Ubiquitination pathway as a target to develop abiotic stress tolerance in rice

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 article 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.

Abbreviations

ROS

reactive oxygen species

Introduction

Rice (Oryza sativa L.) is a species native to tropical regions which has been consumed for nearly 9,000 years.¹ Nowadays, it is considered one of the most important foods, as it feeds approximately half of the world population.² 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, and productivity.^3-5 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 plants.^6,7 One of these mechanisms is the post-translational protein modification through ubiquitination.⁸ This system is involved in several processes, from embryogenesis to senescence,⁹ 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 regulators.¹⁰

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 domains.^11,12 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 degradation.^10,12-14 Ubiquitination is characterized by the combination of ubiquitin in Lys residues of acceptor proteins.⁹ The process occurs through a well-known enzymatic cascade involving 3 enzymes: ubiquitin-activating (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 enzyme.¹⁵

There are currently 7 types of E3 ubiquitin ligases known and these can be subdivided into 2 basic groups dependent on the occurrence of either a ‘Homology to E6-AP C-Terminus’ (HECT) or ‘Really Interesting New Gene’ (RING)/U-box domain.⁹ The HECT domain forms a thiol-ester intermediate E3-ubiquitin on a conserved cysteine before transferring the ubiquitin to the substrate.^16,17 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 action.¹⁶ The RING-containing proteins can either ubiquitinate substrates independently or form multiprotein complexes, such as Skp1-Cullin-F-box (SCF).⁹ Cullin-RING ligases form the largest E3 enzyme family and are composed of many subunits.^12,18 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 protein-protein interactions motifs.

Protein modification process via ubiquitination system is a potential target for developing genetic improvement strategies of plants in different stress conditions.¹³ 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

Many authors suggest that there is a relation between the protein ubiquitination process and the plant responses to different stresses.^20,21 Several transgenic approaches have been tested (Table 1), with different levels of stress tolerance.

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

Gene	Strategy used	Stress condition	Physiological and molecular changes	Characteristics of transgenic plants	Reference
OsSDIR1	↑ expression in rice plants	Drought	Regulation of stomatal opening	Drought treatment at the young seedling stage (4-week old): after 6 days of drought stress and re-watering for 24 h, transgenic rice plants showed 95%–99% survival and control plants showed 0% survival. Drought treatment at the heading stage (70 days old): after 2 cycles of 8 days of drought stress, transgenic rice plants showed 92%–98% survival and control plants showed 15%–21% survival.	6
OsCTR1	↑ expression in Arabidopsisthaliana plants	Drought	Regulation of stomatal opening and phytohormones activity	Drought treatment in 2-week old Arabidopsis plants: after 10 days of drought stress and re-watering for 72 h, transgenic plants showed 74%–95% survival and control plants showed 18% survival.	23
OsRDCP1	↑ expression in rice plants	Drought	Degradation of proteins related to inactivation of hydric stress-related proteins / Increased levels of defense proteins	Drought treatment in 6-week old plants: after 15 days of drought stress and re-watering for 15 days, all the transgenic plants survived and the control plants died.	24
OsBIRF1	↑ expression in Nicotiana tabacum plants	Drought	Reduction in ABA sensitivity in root elongation / Activation of defense responses	Drought treatment at the seed germination stage: transgenic tobacco seeds showed 78% and control tobacco seeds showed 36% of germination.	25
OsDIS1	↓ expression (RNAi) in rice plants	Drought	Degradation of drought-related transcription factors / Activation of the ROS-scavenging pathway / Regulation of stomatal opening	Drought treatment in 4-week old plants: after 9 days of drought stress and re-watering for 4 days, silencing plants showed 75%–90% survival, control plants showed 50% survival, and over-expressing plants showed 20%–30% survival.	27
OsRINGC2-1	↑ expression in Arabidopsis thaliana plants	High salinity	Enhanced root development	High salinity (2 days of submergence in 150 mM NaCl) treatment: transgenic Arabidopsis plants demonstrated longer root lengths than those of control plants.	29
OsHIR1	↑ expression in Arabidopsis thaliana plants	Heavy metal	Binds to OsTIP4;1 (aquaporine) and mediate its proteolysis, decreasing As and Cd uptake	Heavy metal (As and Cd) treatment: transgenic Arabidopsis plants showed decreased As (14% and 46%) and Cd (4% and 12%) accumulation in the shoots and roots, respectively, relative to the control plants.	31
OsHOS1	↓ expression (RNAi) in rice plants	Cold	RNAi::OsHOS1 plants showed a higher (but transient) expression level of OsDREB1A	Cold (10°C) treatment in 2-week old plants: after 24 h of cold, silencing plants did not show increased cold tolerance.	7
OsHCI1	↑ expression in Arabidopsis thaliana plants	High temperature	Mediation of nuclear-cytoplasmic trafficking of nuclear substrate proteins	Arabidopsis plants were subjected to heating to 38°C for 90 min and subsequently cooled for 2 h at room 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.	5
OsDSG1	↓ expression (osdsg1 rice mutant)	Drought and high salinity	Enhanced ABA signaling	High salinity (150 mM NaCl) treatment in rice germinating seeds: osdsg1 mutant germination rate was 5 times higher than for control seeds. High salinity (150 mM NaCl) treatment in 15-days-old seedlings: osdsg1 mutant seedlings showed higher salinity tolerance than control seedlings.Drought treatment in 15-days-old seedlings: osdsg1 mutant seedlings showed higher drought tolerance than control seedlings.	20
OsSIZ1	↑ expression in Agrostis stolonifera plants	Drought and high temperature	Increased photosynthesis / Enhanced root development, water retention and cell membrane integrity	Drought treatment in 10-week old creeping bentgrass plants: after 10 weeks of limited water supply (watered only every 5 days) followed by saturated watering for 2 weeks for recovering, transgenic plants showed less root growth inhibition and produced a much more robust root system with a biomass close to 2.6 times (fresh weight) or 2.1 times (dry weight) that in control plants. High temperature treatment in 10-week old creeping bentgrass plants: after heating at 35–40°C for 12 days, the majority of transgenic plants recovered from the heat-elicited damage and survived the treatment, while all control plants died.	37
OsPUB15	↑ expression in rice plants	Drought and high salinity	Reduced levels of ROS and cell death	Drought treatment in 7-days-old seedlings: after 72 hs under drought, followed by 72 hs recovery, transgenic plants were much less susceptible than control plants.High salinity (250 mM NaCl) 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.	40
OsSRFP1	↓ expression (RNAi) in rice plants	Cold and high salinity	Increased antioxidant enzyme activities and proline concentration	Cold (4°C) 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 survival. High salinity (150 mM NaCl) treatment in 2-week-old seedlings: 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%–74%) than those of control plants (51%).	43

Drought stress

Water limitation damages agricultural crops, especially by causing protein denaturation, decrease in chlorophyll levels, and photosynthesis inhibition, resulting in several restrictions to plant development.^6,22,23 Most studies cover genes related to E3 ligase enzymes, probably because those enzymes, acting together with accessory proteins such as F-box, 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 drought.⁶ 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 opening.²³ 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 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 recover.²⁴ 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 increasing ABA synthesis, making the plants more tolerant to drought during seed germination.²⁵

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 sensitivity.^26,27 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 factors.²⁷

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 (Pyruvate-Phosphate Dikinase1), Cyclin C1-1 and subunit 4 of Cellulose Synthase A proteins, after exposure to stress, aids the adaptation to high salinity in the 3 rice lines assessed. In addition, the polyubiquitination of HSP81-1 and Aldehyde Oxidase 3 proteins showed a relation with low tolerance to high salinity.²⁸

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 OsRINGC2-2 gene is more expressed in the panicles during development.²⁹ Plants over-expressing OsRINGC2-1 submitted to different NaCl concentrations showed an increased tolerance to high salinity stress through the ubiquitination system and enhanced root length.²⁹ 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 proteins.³⁰ 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 pathway.³¹ 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 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 plants.³¹ 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 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 stability.^5,33 Plants are capable of acclimating to survive at extreme temperatures, a process which involves cell membrane protection, solute synthesis and accumulation, and specific enzymatic activities.^7,34 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 stress.³⁵ 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. 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 responses.⁷ 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 ICE1.⁷

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 ubiquitin.^36,37 SUMOylation modulates protein stability, enabling the regulation of several cell processes, such as the enzymatic activity and environmental stress responses in plants.^37,38 Several potential targets of SUMOylation were identified, including proteins involved in the regulation of low temperature responses.⁷ 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 stress.³⁹ BnTR1 is a membrane-bound RINGv (C₄HC₃) 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 tolerance.³⁹ 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-ubiquitination.⁵ OsHCI1 overexpression in Arabidopsis resulted in an increased heat shock tolerance when compared to wild-type plants, suggesting that this gene is involved in the acquired thermal tolerance in plants through the ubiquitination process.⁵ 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 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 stresses.²⁰ 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 germination.²⁰

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 integrity.³⁷

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 stresses.⁴⁰ 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 activity.⁴¹ It was found that OsPUB15 is induced by several stresses and avoid tissue damages.⁴⁰ 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 stresses.^40,42 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 responses.⁴⁰

OsSRFP1 (Stress-related RING finger protein 1), which encodes an E3 ligase, is 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 rice.⁴³ It was shown that decreased OsSRFP1 expression through RNAi makes silenced plants more tolerant to abiotic stresses than wild-type plants.⁴³ 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 osmoregulation.^43,44

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 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 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.

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.

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Felipe Klein Ricachenevsky for critical reading of the manuscript.

Funding

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).