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Ubiquitination pathway as a target to develop abiotic stress tolerance in rice a
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Andressa Dametto , Giseli Buffon , Édina Aparecida Dos Reis Blasi & Raul Antonio Sperotto
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Programa de Pós-Graduação em Biotecnologia (PPGBiotec) and Centro Universitário UNIVATES, Lajeado, RS, Brazil b
Centro de Ciências Biológicas e da Saúde (CCBS), Centro Universitário UNIVATES, Lajeado, RS, Brazil Accepted author version posted online: 03 Aug 2015.
Click for updates To cite this article: Andressa Dametto, Giseli Buffon, Édina Aparecida Dos Reis Blasi & Raul Antonio Sperotto (2015): Ubiquitination pathway as a target to develop abiotic stress tolerance in rice, Plant Signaling & Behavior, DOI: 10.1080/15592324.2015.1057369 To link to this article: http://dx.doi.org/10.1080/15592324.2015.1057369
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Ubiquitination pathway as a target to develop abiotic stress tolerance in rice Andressa Dametto 1; Giseli Buffon1; Édina Aparecida dos Reis Blasi2; Raul Antonio Sperotto 1,2,* 1
Programa de Pós-Graduação em Biotecnologia (PPGBiotec) and 2Centro de Ciências
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Biológicas e da Saúde (CCBS), Centro Universitário UNIVATES, Lajeado, RS, Brazil
*corresponding author E-mail address: [email protected] Tel: 55-51-3714-7000 ext: 5534
Abstract Abiotic stresses may result in significant losses in rice grain productivity. Protein regulation by the ubiquitin/proteasome system has been studied as a target mechanism to optimize adaptation and survival strategies of plants to different environmental stresses. This paper aimed at highlighting recent discoveries about the roles ubiquitination may play in the exposure of rice plants to different abiotic stresses, enabling the development of modified plants tolerant to stress. Responses provided by the ubiquitination process include the regulation of the stomatal opening, phytohormones levels, protein stabilization, cell membrane integrity, meristematic cell maintenance, as well as the regulation of reactive oxygen species and heavy metals levels. It is noticeable that ubiquitination is a potential means for developing abiotic stress tolerant plants, being an excellent alternative to rice (and other cultures) improvement programs. 1
Keywords Cold, drought, heat, heavy metal, protein modification, rice, salinity.
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Abbreviations ROS, reactive oxygen species.
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Introduction Rice (Oryza sativa L.) is a species native to tropical regions which has been consumed for nearly 9,000 years1. Nowadays, it is considered one of the most important foods, as it feeds approximately half of the world population2. The oscillations observed in annual production of this culture derive mainly from abiotic stresses, such as high salinity, drought, extreme temperatures, and chemical toxicity, which limit plant germination, development,
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and productivity3-5. It is estimated that environmental stresses can result in losses of up to 60% in grain productivity and, frequently, restrict the area in which the plant is cultivated 6. Several studies focused on the key mechanisms of environmental stresses responses that can be used in genetic engineering in order to develop tolerant plants6,7. One of these mechanisms is the post-translational protein modification through ubiquitination8. This system is involved in several processes, from embryogenesis to senescence9, being responsible for the removal of abnormal proteins and acting as a short-term regulator, controlling several aspects of the cell metabolism. As such, cells are capable of rapidly responding to the intracellular signals and to changes in environmental conditions through modifications in key regulators10. Ubiquitin is a protein composed of 76 amino acids, found in both the cytosol and the nucleus of eukaryotic cells. It can be covalently bound to other proteins (target proteins) so to regulate stability, function or location of the modified protein. Ubiquitin is recognized by specific receptors that contain one or more ubiquitin-binding domains11,12. Usually, these domains bind to ubiquitin with low affinity, which make this bond highly dynamic. Therefore, the ubiquitin coupling and uncoupling system mediates several cell processes involved in growth and development of plants, such as embryogenesis, photomorphogenesis and hormone regulation. Furthermore, they take part in immune responses, membrane transport, DNA repair, chromatin remodeling and protein degradation10,12-14. Ubiquitination is characterized by the combination of ubiquitin in Lys residues of acceptor proteins9. The 3
process occurs through a well-known enzymatic cascade involving three enzymes: ubiquitinactivating (E1), ubiquitin-conjugating (E2), and ubiquitin-ligase (E3)12. The E2 enzymes play a role in determining the length and topology of ubiquitin networks, while the specificity of the ubiquitin-target protein binding occurs via the E3 enzyme15. There are currently seven types of E3ubiquitin ligases known and these can be subdivided into two basic groups dependent on the occurrence of either a ‘Homology to E6-
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AP C-Terminus’ (HECT) or ‘Really Interesting New Gene’ (RING)/U-box domain9. The HECT domain forms a thiol-ester intermediate E3-ubiquitin on a conserved cysteine before transferring the ubiquitin to the substrate16,17. In contrast, the RING and U box E3s do not form covalent intermediates with ubiquitin. Instead, they appear to function as scaffolds to position substrates in close proximity to an E2-ubiquitin covalent complex, which facilitates the direct transfer of ubiquitin from E2 enzymes to substrates. Despite the lack of sequence homology, the RING and the U box domains display remarkable similarity in structure, suggesting a common mechanism of action16. The RING-containing proteins can either ubiquitinate substrates independently or form multiprotein complexes, such as Skp1-CullinF-box (SCF)9. Cullin-RING ligases form the largest E3 enzyme family and are composed of many subunits12,18. Substrate specificity is provided by the F-box subunit which is anchored to SKP1 via an N-terminal F-box motif19 and target proteins through C-terminal proteinprotein interactions motifs. Protein modification process via ubiquitination system is a potential target for developing genetic improvement strategies of plants in different stress conditions13. In this review, we highlighted the recent findings involving the ubiquitination process in rice and the possibilities of using this complex mechanism to generate abiotic stress tolerant plants. Ubiquitination process in abiotic stress tolerance
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Many authors suggest that there is a relation between the protein ubiquitination process and the responses of the plants to different stresses20,21. Several transgenic approaches have been tested (Table 1), with different levels of stress tolerance.
Drought stress Water limitation damages agricultural crops, especially by causing protein Downloaded by [Monash University Library] at 04:37 26 August 2015
denaturation, decrease in chlorophyll levels, and photosynthesis inhibition, resulting in several restrictions to plant development6,22,23. Most studies cover genes related to E3 ligase enzymes, probably because those enzymes, acting together with accessory proteins such as Fbox, regulate the specificity of the ubiquitin bond to the target proteins, being involved in several processes. Five rice genes that encode E3 ligase enzymes with RING domain (OsSDIR1, OsCTR1, OsRDCP1, OsBIRF1 and OsDIS1) are potential candidates for the development of drought tolerant plants. Due to the fact that it is involved with stomatal control, the OsSDIR1 (Salt- and Drought-induced RING finger 1) overexpression in rice plants and Arabidopsis provided a lower transpiration rate, increasing efficiency in water usage and, consequently, tolerance to drought6. Under drought condition, overexpression of OsCTR1 (Chloroplast targeting RING E3 ligase 1) in Arabidopsis plants enables an increase in responses to dehydration through the regulation of abscisic acid (ABA) synthesis, resulting in the regulation of the stomatal opening23. The OsCTR1 protein can interact with other proteins, such as the chloroplast-localized OsCP12 (Chloroplast protein 12) and OsRP1 (Ribosomal protein 1), being ubiquitinated by OsCTR1 in the cytoplasm, resulting in degradation through ubiquitin/proteasome 26S system. The OsRDCP1 (RING domain containing protein 1) overexpression in rice plants also induces ubiquitination and degradation of proteins responsible for inactivation or degradation of proteins related to drought stress, increasing the levels of stress-response proteins and, consequently, drought 5
tolerance. After 15 days of water stress, transgenic lines were healthy and showed a decrease in damages caused by desiccation, while wild-type plants and osrdcp1 mutants were not able to recover24. As such, OsRDCP1 is probably involved in a subset of physiological responses that neutralize the dehydration stress in rice plants. The OsBIRF1 (BTH-induced RING finger protein 1) overexpression in tobacco plants increased the expression of genes related to oxidative stress, such as APX-, CAT- and GST-encoded antioxidant enzymes, as well as
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increasing ABA synthesis, making the plants more tolerant to drought during seed germination25. Protein ubiquitination can also negatively affect the rice plants responses to stress conditions. Silencing OsDIS1 (Drought-induced SINA protein 1) gene, which acts on the ubiquitination of OsNek6 protein, a Ser/Thr Protein Kinase involved in organ development and cell cycle regulation, provided high drought tolerance in rice plants, while the overexpression promoted plant sensitivity26,27. Thus, OsDIS1 is considered a negative regulator of drought stress signaling as it interferes in reactive oxygen species (ROS) levels, stomatal opening and degradation of drought-related transcription factors27.
Salinity stress High salinity is one of the abiotic stresses that most limits the development of agricultural crops. The relation between ubiquitination and high salinity stress is not yet clear. However, proteins related to responses to high salinity were identified as being regulated by ubiquitination. Liu et al.28 assessed proteins extracted from rice roots in early developmental stage exposed to salinity stress. It was observed that ubiquitination of PPDK1 (PyruvatePhosphate Dikinase1), Cyclin C1-1 and subunit 4 of Cellulose Synthase A proteins, after exposure to stress, aids the adaptation to high salinity in the three rice lines assessed. In
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addition, the polyubiquitination of HSP81-1 and Aldehyde Oxidase 3 proteins showed a relation with low tolerance to high salinity28. The OsRINGC2-1 and OsRINGC2-2 genes encode proteins which present E3 ligase activity at the RING-C2 domain. OsRINGC2-1 is more expressed in roots, while OsRINGC22 gene is more expressed in the panicles during development29. Plants over-expressing OsRINGC2-1 submitted to different NaCl concentrations showed an increased tolerance to
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high salinity stress through the ubiquitination system and enhanced root length29. Probably, OsRINGC2-1 overexpression leads to increased cell division rate or expansion of root cells. Although more in-depth analyses are required, overexpressed genes in the roots with the function of activating the responses to high salinity seem to be important, since the roots are the organs which have the first contact with the stressing environment, being capable of acting in the transmission of signs for the appropriate adaptation of the whole plant.
Heavy metal stress It is believed that protein degradation by the ubiquitin/proteasome 26S system plays an important role in the response to the stress caused by heavy metals in higher plants by acting in the removal of damaged proteins30. The OsHIR1 (Hypersensitive-induced reaction 1) gene, which encodes an E3 ligase with the RING domain, is expressed in rice plants exposed to high concentrations of As and Cd, which are toxic to plants. The expression level of this gene is closely related to the concentration of these metals in the soil. The OsHIR1 protein interacts with the OsTIP4;1 aquaporin, mediating its proteolysis through the ubiquitin/proteasome 26S degradation pathway31. Therefore, according to the authors, the OsHIR1 E3 ligase protein is able to control the absorption of these metals in the plant, probably through the OsTIP4;1 protein regulation via ubiquitination. Even though aquaporin proteins have not been described as As-transporter in plants until now, Zhao et al.32 stated
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that some TIP channels might be permeable to As transport into the vacuoles, due to its pore structure. Moreover, OsHIR1 overexpression in Arabidopsis also increased the tolerance to As and Cd, when compared to wild-type plants31. It is important to highlight that definitive proof of involvement of OsTIP4;1 in As and Cd efflux transport to the vacuole would require the demonstration of its biochemical activity in vitro. Anyway, this was the first study to present an E3 ligase with RING domain conferring tolerance to metals in plants. The
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development of strategies to reduce metal concentrations with the ubiquitin/proteasome 26S system in rice seems to be a promising way to ensure food safety.
Cold and high temperature stresses Stress caused by extreme temperatures disturb the cell homeostasis, resulting in a delay in the plant development, as it affects seed germination, photosynthesis, respiration, and plasma membrane stability5,33. Plants are capable of acclimating to survive at extreme temperatures, a process which involves cell membrane protection, solute synthesis and accumulation, and specific enzymatic activities7,34. Not all mechanisms involved in the acclimation process are known. However, genes related to protein ubiquitination are becoming object of research, aiming to generate cultures tolerant to extreme cold or heat. Transcription factors control the expression of several genes related to the low temperature responses in plants. The Arabidopsis HOS1 (High expression of osmotically responsive gene 1) protein was identified as an E3 ligase that mediates degradation of ICE1 (Inducer of CBF expression 1), a master regulatory protein during low temperature stress35. This was the first time that ubiquitin/proteasome pathway was associated to cold stress responses. Increased expression of cold responsive genes, such as DREB1A/CBF3, member of the well-known DREB1/CBF (Dehydration-responsive element-binding/C-repeat-binding factor) family of transcription factors, is possible through the ICE1 protein stabilization.
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Thus, it is necessary that post-translational modifications occur in ICE1 through phosphorylation or SUMOylation. Recently, it was verified that OsHOS1 rice gene is orthologous of the AtHOS1 Arabidopsis gene, and that both present E3 ligase activity related to the modulation of low temperature responses7. Protein-protein interaction studies revealed that OsHOS1 interacts with OsICE1, modulating its abundance and also the expression of other genes responsive to cold and regulated by ICE17.
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The post-translational modification process called SUMOylation, which involves the function of SUMO (Small Ubiquitin-related MOdifier), is considered an important protein regulatory pathway, which occurs by the conjugation of small modifiers related to ubiquitin36,37. SUMOylation modulates protein stability, enabling the regulation of several cell processes, such as the enzymatic activity and environmental stress responses in plants37,38. Several potential targets of SUMOylation were identified, including proteins involved in the regulation of low temperature responses7. The ICE1 protein stabilization by SUMOylation enables the activation of transcription factor CBF3/DREB1A involved in cold tolerance. The BnTR1 (Thermal Resistance 1) gene, identified in Brassica napus, plays a key role in the response to temperature stress39. BnTR1 is a membrane-bound RINGv (C4 HC3) protein that displays E3 ligase activity in vitro. When overexpressed in B. napus and rice plants, BnTR1 provided thermic tolerance. BnTR1 acts in the regulation of Ca2+ channels, activating transcription factors and heat shock proteins that contribute to plant thermal tolerance39. Another study revealed that OsHCI1 (Heat and Cold Induced 1) gene, which presents E3-ubiquitin ligase activity and is mainly associated to the Golgi apparatus, interacts with several proteins, mediating their transportation from the nucleus to the cytoplasm through mono-ubiquitination5. OsHCI1 overexpression in Arabidopsis resulted in an increased heat shock tolerance when compared to wild-type plants, suggesting that this gene
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is involved in the acquired thermal tolerance in plants through the ubiquitination process5. All of these studies confirm the possibility of developing thermal tolerance in plants through the ubiquitination pathway.
Genes involved in multiple abiotic stresses The expression of some genes related to ubiquitination can influence the tolerance to
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multiple stresses in plants. The OsDSG1 (Delayed Seed Germination 1) gene, which encodes an E3 ligase with RING-finger domain, regulates important responses during the exposure to multiple stresses20. Rice mutant osdsg1 and plants silenced by RNAi are tolerant to high salinity and drought, mediated by enhanced ABA-regulated responses. Furthermore, it was observed a positive correlation between germination rates and OsDSG1 expression levels. The high expression of this gene down-regulates ABA level in seeds, determining the success of germination20. In order to study the feasibility of manipulating SUMO E3 ligases in transgenic cultures, OsSIZ1 rice gene was overexpressed in Agrostis stolonifera plants. The high expression of OsSIZ1 led to an increase in photosynthetic rate and improved the general growth of the plants. Under drought and high temperature stresses, OsSIZ1-overexpressing plants presented higher performance than wild-type plants, showing a stronger growth of the root, higher water retention and higher cell membrane integrity37. Another study verified that OsPUB15 (Plant U-box 15) overexpression, which encodes a cytosolic protein from class II PUB family that contains a U-box domain, is regulated by environmental stresses40. Plant proteins that contain a U-box domain belong to the E3 ligase family that assists the poly-ubiquitination of target proteins in degradation or mono-ubiquitination and subsequent modification of the target proteins localization or activity41. It was found that OsPUB15 is induced by several stresses and avoid tissue
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damages40. OsPUB15 maintains the viability of meristematic cells, protecting them from osmotic stress during germination and growth of rice plants, as well as reducing the levels of ROS produced in the presence of abiotic stresses40,42. OsPUB15 expression levels increased in the presence of hydrogen peroxide (H2O2), high salinity and drought, indicating that PUB15 is an important regulator of ROS levels and stress responses40. OsSRFP1 (Stress-related RING finger protein 1), which encodes an E3 ligase, is
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another gene that can be used for obtaining plants tolerant to multiple abiotic stresses. OsSRFP1 expression is induced by low temperature, drought, high salinity and treatments with ABA and H2O2. The OsSRFP1 protein interferes in the activity of cell-protectant proteins in stressful conditions. Therefore, OsSRFP1 expression plays a negative role in the response to abiotic stresses in rice43. It was shown that decreased OsSRFP1 expression through RNAi makes silenced plants more tolerant to abiotic stresses than wild-type plants43. Silencing this gene increases SOD and CAT activity, intensifying the elimination of ROS and the tolerance to different stresses. It was also verified that silencing OsSRFP1 has led to a higher accumulation of free proline when rice plants are under low temperatures, which supports cell osmoregulation43,44.
Final considerations Ubiquitination process is important in protein degradation/signaling and regulation of several mechanisms related to abiotic stress responses. Modulation of ubiquitination-related gene expression enabled the development of plants that present a high survival rate after exposure to stressful environmental conditions (Table 1). Such genes regulate the responses to environmental adversities, which include regulation of the stomatal opening, phytohormones, protein stabilization, regulation of heat shock proteins, water retention, cell membrane integrity, as well as the regulation of the ROS levels and heavy metals transport. A
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schematic model of physiological and molecular changes found in transgenic plants under different abiotic stresses, after increased or decreased expression of rice genes involved in ubiquitination processes, is shown in Figure 1. Some genes can take part in the responses to multiple abiotic stresses, acting in several adaptive response pathways. In addition, some genes play a negative role in the responses to abiotic stresses, indicating that they can be useful in the development of transgenic plants by gene silencing. Even though some
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transgenic approaches have found promising results, further studies are required in order to elucidate the interaction of the ubiquitination pathway with other signaling proteins in the responses to abiotic stresses in plants, as well as to understand the involved biochemical, molecular and physiological processes.
Acknowledgments Research on Sperotto’s Lab is supported by Centro Universitário UNIVATES, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Rio Grande do Sul (FAPERGS). We thank Felipe Klein Ricachenevsky for critical reading of the manuscript.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
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Table 1. Transgenic approaches tested in the last years with genes involved in ubiquitination processes, which act in rice responses to different abiotic stress conditions.
Physiological Strategy
Stress
and
Characteristics of
used
condition
molecular
transgenic plants
Gene
Reference
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changes Drought treatment at the young seedling stage (4-week old): after 6 days of drought stress and rewatering for 24 h, transgenic rice plants showed 95%-99% survival ↑
and control plants showed 0% Regulation of
OsSDIR1
expression in
Drought
survival.
[6]
stomatal opening Drought treatment at the heading
rice plants
stage (70 days old): after two cycles of 8 days of drought stress, transgenic rice plants showed 92%98% survival and control plants showed 15%-21% survival. Drought treatment in 2-week old ↑ Regulation of
Arabidospis plants: after 10 days of
stomatal opening and
drought stress and re-watering for
phytohormones
72 h, transgenic plants showed
activity
74%-95% survival and control
expression in OsCTR1
Arabidopsis
Drought
[23]
thaliana plants plants showed 18% survival.
↑ OsRDCP1
expression in rice plants
Drought
Degradation of
Drought treatment in 6-week old
proteins related to
plants: after 15 days of drought
inactivation of hydric
stress and re-watering for 15 days,
stress-related proteins
all the transgenic plants survived
/ Increased levels of
and the control plants died.
20
[24]
defense proteins
OsBIRF1
↑
Reduction in ABA
Drought treatment at the seed
expression in
sensitivity in root
germination stage: transgenic
elongation /
tobacco seeds showed 78% and
tabacum
Activation of defense
control tobacco seeds showed 36%
plants
responses
of germination.
Degradation of
Drought treatment in 4-week old
drought-related
plants: after 9 days of drought
transcription factors /
stress and re-watering for 4 days,
Activation of the
silencing plants showed 75%-90%
ROS-scavenging
survival, control plants showed
pathway / Regulation
50% survival, and over-expressing
of stomatal opening
plants showed 20%-30% survival.
Nicotiana
Drought
[25]
↓
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OsDIS1
expression Drought
[27]
(RNAi) in rice plants
↑ OsRINGC21
High salinity (2 days of submergence in 150 mM NaCl)
expression in Enhanced root Arabidopsis
High salinity
treatment: transgenic Arabidopsis
[29]
development thaliana
plants demonstrated longer root
plants
lengths than those of control plants. Heavy metal (As and Cd) Binds to OsTIP4;1
↑
treatment: transgenic Arabidopsis (aquaporine) and plants showed decreased As (14%
expression in mediate its OsHIR1
Arabidopsis
Heavy metal
and 46%) and Cd (4% and 12%)
[31]
proteolysis, thaliana
accumulation in the shoots and decreasing As and Cd roots, respectively, relative to the
plants uptake
control plants. RNAi::OsHOS1 Cold (10oC) treatment in 2-week
↓ plants showed a OsHOS1
old plants: after 24 h of cold,
expression Cold
higher (but transient)
[7] silencing plants did not show
(RNAi) in expression level of
increased cold tolerance.
rice plants
OsDREB1A
↑
Mediation of nuclear-
Arabidopsis plants were subjected
cytoplasmic
to heating to 38°C for 90 min and
trafficking of nuclear
subsequently cooled for 2 h at room
High OsHCI1
expression in temperature Arabidopsis
21
[5]
thaliana
substrate proteins
plants
temperature (24°C). After pretreatment, plants were subjected to heating to 45°C for 3 h and then allowed to recover for 5 days at 24°C. Transgenic plants showed high survival rates (55%-65%) and most control plants did not recover. High salinity (150 mM NaCl) treatment in rice germinating seeds:
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osdsg1 mutant germination rate was five times higher than for control seeds. ↓ OsDSG1
High salinity (150 mM NaCl)
expression
Drought and
Enhanced ABA
treatment in 15-days-old seedlings:
(osdsg1 rice
high salinity
signaling
osdsg1 mutant seedlings showed
[20]
higher salinity tolerance than
mutant)
control seedlings. Drought treatment in 15-days-old seedlings: osdsg1 mutant seedlings showed higher drought tolerance than control seedlings. Drought treatment in 10-week old creeping bentgrass plants: after 10 weeks of limited water supply Increased
(watered only every 5 days)
photosynthesis /
followed by saturated watering for
Enhanced root
2 weeks for recovering, transgenic
development, water
plants showed less root growth
retention and cell
inhibition and produced a much
membrane integrity
more robust root system with a
↑
OsSIZ1
expression in
Drought and
Agrostis
high
stolonifera
[37]
temperature
plants biomass close to 2.6 times (fresh weight) or 2.1 times (dry weight) that in control plants.
22
High temperature treatment in 10week old creeping bentgrass plants: after heating at 35-40oC for 12 days, the majority of transgenic plants recovered from the heatelicited damage and survived the treatment, while all control plants died. Drought treatment in 7-days-old
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seedlings: after 72 hs under drought, followed by 72 hs recovery, transgenic plants were much less susceptible than control ↑ OsPUB15
Drought and
Reduced levels of
plants.
high salinity
ROS and cell death
High salinity (250 mM NaCl)
[40]
expression in rice plants treatment in 7-days-old seedlings: after 24 hs of submergence in salt excess, followed by 72 hs recovery, transgenic plants grew 1.4-fold faster than the control plants. Cold (4oC) treatment in 2-week-old seedlings: after 1 week of cold, followed by 2 weeks recovery, silenced plants showed 55% and control plants showed 20% of ↓
OsSRFP1
expression
Cold and
(RNAi) in
high salinity
Increased antioxidant
survival.
enzyme activities and
High salinity (150 mM NaCl)
proline concentration
treatment in 2-week-old seedlings:
rice plants after 2 weeks of submergence in salt stress, followed by 2 weeks recovery, silenced plants showed longer shoot and root lengths, along with higher survival rates (65%-
23
[43]
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74%) than those of control plants (51%).
24
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Figure 1. Schematic model of physiological and molecular changes found in transgenic plants over-expressing (red color) or down-regulating (green color) rice genes involved in ubiquitination processes, under different abiotic stresses.
25
Plant Signaling & Behavior Publication details, including instructions for authors and subscription information: http://www.tandfonline.com/loi/kpsb20
Ubiquitination pathway as a target to develop abiotic stress tolerance in rice a
a
b
Andressa Dametto , Giseli Buffon , Édina Aparecida Dos Reis Blasi & Raul Antonio Sperotto
ab
a
Programa de Pós-Graduação em Biotecnologia (PPGBiotec) and Centro Universitário UNIVATES, Lajeado, RS, Brazil b
Centro de Ciências Biológicas e da Saúde (CCBS), Centro Universitário UNIVATES, Lajeado, RS, Brazil Accepted author version posted online: 03 Aug 2015.
Click for updates To cite this article: Andressa Dametto, Giseli Buffon, Édina Aparecida Dos Reis Blasi & Raul Antonio Sperotto (2015): Ubiquitination pathway as a target to develop abiotic stress tolerance in rice, Plant Signaling & Behavior, DOI: 10.1080/15592324.2015.1057369 To link to this article: http://dx.doi.org/10.1080/15592324.2015.1057369
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Ubiquitination pathway as a target to develop abiotic stress tolerance in rice Andressa Dametto 1; Giseli Buffon1; Édina Aparecida dos Reis Blasi2; Raul Antonio Sperotto 1,2,* 1
Programa de Pós-Graduação em Biotecnologia (PPGBiotec) and 2Centro de Ciências
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Biológicas e da Saúde (CCBS), Centro Universitário UNIVATES, Lajeado, RS, Brazil
*corresponding author E-mail address: [email protected] Tel: 55-51-3714-7000 ext: 5534
Abstract Abiotic stresses may result in significant losses in rice grain productivity. Protein regulation by the ubiquitin/proteasome system has been studied as a target mechanism to optimize adaptation and survival strategies of plants to different environmental stresses. This paper aimed at highlighting recent discoveries about the roles ubiquitination may play in the exposure of rice plants to different abiotic stresses, enabling the development of modified plants tolerant to stress. Responses provided by the ubiquitination process include the regulation of the stomatal opening, phytohormones levels, protein stabilization, cell membrane integrity, meristematic cell maintenance, as well as the regulation of reactive oxygen species and heavy metals levels. It is noticeable that ubiquitination is a potential means for developing abiotic stress tolerant plants, being an excellent alternative to rice (and other cultures) improvement programs. 1
Keywords Cold, drought, heat, heavy metal, protein modification, rice, salinity.
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Abbreviations ROS, reactive oxygen species.
2
Introduction Rice (Oryza sativa L.) is a species native to tropical regions which has been consumed for nearly 9,000 years1. Nowadays, it is considered one of the most important foods, as it feeds approximately half of the world population2. The oscillations observed in annual production of this culture derive mainly from abiotic stresses, such as high salinity, drought, extreme temperatures, and chemical toxicity, which limit plant germination, development,
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and productivity3-5. It is estimated that environmental stresses can result in losses of up to 60% in grain productivity and, frequently, restrict the area in which the plant is cultivated 6. Several studies focused on the key mechanisms of environmental stresses responses that can be used in genetic engineering in order to develop tolerant plants6,7. One of these mechanisms is the post-translational protein modification through ubiquitination8. This system is involved in several processes, from embryogenesis to senescence9, being responsible for the removal of abnormal proteins and acting as a short-term regulator, controlling several aspects of the cell metabolism. As such, cells are capable of rapidly responding to the intracellular signals and to changes in environmental conditions through modifications in key regulators10. Ubiquitin is a protein composed of 76 amino acids, found in both the cytosol and the nucleus of eukaryotic cells. It can be covalently bound to other proteins (target proteins) so to regulate stability, function or location of the modified protein. Ubiquitin is recognized by specific receptors that contain one or more ubiquitin-binding domains11,12. Usually, these domains bind to ubiquitin with low affinity, which make this bond highly dynamic. Therefore, the ubiquitin coupling and uncoupling system mediates several cell processes involved in growth and development of plants, such as embryogenesis, photomorphogenesis and hormone regulation. Furthermore, they take part in immune responses, membrane transport, DNA repair, chromatin remodeling and protein degradation10,12-14. Ubiquitination is characterized by the combination of ubiquitin in Lys residues of acceptor proteins9. The 3
process occurs through a well-known enzymatic cascade involving three enzymes: ubiquitinactivating (E1), ubiquitin-conjugating (E2), and ubiquitin-ligase (E3)12. The E2 enzymes play a role in determining the length and topology of ubiquitin networks, while the specificity of the ubiquitin-target protein binding occurs via the E3 enzyme15. There are currently seven types of E3ubiquitin ligases known and these can be subdivided into two basic groups dependent on the occurrence of either a ‘Homology to E6-
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AP C-Terminus’ (HECT) or ‘Really Interesting New Gene’ (RING)/U-box domain9. The HECT domain forms a thiol-ester intermediate E3-ubiquitin on a conserved cysteine before transferring the ubiquitin to the substrate16,17. In contrast, the RING and U box E3s do not form covalent intermediates with ubiquitin. Instead, they appear to function as scaffolds to position substrates in close proximity to an E2-ubiquitin covalent complex, which facilitates the direct transfer of ubiquitin from E2 enzymes to substrates. Despite the lack of sequence homology, the RING and the U box domains display remarkable similarity in structure, suggesting a common mechanism of action16. The RING-containing proteins can either ubiquitinate substrates independently or form multiprotein complexes, such as Skp1-CullinF-box (SCF)9. Cullin-RING ligases form the largest E3 enzyme family and are composed of many subunits12,18. Substrate specificity is provided by the F-box subunit which is anchored to SKP1 via an N-terminal F-box motif19 and target proteins through C-terminal proteinprotein interactions motifs. Protein modification process via ubiquitination system is a potential target for developing genetic improvement strategies of plants in different stress conditions13. In this review, we highlighted the recent findings involving the ubiquitination process in rice and the possibilities of using this complex mechanism to generate abiotic stress tolerant plants. Ubiquitination process in abiotic stress tolerance
4
Many authors suggest that there is a relation between the protein ubiquitination process and the responses of the plants to different stresses20,21. Several transgenic approaches have been tested (Table 1), with different levels of stress tolerance.
Drought stress Water limitation damages agricultural crops, especially by causing protein Downloaded by [Monash University Library] at 04:37 26 August 2015
denaturation, decrease in chlorophyll levels, and photosynthesis inhibition, resulting in several restrictions to plant development6,22,23. Most studies cover genes related to E3 ligase enzymes, probably because those enzymes, acting together with accessory proteins such as Fbox, regulate the specificity of the ubiquitin bond to the target proteins, being involved in several processes. Five rice genes that encode E3 ligase enzymes with RING domain (OsSDIR1, OsCTR1, OsRDCP1, OsBIRF1 and OsDIS1) are potential candidates for the development of drought tolerant plants. Due to the fact that it is involved with stomatal control, the OsSDIR1 (Salt- and Drought-induced RING finger 1) overexpression in rice plants and Arabidopsis provided a lower transpiration rate, increasing efficiency in water usage and, consequently, tolerance to drought6. Under drought condition, overexpression of OsCTR1 (Chloroplast targeting RING E3 ligase 1) in Arabidopsis plants enables an increase in responses to dehydration through the regulation of abscisic acid (ABA) synthesis, resulting in the regulation of the stomatal opening23. The OsCTR1 protein can interact with other proteins, such as the chloroplast-localized OsCP12 (Chloroplast protein 12) and OsRP1 (Ribosomal protein 1), being ubiquitinated by OsCTR1 in the cytoplasm, resulting in degradation through ubiquitin/proteasome 26S system. The OsRDCP1 (RING domain containing protein 1) overexpression in rice plants also induces ubiquitination and degradation of proteins responsible for inactivation or degradation of proteins related to drought stress, increasing the levels of stress-response proteins and, consequently, drought 5
tolerance. After 15 days of water stress, transgenic lines were healthy and showed a decrease in damages caused by desiccation, while wild-type plants and osrdcp1 mutants were not able to recover24. As such, OsRDCP1 is probably involved in a subset of physiological responses that neutralize the dehydration stress in rice plants. The OsBIRF1 (BTH-induced RING finger protein 1) overexpression in tobacco plants increased the expression of genes related to oxidative stress, such as APX-, CAT- and GST-encoded antioxidant enzymes, as well as
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increasing ABA synthesis, making the plants more tolerant to drought during seed germination25. Protein ubiquitination can also negatively affect the rice plants responses to stress conditions. Silencing OsDIS1 (Drought-induced SINA protein 1) gene, which acts on the ubiquitination of OsNek6 protein, a Ser/Thr Protein Kinase involved in organ development and cell cycle regulation, provided high drought tolerance in rice plants, while the overexpression promoted plant sensitivity26,27. Thus, OsDIS1 is considered a negative regulator of drought stress signaling as it interferes in reactive oxygen species (ROS) levels, stomatal opening and degradation of drought-related transcription factors27.
Salinity stress High salinity is one of the abiotic stresses that most limits the development of agricultural crops. The relation between ubiquitination and high salinity stress is not yet clear. However, proteins related to responses to high salinity were identified as being regulated by ubiquitination. Liu et al.28 assessed proteins extracted from rice roots in early developmental stage exposed to salinity stress. It was observed that ubiquitination of PPDK1 (PyruvatePhosphate Dikinase1), Cyclin C1-1 and subunit 4 of Cellulose Synthase A proteins, after exposure to stress, aids the adaptation to high salinity in the three rice lines assessed. In
6
addition, the polyubiquitination of HSP81-1 and Aldehyde Oxidase 3 proteins showed a relation with low tolerance to high salinity28. The OsRINGC2-1 and OsRINGC2-2 genes encode proteins which present E3 ligase activity at the RING-C2 domain. OsRINGC2-1 is more expressed in roots, while OsRINGC22 gene is more expressed in the panicles during development29. Plants over-expressing OsRINGC2-1 submitted to different NaCl concentrations showed an increased tolerance to
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high salinity stress through the ubiquitination system and enhanced root length29. Probably, OsRINGC2-1 overexpression leads to increased cell division rate or expansion of root cells. Although more in-depth analyses are required, overexpressed genes in the roots with the function of activating the responses to high salinity seem to be important, since the roots are the organs which have the first contact with the stressing environment, being capable of acting in the transmission of signs for the appropriate adaptation of the whole plant.
Heavy metal stress It is believed that protein degradation by the ubiquitin/proteasome 26S system plays an important role in the response to the stress caused by heavy metals in higher plants by acting in the removal of damaged proteins30. The OsHIR1 (Hypersensitive-induced reaction 1) gene, which encodes an E3 ligase with the RING domain, is expressed in rice plants exposed to high concentrations of As and Cd, which are toxic to plants. The expression level of this gene is closely related to the concentration of these metals in the soil. The OsHIR1 protein interacts with the OsTIP4;1 aquaporin, mediating its proteolysis through the ubiquitin/proteasome 26S degradation pathway31. Therefore, according to the authors, the OsHIR1 E3 ligase protein is able to control the absorption of these metals in the plant, probably through the OsTIP4;1 protein regulation via ubiquitination. Even though aquaporin proteins have not been described as As-transporter in plants until now, Zhao et al.32 stated
7
that some TIP channels might be permeable to As transport into the vacuoles, due to its pore structure. Moreover, OsHIR1 overexpression in Arabidopsis also increased the tolerance to As and Cd, when compared to wild-type plants31. It is important to highlight that definitive proof of involvement of OsTIP4;1 in As and Cd efflux transport to the vacuole would require the demonstration of its biochemical activity in vitro. Anyway, this was the first study to present an E3 ligase with RING domain conferring tolerance to metals in plants. The
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development of strategies to reduce metal concentrations with the ubiquitin/proteasome 26S system in rice seems to be a promising way to ensure food safety.
Cold and high temperature stresses Stress caused by extreme temperatures disturb the cell homeostasis, resulting in a delay in the plant development, as it affects seed germination, photosynthesis, respiration, and plasma membrane stability5,33. Plants are capable of acclimating to survive at extreme temperatures, a process which involves cell membrane protection, solute synthesis and accumulation, and specific enzymatic activities7,34. Not all mechanisms involved in the acclimation process are known. However, genes related to protein ubiquitination are becoming object of research, aiming to generate cultures tolerant to extreme cold or heat. Transcription factors control the expression of several genes related to the low temperature responses in plants. The Arabidopsis HOS1 (High expression of osmotically responsive gene 1) protein was identified as an E3 ligase that mediates degradation of ICE1 (Inducer of CBF expression 1), a master regulatory protein during low temperature stress35. This was the first time that ubiquitin/proteasome pathway was associated to cold stress responses. Increased expression of cold responsive genes, such as DREB1A/CBF3, member of the well-known DREB1/CBF (Dehydration-responsive element-binding/C-repeat-binding factor) family of transcription factors, is possible through the ICE1 protein stabilization.
8
Thus, it is necessary that post-translational modifications occur in ICE1 through phosphorylation or SUMOylation. Recently, it was verified that OsHOS1 rice gene is orthologous of the AtHOS1 Arabidopsis gene, and that both present E3 ligase activity related to the modulation of low temperature responses7. Protein-protein interaction studies revealed that OsHOS1 interacts with OsICE1, modulating its abundance and also the expression of other genes responsive to cold and regulated by ICE17.
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The post-translational modification process called SUMOylation, which involves the function of SUMO (Small Ubiquitin-related MOdifier), is considered an important protein regulatory pathway, which occurs by the conjugation of small modifiers related to ubiquitin36,37. SUMOylation modulates protein stability, enabling the regulation of several cell processes, such as the enzymatic activity and environmental stress responses in plants37,38. Several potential targets of SUMOylation were identified, including proteins involved in the regulation of low temperature responses7. The ICE1 protein stabilization by SUMOylation enables the activation of transcription factor CBF3/DREB1A involved in cold tolerance. The BnTR1 (Thermal Resistance 1) gene, identified in Brassica napus, plays a key role in the response to temperature stress39. BnTR1 is a membrane-bound RINGv (C4 HC3) protein that displays E3 ligase activity in vitro. When overexpressed in B. napus and rice plants, BnTR1 provided thermic tolerance. BnTR1 acts in the regulation of Ca2+ channels, activating transcription factors and heat shock proteins that contribute to plant thermal tolerance39. Another study revealed that OsHCI1 (Heat and Cold Induced 1) gene, which presents E3-ubiquitin ligase activity and is mainly associated to the Golgi apparatus, interacts with several proteins, mediating their transportation from the nucleus to the cytoplasm through mono-ubiquitination5. OsHCI1 overexpression in Arabidopsis resulted in an increased heat shock tolerance when compared to wild-type plants, suggesting that this gene
9
is involved in the acquired thermal tolerance in plants through the ubiquitination process5. All of these studies confirm the possibility of developing thermal tolerance in plants through the ubiquitination pathway.
Genes involved in multiple abiotic stresses The expression of some genes related to ubiquitination can influence the tolerance to
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multiple stresses in plants. The OsDSG1 (Delayed Seed Germination 1) gene, which encodes an E3 ligase with RING-finger domain, regulates important responses during the exposure to multiple stresses20. Rice mutant osdsg1 and plants silenced by RNAi are tolerant to high salinity and drought, mediated by enhanced ABA-regulated responses. Furthermore, it was observed a positive correlation between germination rates and OsDSG1 expression levels. The high expression of this gene down-regulates ABA level in seeds, determining the success of germination20. In order to study the feasibility of manipulating SUMO E3 ligases in transgenic cultures, OsSIZ1 rice gene was overexpressed in Agrostis stolonifera plants. The high expression of OsSIZ1 led to an increase in photosynthetic rate and improved the general growth of the plants. Under drought and high temperature stresses, OsSIZ1-overexpressing plants presented higher performance than wild-type plants, showing a stronger growth of the root, higher water retention and higher cell membrane integrity37. Another study verified that OsPUB15 (Plant U-box 15) overexpression, which encodes a cytosolic protein from class II PUB family that contains a U-box domain, is regulated by environmental stresses40. Plant proteins that contain a U-box domain belong to the E3 ligase family that assists the poly-ubiquitination of target proteins in degradation or mono-ubiquitination and subsequent modification of the target proteins localization or activity41. It was found that OsPUB15 is induced by several stresses and avoid tissue
10
damages40. OsPUB15 maintains the viability of meristematic cells, protecting them from osmotic stress during germination and growth of rice plants, as well as reducing the levels of ROS produced in the presence of abiotic stresses40,42. OsPUB15 expression levels increased in the presence of hydrogen peroxide (H2O2), high salinity and drought, indicating that PUB15 is an important regulator of ROS levels and stress responses40. OsSRFP1 (Stress-related RING finger protein 1), which encodes an E3 ligase, is
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another gene that can be used for obtaining plants tolerant to multiple abiotic stresses. OsSRFP1 expression is induced by low temperature, drought, high salinity and treatments with ABA and H2O2. The OsSRFP1 protein interferes in the activity of cell-protectant proteins in stressful conditions. Therefore, OsSRFP1 expression plays a negative role in the response to abiotic stresses in rice43. It was shown that decreased OsSRFP1 expression through RNAi makes silenced plants more tolerant to abiotic stresses than wild-type plants43. Silencing this gene increases SOD and CAT activity, intensifying the elimination of ROS and the tolerance to different stresses. It was also verified that silencing OsSRFP1 has led to a higher accumulation of free proline when rice plants are under low temperatures, which supports cell osmoregulation43,44.
Final considerations Ubiquitination process is important in protein degradation/signaling and regulation of several mechanisms related to abiotic stress responses. Modulation of ubiquitination-related gene expression enabled the development of plants that present a high survival rate after exposure to stressful environmental conditions (Table 1). Such genes regulate the responses to environmental adversities, which include regulation of the stomatal opening, phytohormones, protein stabilization, regulation of heat shock proteins, water retention, cell membrane integrity, as well as the regulation of the ROS levels and heavy metals transport. A
11
schematic model of physiological and molecular changes found in transgenic plants under different abiotic stresses, after increased or decreased expression of rice genes involved in ubiquitination processes, is shown in Figure 1. Some genes can take part in the responses to multiple abiotic stresses, acting in several adaptive response pathways. In addition, some genes play a negative role in the responses to abiotic stresses, indicating that they can be useful in the development of transgenic plants by gene silencing. Even though some
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transgenic approaches have found promising results, further studies are required in order to elucidate the interaction of the ubiquitination pathway with other signaling proteins in the responses to abiotic stresses in plants, as well as to understand the involved biochemical, molecular and physiological processes.
Acknowledgments Research on Sperotto’s Lab is supported by Centro Universitário UNIVATES, Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Fundação de Amparo à Pesquisa do Rio Grande do Sul (FAPERGS). We thank Felipe Klein Ricachenevsky for critical reading of the manuscript.
Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.
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Table 1. Transgenic approaches tested in the last years with genes involved in ubiquitination processes, which act in rice responses to different abiotic stress conditions.
Physiological Strategy
Stress
and
Characteristics of
used
condition
molecular
transgenic plants
Gene
Reference
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changes Drought treatment at the young seedling stage (4-week old): after 6 days of drought stress and rewatering for 24 h, transgenic rice plants showed 95%-99% survival ↑
and control plants showed 0% Regulation of
OsSDIR1
expression in
Drought
survival.
[6]
stomatal opening Drought treatment at the heading
rice plants
stage (70 days old): after two cycles of 8 days of drought stress, transgenic rice plants showed 92%98% survival and control plants showed 15%-21% survival. Drought treatment in 2-week old ↑ Regulation of
Arabidospis plants: after 10 days of
stomatal opening and
drought stress and re-watering for
phytohormones
72 h, transgenic plants showed
activity
74%-95% survival and control
expression in OsCTR1
Arabidopsis
Drought
[23]
thaliana plants plants showed 18% survival.
↑ OsRDCP1
expression in rice plants
Drought
Degradation of
Drought treatment in 6-week old
proteins related to
plants: after 15 days of drought
inactivation of hydric
stress and re-watering for 15 days,
stress-related proteins
all the transgenic plants survived
/ Increased levels of
and the control plants died.
20
[24]
defense proteins
OsBIRF1
↑
Reduction in ABA
Drought treatment at the seed
expression in
sensitivity in root
germination stage: transgenic
elongation /
tobacco seeds showed 78% and
tabacum
Activation of defense
control tobacco seeds showed 36%
plants
responses
of germination.
Degradation of
Drought treatment in 4-week old
drought-related
plants: after 9 days of drought
transcription factors /
stress and re-watering for 4 days,
Activation of the
silencing plants showed 75%-90%
ROS-scavenging
survival, control plants showed
pathway / Regulation
50% survival, and over-expressing
of stomatal opening
plants showed 20%-30% survival.
Nicotiana
Drought
[25]
↓
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OsDIS1
expression Drought
[27]
(RNAi) in rice plants
↑ OsRINGC21
High salinity (2 days of submergence in 150 mM NaCl)
expression in Enhanced root Arabidopsis
High salinity
treatment: transgenic Arabidopsis
[29]
development thaliana
plants demonstrated longer root
plants
lengths than those of control plants. Heavy metal (As and Cd) Binds to OsTIP4;1
↑
treatment: transgenic Arabidopsis (aquaporine) and plants showed decreased As (14%
expression in mediate its OsHIR1
Arabidopsis
Heavy metal
and 46%) and Cd (4% and 12%)
[31]
proteolysis, thaliana
accumulation in the shoots and decreasing As and Cd roots, respectively, relative to the
plants uptake
control plants. RNAi::OsHOS1 Cold (10oC) treatment in 2-week
↓ plants showed a OsHOS1
old plants: after 24 h of cold,
expression Cold
higher (but transient)
[7] silencing plants did not show
(RNAi) in expression level of
increased cold tolerance.
rice plants
OsDREB1A
↑
Mediation of nuclear-
Arabidopsis plants were subjected
cytoplasmic
to heating to 38°C for 90 min and
trafficking of nuclear
subsequently cooled for 2 h at room
High OsHCI1
expression in temperature Arabidopsis
21
[5]
thaliana
substrate proteins
plants
temperature (24°C). After pretreatment, plants were subjected to heating to 45°C for 3 h and then allowed to recover for 5 days at 24°C. Transgenic plants showed high survival rates (55%-65%) and most control plants did not recover. High salinity (150 mM NaCl) treatment in rice germinating seeds:
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osdsg1 mutant germination rate was five times higher than for control seeds. ↓ OsDSG1
High salinity (150 mM NaCl)
expression
Drought and
Enhanced ABA
treatment in 15-days-old seedlings:
(osdsg1 rice
high salinity
signaling
osdsg1 mutant seedlings showed
[20]
higher salinity tolerance than
mutant)
control seedlings. Drought treatment in 15-days-old seedlings: osdsg1 mutant seedlings showed higher drought tolerance than control seedlings. Drought treatment in 10-week old creeping bentgrass plants: after 10 weeks of limited water supply Increased
(watered only every 5 days)
photosynthesis /
followed by saturated watering for
Enhanced root
2 weeks for recovering, transgenic
development, water
plants showed less root growth
retention and cell
inhibition and produced a much
membrane integrity
more robust root system with a
↑
OsSIZ1
expression in
Drought and
Agrostis
high
stolonifera
[37]
temperature
plants biomass close to 2.6 times (fresh weight) or 2.1 times (dry weight) that in control plants.
22
High temperature treatment in 10week old creeping bentgrass plants: after heating at 35-40oC for 12 days, the majority of transgenic plants recovered from the heatelicited damage and survived the treatment, while all control plants died. Drought treatment in 7-days-old
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seedlings: after 72 hs under drought, followed by 72 hs recovery, transgenic plants were much less susceptible than control ↑ OsPUB15
Drought and
Reduced levels of
plants.
high salinity
ROS and cell death
High salinity (250 mM NaCl)
[40]
expression in rice plants treatment in 7-days-old seedlings: after 24 hs of submergence in salt excess, followed by 72 hs recovery, transgenic plants grew 1.4-fold faster than the control plants. Cold (4oC) treatment in 2-week-old seedlings: after 1 week of cold, followed by 2 weeks recovery, silenced plants showed 55% and control plants showed 20% of ↓
OsSRFP1
expression
Cold and
(RNAi) in
high salinity
Increased antioxidant
survival.
enzyme activities and
High salinity (150 mM NaCl)
proline concentration
treatment in 2-week-old seedlings:
rice plants after 2 weeks of submergence in salt stress, followed by 2 weeks recovery, silenced plants showed longer shoot and root lengths, along with higher survival rates (65%-
23
[43]
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74%) than those of control plants (51%).
24
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Figure 1. Schematic model of physiological and molecular changes found in transgenic plants over-expressing (red color) or down-regulating (green color) rice genes involved in ubiquitination processes, under different abiotic stresses.
25
