3.2.6. Salicylic acid
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It has been considered that SA plays a crucial role in the plant energy status,
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translocation and storage of assimilates in response to biotic and abiotic factors. Carbohydrate metabolism in both source and sink tissue was regulated by SA activating plant defence response against pathogens and other biotic stresses through increasing invertase activity or vice versa (Ehness and Roitsch, 1997; LeClere et al., 2008). Foliar
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SA (10-6-10-4 M) sprays decreased hexose concentrations in the leaf but increased them in fruits of salinized pepper plants, with concomitant increases in sink strength and fruit
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growth (Elwan and El-Hamahmy, 2009). Increased sink strength by exogenous application of SA was also found in wheat (Arfan et al., 2007) and maize (Gunes et al., 2007). Further, SA can interact with other hormones in regulating source-sink
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relationships under stress conditions (Iqbal et al., 2011). For example, constitutive
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overexpression of a GA-responsive gene FSGASA4 in Arabidopsis plants subjected to salt stress increased not only the endogenous levels of SA, but also the expression of
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ICS1 and NPR1 genes involved in SA biosynthesis and action, respectively (AlonsoRamírez et al., 2009a; b). This hypothesis was supported by the finding that sid2 mutants, impaired in SA biosynthesis, were more sensitive to salt stress than the wild
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type without any apparent effect of exogenous application of GAs (Alonso-Ramírez et al., 2009b).
3.2.7. Ethylene
Although ethylene can stimulate the growth of certain plant organs under specific circumstances (i.e. internode extension of deep-water rice), it is often regarded as a growth inhibitor (Pierik et al., 2007). Exogenous application of the ethylene-releasing compound ethephon decreased both root and fruit sink strength in salinized tomato plants by decreasing invertase activity (Albacete et al, unpublished results). Thus, researchers have pursued chemical, genetic, and even microbiological ways of decreasing crop ethylene production. In this sense, some rhizosphere bacteria use ACC, the immediate precursor of ethylene in higher plants, by hydrolysing it through the enzyme ACC deaminase into α-ketobutyrate and ammonia, and in this way promote 32
ACCEPTED MANUSCRIPT sink strength and root growth by lowering endogenous ethylene levels in the microrhizo environment (Hayat et al., 2010). Different biotechnological strategies have been also use to reduce ethylene biosynthesis and perception (reviewed by Stearns and Glick
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(2003)), mainly focused on the ACC deaminase gene, and have been shown in many
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cases to regulate carbon metabolism and sink strength. Additionally, attempts were made to manipulate the growth and development of the pollen grains by exogenous
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application of chemicals regulating formation/action of ethylene. For example, application of ethylene action (AgNO3) and synthesis inhibitor (Co(NO3)2, paclobutrazol and uniconazole) improved grain setting in rice (Naik and Mohapatra,
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1999), related to enhanced activities of acid invertases and sucrose synthase.
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3.2.8. Strigolactones
The novel class of plant hormones strigolactones (SLs) has a clear impact in source-sink relations, since they modulate shoot branching and height and could have an important
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implication in crop productivity in diverse species (de Saint Germain et al., 2013). For
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example, the reduction in SL production in rice mutants affected in carotenoid-derived MAX/RMS/D (more axillary branching) pathway resulted in increased tillers and
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reduced plant height and spikelet fertility (Lin et al., 2009; Liu et al., 2013) Although the effect of SL on stem elongation seems to be independent of GAs (de Saint Germain et al., 2013), strong evidence supporting the role of SLs in the regulation of growth
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redistribution in the shoot by rapidly modulating auxin transport (Shinohara et al., 2013) has been provided. This SL-mediated regulatory effect is important to coordinate biomass reallocation in response to stress, since auxin has been proposed to mediate the systemic growth coordination by sensing the environmental cues. In particular, a positive feedback mechanism that establishes auxin flow from active shoot meristems (auxin sources) to the roots (auxin sinks) has been proposed to mediate competition between shoot meristems and to balance shoot and root growth (Shinohara et al., 2013). Indeed, this regulatory mechanism might be responsible for the plant response to nutrient starvation, water and saline stresses, since an induction in SL production would minimize shoot branching, increase the root/shoot ratio and promote root colonization by AM fungi, thus alleviating the stress impact (Aroca et al., 2013; Xie and Yoneyama, 2010). Further research is required to elucidate the potential of SL manipulation in order
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ACCEPTED MANUSCRIPT to improve crop productivity by altering biomass allocation under abiotic stress conditions.
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3.3. Root-sink strength to improve stress adaptation
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Many genes having a positive effect on abiotic stress tolerance seem to have a direct or
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indirect effect on source-sink relations, including the roots. Although enhanced root growth could be initially considered as a waste of assimilates to the detriment of shoot growth and yield, it has been hypothesized that modified sucrose allocation to the roots in plants growing under saline or drought stress may be an adaptive response to recover
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the functional equilibrium of the plant under the new conditions (Albacete et al., 2008; Balibrea et al., 2000; Sharp et al., 2004; Siahpoosh et al., 2012). Indeed, root-specific
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traits can be exploited to improve resource (water and nutrients) capture and plant development under resource-limited conditions (Ghanem et al., 2011b). The reduction of root sucrose levels in sensitive rice cultivars after exposure to high-salt
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concentrations was associated with a complex metabolic depletion syndrome
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(Siahpoosh et al., 2012). Because sucrose transporters were previously implied to be part of the rice salt acclimation process (Shinozaki and Yamaguchi-Shinozaki, 2007),
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the increased supply of assimilates to the root organ (i.e. manipulation of the sucrose transporters and/or invertases) should be considered in breeding programs for increasing salt tolerance, which would help to maintain root functioning (i.e. toxic ions exclusion)
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and adaptation to stress (Ghanem et al., 2011a; Ghanem et al., 2011b; Pérez-Alfocea et al., 2010; Siahpoosh et al., 2012). Increased assimilate partitioning to the roots of salinized tomato plants can be explained by changes in cwInv activity in both roots and leaves (Albacete et al., 2008), as reported in CK-deficient tobacco plants (Werner et al., 2008). This enzyme regulates sucrose transport by controlling the apoplastic unloading step from phloem (Roitsch et al., 2003). However, although CKs are major hormones establishing sink activity through the co-ordinated induction of cwInv and hexose transporters in order to enhance phloem carbohydrate supply to actively growing tissues (Roitsch et al., 2000), this response seems to depend on the tissue, and CKs are unlikely to be directly responsible for the cwInv induction in the roots. This idea is supported by the induction or maintenance of this enzyme activity reported in roots of CKXoverexpressing tobacco plants with lowered total and bioactive CKs (Werner et al., 2001). 34
ACCEPTED MANUSCRIPT However, IAA seems to induce both sink (sucrose allocation) and cwInv activities in both young leaves and roots (Albacete et al., 2008; Roitsch et al., 2003), supporting its role in the relative increase in root sink strength under drought and
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salinity. Indeed, lateral root initiation is suppressed by ABA signals while auxin is a
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crucial phytohormone for the initiation of lateral roots by promoting lateral root formation under water stress (Seo et al., 2009). Several MYB genes have been identified
The
constitutive
expression
of
the
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for their important roles in auxin-induced lateral root formation (Dubois et al., 2013). R2R3-MYB
transcription
factor
gene
35S::MdSIMYB1 from apple (Malus × domestica) in tobacco and apple resulted in
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ABA- and NaCl- insensitive germination and enhanced tolerance to high salinity, drought and cold by up-regulating the stress-responsive genes NtDREB1A, NtERD10B
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and NtERD10C (Wang et al., 2013). Transgenic tobacco plants also exhibited increased root sink strength (measured as biomass allocation) because of enhanced expression of the auxin-responsive genes NtIAA4.2, NtIAA4.1, and NtIAA2.5 under stress conditions.
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Gene transcripts were abundant in the leaves, flowers, and fruits compared to other
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organs, and were induced by abiotic stresses and plant hormones. Dexamethasone-induced expression of the Arabidopsis HARDY (HRD) gene,
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encoding for AP2/ethylene response factor (ERF)-like transcription factor that belongs to the BREB/CRB family in rice, improves WUE by enhancing photosynthetic assimilation (40-55% more than WT under control and drought conditions, respectively)
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and reducing transpiration, thus increasing both intrinsic (photosynthetic rate/stomatal conductance) and agronomical (biomass/water use) WUE (Karaba et al., 2007). Although no yield data were provided, these drought-tolerant low-water-consuming rice plants exhibit increased shoot biomass under well watered conditions and an adaptive increase in root biomass under drought stress (soil at 70% of field capacity) without growth penalty under control conditions. The HRD gene was identified by a gain-offunction Arabidopsis mutant hrd-D having roots with enhanced strength, branching and cortical cells, which exhibited drought resistance and salt (300 mM NaCl) tolerance accompanied by enhanced expression of abiotic stress-associated genes. HRD overexpression in Arabidopsis produced thicker leaves with more chloroplast-bearing mesophyll cells and, in rice, leaf biomass and bundle sheath cells were increased which probably contributed to the enhanced photosynthetic assimilation and efficiency. The mechanism(s) involved in regulating these responses remains unknown, but gene 35
ACCEPTED MANUSCRIPT clusters repressed under drought are up-regulated by HRD, suggesting a protective influence on essential processes, such as protein biosynthesis and carbohydrate metabolism (Karaba et al., 2007). Increased photosynthesizing area and carbon
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assimilation (better mesophyll efficiency) would significantly improve canopy
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photosynthesis, enhancing the capacity of both source and sink tissues and resulting in high biomass. Therefore, the increase in root-sink activity is an example not only of
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adaptation to drought and salinity, but also of source activity maintenance, as indicated by the unaltered maximum quantum yield of PSII (Fv/Fm), with a potential positive
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impact on yield stability. 3.4. Increasing sink number
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Modifying plant architecture, which implies alterations in source-sink balance, can potentially increase crop yield also under suboptimal conditions, as stated above. Since grain yield is controlled by QTLs derived from natural variations in many crop plants,
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fruit/grain number and weight are the most important traits in determining crop
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productivity. Three promising recent examples alter the plant architecture through modifying shoot meristematic sink activity. Grain number 1a (Gn1a) is a major QTL of
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grain number in rice that encodes the CK-degrading enzyme OsCKX2. This gene causes CK accumulation in the inflorescence meristem and increases the number of reproductive organs, increasing grain number per panicle (45%) and per plant (34%) in
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the near isogenic line (NIL)-Gn1a with respect to the Koshihikari genetic background (Ashikari et al., 2005). Similarly, the gain-of-function mutation allele of DEP1 (Dense and Erect Panicle1), coding for a truncated phosphatidylethanolamine-binding proteinlike domain protein, enhances meristematic activity and reduces length of the inflorescence internode resulting in three dominant pleiotropic phenotypes: dense and erect panicle, increased number of grains per panicle and a consequent increase in grain yield (40%), although they also have decreased grain weight (Huang et al., 2009). More recently, a point mutation in the OsSPL14 (Souamosa Promoter Binding Protein-Like 14 gene), located in the IPA1 (ideal plant architecture) QTL, perturbs the microRNA OsmiR156-directed regulation of OsSPL14, generating an ideal rice plant with a reduced tiller number, increased lodging resistance and more and bigger grains per panicle (Jiao et al., 2010). It was demonstrated that introducting the OsSPL14 ipa1 allele into the common japonica rice variety Xiushui 11 cultivated in the South of China increased the 36
ACCEPTED MANUSCRIPT grain yield by more than 10% in the test plot (Jiao et al., 2010). The yield stability of those high yielding lines under changing environments needs to be addressed given the critical role of CKs and microRNAs in both plant development and response to abiotic
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stresses (see Lima et al. (2011), for a review).
4. Conclusions
Figures 1 and 2 summarize the different hormonal and metabolic factors that regulate
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source-sink relations with demonstrated or potential impact on plant performance under abiotic stresses that have been addressed in this review. Since salinity and drought
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decrease crop productivity by reducing the activity of the source leaves, and/or the number and size of the harvestable sink tissues, maintaining source and sink activities is the best strategy to preserve crop productivity. However, the reallocation of assimilates
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between different sink organs also represents an adaptive response to stress in terms of
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plant survival and/or growth (and yield) stability. Delayed senescence with concomitant growth penalty (plant survival) could help to withstand transient stress but will not
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guarantee food security under permanent environmental constraints. Nevertheless, since the physiological plant adaptation has an energetic cost, yield stability will probably require an increase in either photosynthetic and/or adaptability efficiencies. Photosynthetic efficiency (source activity) can be optimized in an integrated process
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with stomatal opening, assimilate transport and sink activity (optimization of sourcesink relations), probably requiring a modelling approach. However, Omics technologies have revealed hundreds of genes involved in adaptation and maintenance of source activity by delaying senescence, but in detriment of growth (sink activity). Ideally, maintaining or increasing sink activity in the plant (individual or collectively) will help to avoid initial photoinhibition and premature stress-induced senescence, thus maintaining assimilate production and transport in/from the source tissues. For this reason it is essential to gain insights into the factors regulating sink activity (i.e. at inflorescence setting, flower fertilization and fruit/grain filling). Hence, the discovery of any novel gene involved in stress adaptation in model species should include an investigation on sink activity and, ultimately, on yield performance in crop plants. Increasing sink activity in the reproductive organs by either (i) the overexpression of sucrolytic activities, for example under the control of a fruit/grain specific promoter, or 37
ACCEPTED MANUSCRIPT (ii) the positive (i.e. CKs, GAs) or negative (i.e. ethylene) modulation of hormonal factors regulating the sink number and activity, could be useful strategies for improving yield under suboptimal abiotic stress conditions. Other specific metabolic alterations
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(i.e. carbon metabolism) can also improve the photosynthesis and WUE under resource
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limiting conditions. Finally, some of those factors seem to be able to simultaneously increase or maintain both source and sink activities (i.e. CK signalling; cell wall
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invertase) under stress, thus minimizing the negative impact on yield. Root-targeted strategies could be used to optimize source-sink relations through manipulating root-to-
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shoot hormonal and genetic (ie. miRNA) communication.
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Acknowledgements
AA is very grateful to CSIC (Spain) for a postdoc research grant (I3P program). The
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authors thank Dr Ian C Dodd for revising the English version of the manuscript and for
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constructive criticism, the European Commission (ROOTOPOWER Contract # 289365), the EU COST office (action FA1204 on vegetable grafting), the Fundación
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Séneca de la Región de Murcia, Spain (project 08712/PI/08), and the Spanish MINECO-FEDER (AGL2011-27996/AGR) for support of source-sink relations and
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hormonal signalling research.
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ACCEPTED MANUSCRIPT Legend to figures
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Figure 1. Metabolic (right) and hormonal (left) factors regulating source-sink relations that could influence plant growth, adaptation and yield under drought and salinity. See main text and Tables for explanation. Arrow or bar heads indicate positive and negative regulation, respectively. Abbreviations: ACCd, ACC deaminase; AGPs, ADP-glucose pyrophosphorylase; AREB/DREB, abscisic acid/dehydration responsive element binding transcription factors; CBF2, C-repeat/dehydration-responsive element binding factor; CCD, carotenoid cleavage deoxygenase; CIN1, cell wall invertase (cvInv); CKX, cytokinin oxidase; CSA, MYB domain protein; CYP, sterol C-22 hydroxylase; DDF, Dwarf and delayed flowering gene; ERF, ethylene response factor; G1-1/2, genes involved in sink activation; GAMT, gibberellin methyl transferase; GAox, gibberellin oxidase; GIF1, Grain Incomplete Filling 1, cell wall invertase; GPT, glucose-6phosphate/translocator; HRD, AP2/ethylene response factor-like transcription factor; IAA, indole 3-acetic acid responsive genes; IPT, isopentenyltransferase; IVR1, anther cell wall invertase; JMT, jasmonic acid carboxyl methyltransferase; LOS5,molybdenum-cofactor sulfurase; MADS, regulator of starch synthesis; MAX, more axillary growth; MYB, transcription factor; NCED, 9-cis-epoxycarotenoid dioxygenase; NTT1, adenilate translocator; ORF, rolB-like part gene PEPC; phosphoenolpyruvate carboxylase; PIN, indole 3-acetic acid transporter; SPS, sucrose phosphate synthase; SSIV, starch synthase; SUC2, phloem-localized sucrose/H+ symporter; SUS, sucrose synthase; SUT, sucrose transporter; TDY1, phloem-expressed transmembrane protein; TZF, CCCH-type zinc finger transcription factor.
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Figure 2. Interactions between hormones and carbon metabolism in the regulation of source and sink activities, phloem assimilate (un)loading and transport, and their relative influence on plant survival (only source activity is required) and on growth/yield (sink activity and transport from the source are also required) related processes under drought and salinity conditions. See main text and Tables for explanation, and legend to Figure 1 for abbreviation list. Arrow or bar heads indicate positive and negative regulation, respectively.
39
ACCEPTED MANUSCRIPT Table 1 List of genes related to source strength, and their effect on growth, plant survival and/or yield Effects on growth/survival/yield
CBF2
Overexpression of CBF2 in Arabidopsis (Luo et al., 2013; Sharabi-Schwager et al., 2010a)
Delay of senescence by interaction with ABA-, JA-, SA- and ethylene-related genes
CBF1, CBF2, CBF3
Overexpression of CBF1, CBF2, and CBF3 in Arabidopsis (Gilmour et al., 2004; Liu et al., 1998)
Growth retardation and dwarf phenotype by accumulation DELLAs, incompatible with yield promotion
ERF6
ERF6 overexpression in Arabidopsis (Skirycz et al., 2011)
Hypersensitive to osmotic stress, and inhibition of cell proliferation and growth via GA degradation by GA2-oxidase6
ERF5, ERF6
erf5erf6 loss-of-function double mutants (Dubois et al., 2013)
Arabidopsis
Less affected by salinity, but no clear effect on growth
SlERF5
SlERF5 overexpression in tomato under drought and salinity stress (Pan et al., 2012)
Increased tolerance to drought and salinity, increasing plant survival but without additional effect on growth
OsTZF1
Overexpression of OsTZF1in rice (Jan et al., 2013)
Delay of salt-, JA-, and ABA-induced senescence, and improved tolerance (plant survival) to high salinity and drought
SlAREB1
Overexpression in tomato of the AREB1 transcription factor implicated in the expression of ABA-related genes (Orellana et al., 2010b)
Increased plant survival under drought and salinity stress through senescence-delaying and water-conservative mechanisms
SAG12::IPT
Overexpression of the IPT gene under the control of senescence promoter SAG12 in tobacco plants (Cowan et al., 2005)
Increased CK content in SAG12::IPT plants impacts source-sink relations
PSARK::IPT
Overexpression of the IPT gene in rice (Peleg et al., 2011)
SAG39::IPT
Identification and application of a rice senescenceassociated promoter (Liu et al., 2010)
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Gene Description of the study Hormone-related genes
Overexpression of genes encoding sterol C-22 hydroxylases that control brassinosteroid levels in rice (Wu et al., 2008)
Source-sink modifications and strong sink capacity of PSARK::IPT plants during water stress SAG39::IPT plants show increased yield and rapid sugar reutilization at the onset of leaf senescence Brassinosteroids stimulates the flow of assimilates from the source to the sink, increasing grain yield
Carbon metabolism-related genes
ADP-Glucose pyrophosphorylase mutation (agpsm1) in maize (Schlosser et al., 2012)
Leaf maize starch mutation diminished field growth and productivity
C-metabolism and other genes
Transcriptome profiling in Vitis vinifera berries (Pastore et al., 2011)
Increasing source-sink ratio induces extensive transcriptome reprograming
AGPase, SUS, SUT1, SUT2, SPS
Sucrose metabolism genes under sink limitation in Citrus (Nebauer et al., 2011)
Inhibition of sucrose synthesis and transport genes, and increase in AGPase gene
SSIV
Overexpression of starch synthase SSIV in Arabidopsis and potato (Gámez-Arjona et al., 2011)
SSIV overexpression lead to accumulation of starch in source and sink organs, increasing growth rate and yield
OsSUT2
Study of ossut2 rice mutants (Eom et al., 2011)
Essential role of SUT2 in sugar export from source leaves to sink organs
AtSUC2
Characterization of a phloem-localized sucrose/H+ symporter AtSUC2 in Arabidopsis (Srivastava et al., 2009)
AtSUC2 overexpression in mutants restores growth and carbon partitioning
SUT1
Function of sucrose transporter1 gene in maize using sut1 mutants (Slewinski et al., 2009)
Sut1 mutants have altered biomass partitioning and reduced phloem loading of sucrose
AtSUC2
Functional
sucrose/H+
Overexpression of AtSUC2 supports a role in retrieval but not efflux along the phloem
AC
AGPS
characterization
of
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Array 128 genes related to photosynthesis and Cmetabolism
Changes in expression of photosynthesis and Cmetabolism related genes (McCormick et al., 2008)
Sink demand limits source activity through a kinase-mediated sugar signaling mechanism
SPS1, RSUS1
Changes in sucrose phosphate synthase (SPS1) and sucrose synthase (RSUS1) genes under elevated CO2 in rice (Li et al., 2008)
Important role of SPS in regulating leaf sink to source transition under elevated CO2 conditions
SPS
Overexpression of SPS in potato under field conditions (Ishimaru et al., 2008)
SPS overexpression improves supply of photosynthate from source (leaves) to sink (tubers), increasing yield
TPS1
Constitutive overexpression of TPS1 in tomato (Cortina and Culiáñez-Macià, 2005)
Improved plant survival under salinity, drought, and oxidative stress, but abnormal growth (pleiotropic changes)
ostA, ostB
Constitutive overexpression of a synthetic fusion ostA and ostB in potato (Jang et al., 2003) and alfalfa (Suárez et al., 2009)
Stunted plant growth.
ostA, ostB
Light regulated or ABA-inducible expression of ostA and ostB fusion (Garg et al., 2002)
Increased tolerance to drought and salinity in terms of vegetative growth, with higher photosynthetic capacity
PEPC
Overexpression of PEPC in rice (Ding et al., 2013)
Effective when photosynthesis is limited. Higher photosynthesis and final grain yield under drought stress
CIN1
CIN1 overexpression under the control of senescence-associated promoter SAG12 (Lara et al., 2004)
Increased source senescence.
CIN1
CIN1 overexpression under the control of a putative fruit specific promoter (Albacete et. al, unpublished)
Increased WUE and tolerance to drought due to increased photosynthetic capacity and lower water use
Ascorbate oxidase RNAi in tomato (Garchery et al., 2013)
Diminution of ascorbate oxidase affects Callocation and increases tomato yield under drought stress
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Other genes
TDY1
T
symporter in Arabidopsis (Srivastava et al., 2008)
strength
and
delayed
Identification and characterization of a collection of phloem-expressed transmembrane (tdy1) maize mutants (Ma et al., 2009)
TDY1 functions in carbon partitioning by promoting phloem loading
rolB-like part of ORF8 gene
Overexpression of ORF8 in tobacco (Umber et al., 2002)
Transgenic plants reduces sucrose export from source leaves to sink organs
SAGs, SEN1
Expression of different senescence associated genes in Arabidopsis (Schenk et al., 2005)
Regulatory expression of SAGs by senescence and stress with reduction of photosynthetic activity
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Effects on growth/survival/yield
DDF1
ga2ox7-2 Arabidopsis mutant in DDF1overexpressing background (Magome et al., 2008)
ga2ox7-2 mutant suppress dwarf genotype of DDF1-overexpressing plants under salt stress, indicating key role in salt-mediated growth impairment
DREB1A
Overexpression of AtDREB1A in tobacco (Cong et al., 2008) and SlDREB in tomato (Li et al., 2012)
GA-deficient dwarf genotypes, with increased root sink growth and tolerance to drought and salinity
AtGAMT1A
Overexpression of AtGAMT1A implicated in the degradation of GAs in tomato (Nir et al., 2013)
Semi-dwarfed genotype and increased tolerance to drought stress
CKX
CKX overexpression in tobacco (Werner et al., 2008)
Decreased shoot sink activity due to reduced cell number in meristems and sucrolytic activities
35S::IPT
35S::IPT tomato rootstocks (Ghanem et al., 2011a)
Grafting WT plants onto 35S::IPT rootstocks increased fruit yield under salinity
SAG12::IPT
Overexpression of SAG12::IPT (Swartzberg et al., 2006)
tomato
Higher fruit weight due to increased sink assimilate import and delayed leaf senescence
IPT
IPT overexpression in axillary buds of tobacco (Guivarc'h et al., 2002)
Cytokinins play a role in tuberization and sink formation
AUX/IAA, AXR1, RBX1like E3
Transcriptomic study on hybrid embryos of Vicia faba (Meitzel et al., 2011)
Regulatory model on seed carbon allocation and growth of auxin-related genes
LOS5
Overexpression of the LOS5 gene encoding a key enzyme of ABA biosynthesis (Xiao et al., 2009)
Increased spikelet fertility and subsequent improved yield under water stress
LeNCED1
LeNCED1 overexpression in tomato (Thompson et al., 2007)
Growth maintenance under drought stress, but growth penalty under control conditions
Ubi1::AtJMT
AtJMT overexpression in rice, encoding JA carboxyl methyltransferase (Kim et al., 2009)
Lower spikelet number and yield under drought stress
35S::AtJMT
Constitutive overexpression Arabidopsis (Cipollini, 2010)
in
Reduced yield due to a reduction in seed weight
Overexpression of genes encoding sterol C-22 hydroxylases that control brassinosteroid levels in rice (Wu et al., 2008)
Brassinosteroids stimulates the flow of assimilates from the source to the sink, increasing grain yield
SC R
IP
T
Gene Description of the study Hormone-related genes
NU
Table 2 List of genes related to sink strength, and their effect on growth, plant survival and/or yield.
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MA
in
ZmCYP, AtCYP, OsCYP
of
AtJMT
Carbon metabolism-related genes
Regulatory expression of the anther cell wall invertase gene IVR1 in wheat (Koonjul et al., 2005)
Higher expression of IVR1 in anthers, increased sink strength and cell wall invertase in tolerant wheat cultivars to drought stress
OsINV4, OsMST8
Expression of cell wall invertase and sugar transporter genes in rice (Oliver et al., 2005)
Increased expression in sink organs, existing a correlation between tissue sink activity and tolerance to drought and cold stress
GIF1
Cell wall invertase gene GIF1 overexpression driven by its native promoter in rice (Wang et al., 2008)
Increased sink strength during grain filling and final yield
CIN1
Overexpression of CIN1 gene under the control of a putative fruit-specific promoter in tomato (Albacete et al., unpublished)
Specific increase in fruit sink activity, reduced flower abortion, and higher fruit yield under salinity
GPT, NTT1
Simultaneous overexpression of glucose-6phosphate/translocator (GPT) and adenilate translocator (NTT1) genes in potato (Zhang et al., 2008)
Overriding co-limiting import of carbon and energy into tubers increases starch content and yield
ISI1
Study of Arabidopsis mutants of the impaired sucrose induction1 gene (Rook et al., 2006)
Isi1 mutants do not utilize available sugars efficiently due to a lack of control of sugarresponsive gene expression
Wheat isogenic lines containing the Nax2 locus encoding the HKT1 Na+ transporter (Munns et al., 2012)
Reduced Na+ in flag leaf and flower abortion, and increased photosynthetic activity during grain filling and yield.
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IVR1
Other genes HKT1
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ACCEPTED MANUSCRIPT QTL for salt tolerance SKC1 in rice, encoding a Na+ transporter (Ren et al., 2005)
Higher salt tolerance but no practical results in terms of improved yield
MdSIMYB1
Overexpression of the MdSIMYB1 transcription factor in tobacco and apple (Wang et al., 2013)
Increased resistance to salt, drought and cold stress, and biomass partitioning to the root
HRD
Overexpression of the HRD transcription factor in Arabidopsis and rice (Karaba et al., 2007)
Increased root sink strength and biomass under drought stress, but no yield data.
Gn1a
QTL of grain number in rice that encodes OsCKX2 (Ashikari et al., 2005)
Enhanced grain number per panicle and per plant, increasing final yield
DEP1
Gain-of-function mutation of allele DEP1 (Huang et al., 2009)
Dense panicle, increased number of grains and consequent increase in grain yield
OsSPL14
Point mutation in OsSPL14 in rice (Jiao et al., 2010)
Ideal rice with reduced tiller number, more and bigger grains per panicle, and increased yield
TDY1
Identification and characterization of a collection of phloem-expressed transmembrane (tdy1) maize mutants (Ma et al., 2009)
TDY1 functions in carbon partitioning by promoting phloem loading
rolB-like part of ORF8 gene
Overexpression of ORF8 in tobacco (Umber et al., 2002)
Transgenic plants reduces sucrose export from source leaves to sink organs
SAGs, SEN1
Expression of different senescence associated genes in Arabidopsis (Schenk et al., 2005)
Regulatory expression of SAGs by senescence and stress with reduction of photosynthetic activity
R2R3 MYB transcription factor, MST8
Characterization of carbon starved anther (csa) rice mutant encoding a MYB Domain Protein (Zhang et al., 2010)
CSA is a key transcriptional regulator for sugar partitioning in rice during male reproductive development
G1-1, LeG1-1, LeG1-2
Changes in G1-1 expression in potato tubers and LeG1-1 and LeG1-2 expression in tomato seeds (Agrimonti et al., 2007)
G1-1 and its homologs in tomato are involved in maintenance of sink function of potato tubers and tomato seeds
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SKC1
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