New challenges in plant aquaporin biotechnology.

ARTICLE IN PRESS

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Plant Science xxx (2013) xxx–xxx

Contents lists available at ScienceDirect

Plant Science journal homepage: www.elsevier.com/locate/plantsci

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Review

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New challenges in plant aquaporin biotechnology

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Maria del Carmen Martinez-Ballesta ∗ , Micaela Carvajal Department of Plant Nutrition, Centro de Edafología y Biología Aplicada del Segura – CSIC, Campus de Espinardo, 30100 Murcia, Spain

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a b s t r a c t

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Article history: Received 24 July 2013 Received in revised form 4 December 2013 Accepted 5 December 2013 Available online xxx

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Keywords: Aquaporin Carbon nanotubes Stress resistance Water use efficiency

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Contents

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Recent advances concerning genetic manipulation provide new perspectives regarding the improvement of the physiological responses in herbaceous and woody plants to abiotic stresses. The beneficial or negative effects of these manipulations on plant physiology are discussed, underlining the role of aquaporin isoforms as representative markers of water uptake and whole plant water status. Increasing water use efficiency and the promotion of plant water retention seem to be critical goals in the improvement of plant tolerance to abiotic stress. However, newly uncovered mechanisms, such as aquaporin functions and regulation, may be essential for the beneficial effects seen in plants overexpressing aquaporin genes. Under distinct stress conditions, differences in the phenotype of transgenic plants where aquaporins were manipulated need to be analyzed. In the development of nano-technologies for agricultural practices, multiple-walled carbon nanotubes promoted plant germination and cell growth. Their effects on aquaporins need further investigation. © 2013 Published by Elsevier Ireland Ltd.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Plant responses to aquaporin suppression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Plant responses to aquaporin overexpression. Cases of successful improvement of plant tolerance to abiotic stress and causes of failure . . . . . . 3.1. PIP manipulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. TIP manipulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Innovative approaches affecting plant aquaporins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conflict of interest . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction The importance of water movement in living organisms has led to studies of water transport through biological membranes, a matter of ongoing investigations. Aquaporins, proteins belonging to MIP (Membrane Intrinsic Proteins) family facilitate the bidirectional transport of water through biological membranes. However, other molecules such as glycerol, ammonia, boric acid, silicic acid, CO2 , or even arsenic acid can also be transported [1–3]. They perform fundamental functions in plants, especially related to the adaptation under variable environments [4–6]. Aquaporins are classified into five families depending on membrane localization and amino acid sequence: PIPs, plasma

∗ Corresponding author. Tel.: +34 968 39 62 29; fax: +34 968 39 63 13. E-mail addresses: [email protected] (M.d.C. Martinez-Ballesta), [email protected] (M. Carvajal).

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membrane-; TIPs, tonoplast-; NIPs, nodulin-26-like-; SIPs, small and basic-; and XIPs, uncharacterized-intrinsic proteins. Reverse genetics and overexpression are effective tools in plants, for investigating the physiological functions of each aquaporin isoform and for understanding their roles in water transport and abiotic stress responses [7]. Under abiotic stress, such as water deficit or salinity, stomata closure is considered to be the first mechanism that the plant employs to preserve water. However, the level of productivity is related to the higher rate of transpiration and leaf growth [8]. Thus, an optimal balance between water status, nutrient uptake, photosynthesis, and transpiration rate is needed in plants. Enhancing water absorption by the roots is one of the main mechanisms by which plants can maintain their water content under stress conditions [9] and aquaporins are related to the regulation of the hydraulic conductivities that finally affect plant water uptake ability. For this reason, transgenic plants overexpressing aquaporins in their roots were obvious candidates as breeding

0168-9452/$ – see front matter © 2013 Published by Elsevier Ireland Ltd. http://dx.doi.org/10.1016/j.plantsci.2013.12.006

Please cite this article in press as: M.d.C. Martinez-Ballesta, M. Carvajal, New challenges in plant aquaporin biotechnology, Plant Sci. (2013), http://dx.doi.org/10.1016/j.plantsci.2013.12.006

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ARTICLE IN PRESS M.d.C. Martinez-Ballesta, M. Carvajal / Plant Science xxx (2013) xxx–xxx

targets to improve abiotic stress tolerance. However, these transformed plants failed to cope with mild and severe stresses [10–12] which focused research efforts on the ability of aquaporins – to increase water use efficiency (WUE). It has been suggested that increases in both carbon gain and WUE are the most efficient strategy that allows plants to cope with abiotic stress [13,14]. Thus, any factor involved in both processes could be a main target for manipulation. For this reason, aquaporins, having the dual function of water and CO2 transport, as well as alternative isoforms to PIP, such as TIPs that allow a tight cell osmoregulation under stress, are now objectives for breeding. The search for new nanomaterials has recently brought surprising results in the area of plant biotechnology. The new results at the nanoscale may provide solutions to problems concerning plant physiology focusing on aquaporins [15]. The aim of the present review is, therefore, to explore the recent advances in the role of specific aquaporin isoforms, through the study of aquaporin suppression in transformed plants. The review also underlines the successful cases for plant tolerance to stress achieved through aquaporin overexpression, as well as an analysis of the causes of failure, in particular cases. The future perspectives for the use of new materials are also discussed.

2. Plant responses to aquaporin suppression The cellular and tissue hydraulic function of each aquaporin isoform has been studied using genetic manipulation during the last decade. The development of knockout plants for aquaporin genes PIP or TIP has been important for allowing measurements of water permeabilities and ion transport compared to those of wild type. These mutations have exerted a wide range of different effects on transformants sometimes difficult to explain due to the complexity of the maintenance of plant cell homeostasis. Physcomitrella patens pip2;1 and pip2;2 knockouts decreased internal protoplast osmotic water potential under moderated water stress, whereas wild type plants had no differences in water potential between the inside of the cell and the external medium [16]. They concluded that, under moderate water stress, these two aquaporins may act as constitutive isoforms for water transport, delaying water loss by a facilitated water flux through the cell surface that is in contact with the liquid water phase. Arabidopsis has been used for the majority of the aquaporins knockout studies. Thus, Arabidopsis T-DNA mutants, lacking both AtTIP1;1 and AtTIP1;2 were without phenotypic differences compared to the wild type [17,18]. The authors proposed that these isoforms are not essential aquaporins under optimal growth conditions – since other members of the TIP family may facilitate the water flow through the tonoplast – but AtTIP1;1 and AtTIP1;2 appear to be indispensable under environmental stress [18]. These results question the previous use of interference RNAs (RNAi) as tools to characterize the function of specific aquaporin genes, since an artifact effect, due to the silencing of non-target transcripts, was associated with senescence and plant death after AtTIP1;1 suppression [19]. In this regard, the use of antisense constructions has been applied as an effective approach to characterize the role of aquaporins as water channels [20]. Plants with knockouts of AtPIP2;2, another abundant plasma membrane aquaporin, were compared with wild type plants [21,22]. There was similar osmotic water transport in roots, but lower hydrostatic root water transport in the mutants, which could have significant repercussions for the whole plant hydraulics. However, results with Atpip1;2 suppression revealed that this aquaporin affected root hydrostatic hydraulic conductivity but osmotic water transport was not altered. The role of this isoform in mesophyll water transport was also demonstrated contributing

approximately 20% of hydraulic conductivity of leaf rosettes [23]. Similarly, AtPIP2;1 contributed to water flux into the Arabidopsis rosette and both AtPIP2;1 and AtPIP2;2 isoforms may participate in water relocation from the roots to the leaves [22]. Other aquaporin functions were proposed in Arabidopsis leaf tissues, based on the study of knockout plants. Thus, the expression of three PIP aquaporins (AtPIP2;1, AtPIP1;2 and AtPIP2;6) in leaf veins contributed to growth, due to the effect on total rosette water transport [24]. Conversely, antisense NtAQP1 well watered tobacco plants did not have a characteristic phenotype, but under stress this isoform seems to be involved in the reduction of root hydraulic resistance, resulting in enhanced cell water permeability [25]. Aquaporins have important functions as CO2 transporters [13,26–28]. However, few reports have demonstrated whether specific aquaporin suppression limits CO2 transport. Such studies may be confounded by collateral effects of the mutation on plant anatomy and by the pleiotropic effects on closely related genes. Internal CO2 conductance and leaf photosynthesis were strongly decreased in T-DNA insertion lines of the Atpip1;2 knockout [29]. However, when the authors produced the mutation in the AtPIP2;3 isoform, which is not a CO2 transporter, no effect on the CO2 related processes was found. These results show that the AtPIP1;2 aquaporin is highly involved in the transport of CO2 within the cell and that this is a rate limiting process. Also, EgPIP1 and EcPIP2 expression in Eucalyptus trees, was co-suppressed by a Raphanus sativus aquaporin (RsPIP1;1). A low rate of CO2 assimilation was observed which decreased the rate of photosynthesis in these knockdown mutants [30]. Nodulin-26-like intrinsic proteins (NIPs), a subfamily of the aquaporins proteins, facilitate the transport of silicic acid Si(OH)4 (AtNIP2;1) [31], boric acid (AtNIP5;1) [32], and arsenic acid (AtNIP7;1) [33]. Their transport functions have been assessed by direct mutagenesis and by expression in oocytes. Thus, lower B uptake was observed in two T-DNA insertion mutant lines of AtNIP5;1 with respect to the non-transformed plants. The substitution of a residue in the aromatic/arginine region in the fifth helix position of two NIP aquaporins; the silicic acid (Si) transporter OsLsi1 (OsNIP2;1) from rice and the boric acid (B) transporter AtNIP5;1 from Arabidopsis; completely inhibited the transport of B and Si but not that of As in Xenopus oocytes. Osmotic water permeability in individual aquaporin knockout cells decreased in comparison with the wild type [34], as well as in protoplasts isolated from knockout plants [20,24,25,16,35]. This indicates that aquaporins are involved in adaptation in response to stresses, and maintaining the homeostasis – necessary for growth. Finally, transcription factors were identified that modulate the expression of aquaporin genes, and other genes. These transcriptions factors may be useful in the study of the molecular basis of plant water homeostasis in response to stress, as well as the signaling network involved in this stress response. The use of Q2 knockouts of these transcriptions factors, that regulate different genes involved in the stress response offers, in this sense, a biotechnological approach to the improvement of crop tolerance. Microarray transcriptional profiling of the double knockout lines and overexpression of the transcription factors RAP2.4B and RAP2.4, belonging to the abiotic stress associated DREB A-6 clade in Arabidopsis, were used to ascertain the mechanisms of aquaporin regulation in response to abiotic stress [36]. Double knockout line of rap2.4b and rap2.4 when growing under drought stress, had downregulated expression of eight aquaporin genes. Overexpression of the rap2.4 gene resulted in the upregulation of six aquaporin genes. This indicated that aquaporins were positively regulated by RAP2.4B and RAP2.4 as part of the very early response to dehydration. There is a discrepancy between these findings and other work that found the opposite result (downregulation) for the same genes,


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namely AtPIP2;1, AtPIP2;2, AtPIP2;3, AtTIP1;1, and AtTIP2;2 [37], pointed that the age of the plants, growing media and different duration of drought stress are important issues that contributed to the different results.

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3. Plant responses to aquaporin overexpression. Cases of successful improvement of plant tolerance to abiotic stress and causes of failure

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3.1. PIP manipulation

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Aquaporins have been used as target proteins in genetic manipulation in attempts to improve plant water relations under environmental stress, with numerous examples in the literature (Tables 1 and 2). Thus, Vicia faba PIP1 (VfPIP1) expression in transgenic Arabidopsis thaliana improved drought resistance by a decrease of water loss through transpiration, due to an inducement of stomatal closure [38]. This control of the stomatal behavior was also described when SlTIP2;1 was overexpressed in tomato plants, conferring resistance to salt and water stress [39]. Most VfPIP1 expression level was found in roots [38]. Therefore, a hydraulic signal between root and shoot could exist. Deeper knowledge of root water uptake and transport is necessary. In the same way, overexpression of RWC3 in transgenic lowland rice (Oryza sativa L.) plants [40] improved resistance to osmotic stress imposed by polyethylene glycol (PEG) through higher root osmotic hydraulic conductivity and lower leaf water potential. Similarly in rice, increased root hydraulic conductivity conferred salt stress resistance after the moderate overexpression of OsPIP1;1 [41]. Nevertheless, plant aquaporin overexpression did not always improve abiotic stress tolerance [10–12,42]. Arabidopsis and tobacco plants overexpressing Arabidopsis PIP1;4 or PIP2;5 had increased water flow in individual root cortical cells under cold stress, along with an increased percentage of germination, relative to the non-transformed plants. These transformants had increased water loss, under drought conditions, together with reductions in growth, with lower germination rates [43]. Similarly, the overexpression of an Arabidopsis aquaporin gene (AtPIP1b) in Nicotiana tabacum under favorable conditions resulted in increased growth rates, gas exchange and stomatal density in plants, but under drought stress it provoked faster wilting [10]. Different responses to stress were observed after overexpression of AtPIP1b, RWC3 and NtAQP1 isoforms in tobacco, rice or Arabidopsis plants in spited of a high homology between their amino acidic sequences [38]. These distinct responses indicate a complex aquaporin regulation mechanism, where the increased or decreased expression of a PIP gene, in a spatial and temporal manner, is required to cope with stress. Anatomical differences may results from aquaporin gene overexpression. The stomatal density was increased in transgenic AtPIP1b-tobacco plants, enhancing transpiration, whereas it remained unmodified in transgenic NtAQP1-Arabidopsis plants [44], relative to the wild type. Greater water loss may occur under stress condition, at higher stomatal density. By contrast, reduced stomatal density contributed to improved WUE under salt stress in basil (Ocimum basilicum L.) [45] and strawberry (Fragaria × ananassa Duch.) preventing an excessive water loss [46]. Therefore, morphological changes resulted critical for plant adaptation to salinity and a correlation between plant tolerance and reduced transpirational flux – due to low stomatal density – was established [46]. A reduction in the root/shoot fresh weight ratio was also observed in transgenic AtPIP1b – tobacco plants, with respect to non-transformed control plants, whereas plants overexpressing VfPIP1 had longer lateral and primary roots than controls [38],

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which affected root hydraulic conductivity. Therefore, root growth is directly related to the whole plant water transport efficiency, highlighting that the phenotypic changes produced by aquaporin overexpression should be considered as a key factor that influences stress tolerance, in addition to the nature, level, and duration of the stress. In addition, differential PIP functionality cannot be discounted in distinct transgenic plants as being due to the interaction with other membrane aquaporins, since co-expression and formation of heterotetramers between PIP1 and PIP2 has been attributed to isoforms functionality [47]. Also, the overexpression of a foreign aquaporin may improve stress tolerance through cooperation with endogenous aquaporins. Thus, the overexpression of durum wheat TdPIP1;1 in tobacco plants enhanced stress tolerance through increased root growth and leaf area of the plants, even though this isoform showed no functionality when expressed in Xenopus oocytes [48]. In a similar manner, overexpression of the Brassica napus BnPIP1 gene in transgenic tobacco increased water stress tolerance at the whole-plant level, by increasing the water conductance at the cellular level [49]. This positive effect may be related to the formation of heterotetramers, but it is not clear whether physical contact between ectopic and endogenous proteins occurred. Also, the expression in one plant species of an aquaporin gene from different species may differentially affect the expression patterns and distribution of endogenous aquaporin genes, depending on the target PIP isoform as well as the nature of the foreign protein, conditioning the response to stress [43]. Thus, the expression of CfPIP2;1, a figleaf gourd (Cucurbita ficifolia) aquaporin gene, increased the survival rate of Arabidopsis plants under drought [50]. The expression of cucumber (Cucumis sativus) aquaporin genes CsPIP1;1, or CfPIP2;1 also enhanced the seed germination under high salinity. The authors concluded that, the PIP2 subfamily was affected more by ectopic expression under environmental stress than the PIP1 members. The endogenous expression patterns of CfPIP2;1 and CsPIP1;1 under drought stress could be reflected in their influence as ectopic aquaporins. The nature of stress also influenced the expression patterns of each individual isoform [50]. However, there was no clear evidence whether the foreign aquaporin specifically modified the endogenous Arabidopsis PIP response to stress or whether it first altered the endogenous PIP expression and that this modification was responsible for the response to stress. The overexpression of the exogenous RsPIP2;1 in Eucalyptus trees induced higher CO2 assimilation under normal conditions, increasing the water use efficiency when compared with nontransformed lines. However, RsPIP2;1 had no water channel activity when expressed in yeast [51]; thus, it may have acted as a functional water channel when it was expressed with the endogenous Eucalyptus PIP2 [30]. It has been suggested that dual activity of aquaporin isoform NtAQP1 attenuates the reduction of root hydraulic conductivity and maintains leaf CO2 assimilation in tomato plants overexpressing NtAQP1 under salt (100 mM NaCl)-stress. Thus, an increased WUE contributed to the enhancement of plant biomass and yield under salinity in overexpressing plants, compared to the nontransformed controls [52]. In fact, a role for NtAQP1 has been proposed in leaf CO2 transport [26]. A new aquaporin McMIPB has recently been identified in Mesembryanthemum crystallinum. It enhanced CO2 diffusion, mesophyll and stomatal conductance, leaf photosynthesis, and plant growth under well-watered conditions [53]. The protein was classified as a PIP1 that may possibly interact with PIP2 aquaporins as hetero-tetramers. Thus, the coexpression of PIP2 and PIP1 may increase the activity of PIP1 not only for water flow but also for CO2 diffusion [54]. However, under prolonged soil water deficit, the overexpression of McMIPB resulted in a decreased rate of photosynthesis. The induction of


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Table 1 Examples in the literature of success and failure regarding the improvement of plant tolerance to abiotic stress, for the model plant Arabidopsis thaliana.

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Aquaporin gene isoform

Aquaporin overexpressing plant

Promoter

Stress condition

Response

Reference

AtPIP1;b VfPIP1 AtPIP1;4, AtPIP2;5

Nicotiana tabacum Arabidopsis thaliana Arabidopsis thaliana, Nicotiana tabacum

35S 35S 35S

More sensitive Resistance No effect No effect No effect

Aharon et al. [10] Cui et al. [39] Jang et al. [43]

PgTIP1

Arabidopsis thaliana

35S

Resistance More sensitive Resistance More sensitive

Peng et al. [59]

At TIP5;1

Arabidopsis thaliana

35S

Salinity (90 mM NaCl, 40 d) Drought (soil drying by withholding water) Water stress (100–400 mM mannitol 12–24 h, withholding water 15 d) Salinity (50 mM NaCl 14 d) Cold (10 ◦ C, 24 h) Salinity (100 mM, 1 week) Drought (withholding irrigation for 10 d, 10 cm deep pots) Drought (withholding irrigation for 4 weeks, 45 cm deep pots) Cold (−6, −7, −8 and −9 ◦ C) Boron toxicity (3 mmol/L boric acid, 18 d)

Resistance

Pang et al. [60]

McMIPB expression ameliorated the decreases of stomatal conductance and photosynthesis rate under water deficit compared with those observed in non-transformed plants [53]. Similarly, the overexpression in rice of the aquaporin gene, HvPIP2;1,- from barley resulted in a greater sensitivity to salinity [52], even though the overexpression of the same barley gene enhanced internal CO2 conductance and CO2 assimilation [55]. However, in both McMIPB and HvPIP2;1 overexpressing plants, a reduced shoot/root biomass ratio was observed under stress. An inability of root water uptake to satisfy the water demand of the aerial parts cannot be discounted. It has been suggested that the overexpression of aquaporin isoforms that are induced by stress may lead to plant stress resistance [52]. By contrast, the overexpression of aquaporins that are down-regulated by stress resulted in greater damage than in non-transformed plants under stressful conditions. The loss of down-regulation in the leaf tissues may increase cell water loss through the stomata. They demonstrated that the control of stomatal conductance and the net photosynthetic rate by NtAQP1 was independent of the root signals [52]. Therefore, elucidation of how root-to-shoot signals may influence the effect of aquaporin overexpression in the roots on the behavior of stomata could also provide an indication about the transgenic plant response to stress. The treatment of grape (Vitis vinifera) overexpressing the VvPIP2;4N aquaporin with ABA – to close stomata – prevented the negative effect of the root overexpression of VvPIP2;4N on plant water status under water stress [11]. They proposed that alternative mechanisms to the hydraulic conductance of cell membranes may operate that control water flow through the plant under water stress. In fact, it was recently observed that the overexpression of wheat TaAQP7 gene, in tobacco increased the activity of detoxification enzymes such as SOD and CAT, improving the antioxidant defense system

and reducing the H2 O2 levels under drought/osmotic stress [56]. TaAQP7 encodes a PIP2 subgroup of aquaporins in wheat and additional mechanisms, other than the well-known improvement of WUE and plant water retention, may contribute to the abiotic stress tolerance of transgenic plants overexpressing aquaporins. It has also been suggested that there are anatomical modifications at the leaf [55] and root level [10] under abiotic stress conditions, when aquaporin levels exceed an expression threshold, affecting the whole plant water balance. In accordance with this, low or moderate overexpression of OsPIP1;1 in rice enhances plant resistance to salt stress. OsPIP1;1 expression naturally increased in leaves but was reduced in roots under salinity. However, high overexpression of OsPIP1;1 produced sterile rice seeds – through a reduction of reproductive growth – and OsPIP1;1 function as a component of water conductance was rather limited [41]. A disruption of the plasma membrane structure by OsPIP1;1 with subsequent alteration of the endogenous PIP, has been proposed [41]. Thus, the ability of each overexpressed aquaporin isoform to confer (or not) resistance to a specific stress may depend on its contribution to the plant control of water loss by transpiration or its ability to maintain CO2 assimilation or increase water uptake (Fig. 1). The mode of action of a particular isoform on other endogenous aquaporins may produce an overall response to the stress.

3.2. TIP manipulation

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It has been predicted that the selection of the suitable aquaporin isoforms for genetic manipulation is a decisive factor for success in improving abiotic stress tolerance [39]. The TIPs (Tonoplast Intrinsic Proteins) are considered as important elements of

Table 2 Examples in the literature of success and failure regarding the improvement of plant tolerance to abiotic stress, for different crops. Aquaporin gene isoform

Aquaporin overexpressing plant

Promoter

Stress condition

Response

Reference

VvPIP2;4N

Vitis vinifera

35S

More sensitive

Perrone et al. [11]

StPIP1

Nicotiana tabacum

More sensitive

Wu et al. [60]

RWC3 OsPIP1;1 SlTIP2;2 NtAQP1

Oryza sativa Oryza sativa Solanum lycopersicum Solanum lycopersicum

35S rd29A SWPA2 35S EVO205 35S

Water stress (soil drying by withholding water, 14 d) Water stress (25% PEG 6000)

HvPIP2;1 TdPIP1;1

Oryza sativa Nicotiana tabacum

35S –

Nicotiana tabacum Nicotiana tabacum

35S 35S

Resistance Resistance Resistance Resistance Resistance More sensitive Resistance Resistance Resistance Resistance Resistance

Lian et al. [40] Liu et al. [41] Sade et al. [57] Sade et al. [58] Sade et al. [52] Katsuhara et al. [42] Ayadi et al. [49]

BnPIP1 TaAQP7

Water stress (PEG treatment, 10 h) Salinity (100 mM NaCl, 14 d) Salinity (180–200 mM NaCl) Drought (30–35% soil volumetric water content) Salinity (100 mM, 3 d) Salinity (100 mM NaCl, 2 weeks) Salinity (250 mM NaCl, 30 d) Water stress (300 mM mannitol, 30 d) Drought (stop irrigation, 20% PEG8000) Drought (water deprivation, 20 d) Osmotic stress (150–300 mM mannitol, 7–12 d)

Yu et al. [50] Zhou et al. [56]


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Fig. 1. Effects of aquaporins overexpression conferring plant resistance or sensitivity to abiotic stress. Overexpression of a specific aquaporin isoform may provide resistance by higher water uptake at the root level, lower water loss by transpiration, and/or maintained CO2 assimilation. When overexpression exceeds a threshold in the plant, as in the case of OSPIP1;1, no involvement in plant water hydraulics is observed.

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the mechanism that controls cell water homeostasis through the fast water exchanges between the vacuole and cytoplasm [39,57]. An efficient computational approach in which the expression patterns for different aquaporin gene families were considered together with plant anatomy, developmental stage, and abiotic stress, was used to predict aquaporin isoform candidates for plant abiotic stress improvement [39]. They then demonstrated that the overexpression of SlTIP2;2 in tomato (Solanum lycopersicum L.) attenuated the reduction of plant transpiration under stress, allowing an adequate balance between CO2 uptake and water and nutrient supply. The experiments were carried out in the field under salt (180–200 mM NaCl) and drought (plants irrigated once a week until soil saturation) conditions and the duration to exposure to the stress and the stress intensity were important factors to determining the plant recovery rate. This behavior was attributed to the high tonoplast osmotic water permeability that enhanced the osmoregulation ability of the vacuole. Previously, overexpression of the tonoplast aquaporin, PgTIP1 from Panax ginseng, in Arabidopsis plants increased plant growth, but only under optimal conditions or very low drought stress [58]. The mechanisms by which the overexpression of different PIP and TIP subfamilies had distinct effects on the stress response need further investigation, but the rapid water exchange in the vacuole of plants overexpressing TIPs may facilitate greater Na+ accumulation in the case of salt stress. The selection of aquaporin isoforms involved not only in water transport, but which also influence processes such as ion transport may improve stress tolerance. Thus, the overexpression of tonoplast aquaporin TIP5;1 in Arabidopsis increased tolerance to high levels of borate, through the involvement of this isoform in borate compartmentation in the vacuole [59]. In a similar way, the overexpression of wheat nodulin 26-like intrinsic protein gene, TaNIP, in Arabidopsis plants favored Na+ extrusion from the cytoplasm to the extracellular matrix, whereas it increased K+ and Ca2+ contents in the plant tissues, hence improving salinity tolerance above non-transformed plants [60].

Differences in the experimental design, the nature, intensity and duration of the stress and the developmental stage of the plants, must be considered in plants overexpressing aquaporins response to abiotic stress. For example, Arabidopsis plants overexpressing PgTIP1 were resistant to water stress compared to the non-transformed plants when they were grown in 45 cm deep pots, whereas they were more sensitive when grown in 10 cm deep pots, as consequence of faster drying of the rooting medium [58]. 4. Innovative approaches affecting plant aquaporins It is well documented that alterations of aquaporin function and/or expression affect human health, and natural products from plants or drugs can prevent diseases by acting upon aquaporins (for review see [61]). Similarly, exogenous compounds such as thiourea [62], dopamine [63], glycine-betaine [64], and sinigrine [65] can down or up-regulate aquaporins in Brassica juncea, O. sativa, Zea mays, and Brassica oleracea, plants respectively. The aquaporin expression pattern conferred on all the above species were related to greater stress tolerance. Also, the interest in the biological and biomedical effects of new nanomaterials has recently increased. Nanoscale materials interact with animal tissues (even tumor cells; [66]) and plant cells [67] by penetration through the cell wall and membranes or allowing the delivery of biological molecules into plant cells. Carbon nanotubes have been particularly studied, due to their unique physical, chemical, and electronic properties for biotechnological applications – such as biomolecule delivery and tissue engineering [67–69] – more than other nanoparticles such as nano-TiO2 and nano-Al [70,71]. Single or multiple-walled carbon nanotubes are often toxic to humans [72], while their application to plants has beneficial effects. The plant–nanoparticle interactions are quite complex and require further investigation. However, there are some evidence for an effect on gene transcription, positively affecting plant response growth [69,73,74]. Multiple-walled carbon nanotubes are taken up by tomato plants, increasing their total gene expression and


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particularly aquaporins [75]. The effects of carbon based structures were compared: activated carbon, graphene-with single and multiple-walled carbon nanotubes. There was an increase in growth due to the carbon nanotubes treatments, especially in the multi-wall treated plants [15,69,73,74]. The authors also suggested that the increase in aquaporin expression had an impact on the germination and growth. It was concluded that the ability of multiple-walled carbon nanotubes to penetrate cell walls [69] and their promotion of cell division/extension, involving aquaporins, needs further in depth investigation of the mechanisms and signaling pathways and cascades involved. All these preliminary results open a new research area at the interface between nanomaterials and plant biology, with multiple possibilities in the genetic engineering and improvement of plant stress tolerance that could be beneficial to the development of new biotechnologies. However, the safety profile has to be defined and the environmental and health risk evaluated before considering the extended use of nanotubes in the agri-food sector. 5. Concluding remarks

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The development of knockout and overexpressing aquaporin mutants has been an efficient tool for studying the contribution of individual isoforms to plant water flow. In general, aquaporins may delay water loss by promoting cell water retention under unfavorable conditions. It has been determined using reverse genetic approaches that some TIP and PIP members seems to be redundant in optimal culture conditions but are essential under environmental stress, with specific isoforms making a significant contribution to whole plant hydraulics. However, different factors have to be taken into account for successful plant tolerance of abiotic stress when aquaporins are overexpressed. Thus, distinct phenotypes of aquaporin overexpressing plants may depend on the combination of water transport ability, transpiration rate, and stomatal aperture – which are in turn related with plant anatomy. It seems that the overexpression of aquaporins that are down-regulated under stress fails to confer tolerance. The influence of ectopic expression on endogenous aquaporins and the formation of active heteromers need further investigation, as the interactions between different aquaporin isoforms and their coupled response/functionality under stress conditions still need to be elucidated. The multiplicity of aquaporin isoforms and their involvement in the response to stress depend on the nature, intensity and duration of the stress, necessitating a selection “on demand” of the aquaporin genetic manipulation to solve a specific environmental problem for each particular cultivar. Finally, the use of carbon nanotubes offers a new challenge to be investigated. The application of nanotechnology to crops in order to improve their responses to different environmental stresses represents a new research area.

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Conflict of interest

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No conflict of interest. Acknowledgments

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The authors thank Dr. David J. Walker for correction of the written English in the manuscript. M.C. Martínez-Ballesta thanks the Spanish Ministerio de Ciencia e Innovación for funding through the “Ramón y Cajal” program [Ref. RYC-2009-04574].

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