Rice OVERLY TOLERANT TO SALT 1 (OTS1) SUMO protease is a positive regulator of seed germination and root development

ABSTRACT

Salinity is one of the major environmental stresses affecting rice production worldwide. Improving rice salt tolerance is a critical step for sustainable food production. Posttranslational modifications of proteins greatly expand proteome diversity, increase functionality and allow quick responses to environmental stresses, all at low cost to the cell. SUMO mediated modification of substrate proteins is a highly dynamic process governed by the balance of activities of SUMO E3 ligases and deconjugating SUMO proteases. In recent years, SUMO (Small Ubiquitin like Modifier) conjugation of proteins has emerged as an influential regulator of stress signaling in the model plant Arabidopsis. However SUMOylation remain largely under studied in crop plants. We recently identified the SUMO protease gene family in rice and demonstrated a role for OsOTS1 SUMO proteases in salt stress. Interestingly, rice plants silencing OsOTS1 also show significantly reduced germination rate. Knockdown of OsOTS1 gene expression affects root growth by primarily reducing cell size rather than cell division.

SUMO is a small polypeptide of approximately 100-115 amino acids first identified in tomato plants.¹ SUMOylation is the process of attaching SUMO to target substrates. It may act as a quick response mechanism to modify the behavior of substrate proteins under stress and is proving to be a major post-translational regulator in plants and other eukaryotic organisms.^2-6 Like ubiquitination SUMO conjugation in plants is facilitated by the sequential activity of 3 enzymes (E1, E2 and E3). In Arabidopsis, the E1 SUMO-Activating Enzymes AtSAE1 and AtSAE2, act as a heterodimer, responsible for adenylation mediated ATP-dependent thiol-ester bond formation between SAE2 and SUMO. Transesterification results in the transfer of SUMO to the E2 SUMO-Conjugating Enzyme, AtSCE1. AtSCE1 finally catalyzes SUMO isopeptide bond formation to target proteins, in conjunction with E3 SUMO ligases HIGH PLOIDY 2 AtHPY2/AtMMS21, or SAP and MIZ1 AtSIZ1.^2-6 SUMOylation is a dynamic process that is altered during biotic or abiotic stresses and can change the stability of proteins or interfere in protein-protein interactions therefore alter target protein functionality.⁷

In addition to Arabidopsis, SUMO gene families have been identified in crop plants such as rice, maize, wheat and sorghum.^8-10 SUMO proteases act by reversing the SUMOylation process resulting in the removal of SUMO from its target.¹¹ Generally, proteases that are responsible for the removal of SUMO are said to do so by a process called deSUMOylation, as opposed to SUMO proteases that cause SUMO maturation. DeSUMOylating proteases cleave precisely between the terminal Glycine of SUMO that is conjugated to the substrate, releasing free SUMO from the target protein ready for further conjugation cycles. These proteases play critical roles in maintaining the equilibrium in SUMO signaling.¹² SUMO proteases remain largely understudied especially in crop plants. Recent investigations into the activities of SUMO proteases in plants have begun to contribute toward our understanding of the role of SUMO in plant stress. The seven identified SUMO specific proteases in Arabidopsis are EARLY IN SHORT DAYS 4 (ESD4), ULP1a/ESD4 LIKE SUMO PROTEASE (ELS1), ULP1b, ULP1c/OVERLY TOLERANT TO SALT 2 (OTS2), ULP1d/OTS1 ULP2a and ULP2b.^13-16

Recently, we identified the SUMO protease gene family in rice and demonstrated that one of its members; OsOTS1 has a crucial role in salinity tolerance.¹⁷ Like AtOTS1, OsOTS1 is localized in nucleus, and RNAi lines of OsOTS1 show severe sensitivity to salt. Salt stress induces a dose dependant accumulation of SUMO conjugated proteins in rice. OsOTS1-RNAi lines, besides showing less chlorophyll content in adult plants, were also less able to activate antioxidant systems during high salinity. Our data showed that OTS family of SUMO proteases had a significant role in growth and development of rice in high salinity.

Seed germination is a complex developmental process involving various physical and biochemical cues such as water, light and phytohormones.^18-19 It has been reported that seed germination is sensitive to different abiotic stresses including salinity. In comparison with the vector only control, seed germination of OsOTS-RNAi lines were inhibited significantly by 150mM NaCl salt stress treatment (Fig. 1). Under optimal conditions, over 99% of seeds from vector control plants, OsOTS1-OX and OxOTS-RNAi seeds germinated after 8 d. When exposed to 150mM NaCl, the percentage of germinating seeds from OsOTS-RNAi lines was significantly lower. The average germination rate in OsOTS-RNAi transgenic lines was about 50% less than the vector only control and OsOTS1-OX plants (Fig. 1). The decrease in germination rate particularly in salt stress conditions may be due to the fact that seeds seemingly develop an osmotically enforced dormancy under water limiting conditions.²⁰ It is postulated that this may be due to the combined effect of high osmotic potential and specific ion toxicity.²¹ High accumulation of Na⁺ in saline soils causes lowering of water potential and thus makes the seeds unable to imbibe water necessary for germination from dryer soils.²²

Figure 1.

Effect of salt strees on seed germination of WT (Nilpponbare) with empty vector, OsOTS1-OX and OsOTS-RNAi line in MS plates with 150nM of NaCl, Percentage of seed germination was calculated different days interval.

Salinity caused a significant reduction in root length of OsOTS1-RNAi transgenic plants compared to the control carrying the empty vector.¹⁷ We then focused on the cellular mechanism underlying the salt induced reduction of root lengths in OsOTS-RNAi transgenic plants. To determine whether the reduced growth effects observed in OsOTS-RNAi roots was due to a reduction in either cell number or cell size or both we prepared longitudinal sections of roots from vector only, OsOTS1-OX and OsOTS-RNAi plants and examined them using light microscopy. The cell size in the elongation zone of OsOTS1-RNAi transgenic roots was significantly smaller than those of vector only and OsOTS1-OX plants. (Figs. 2A-B). The average cell length in the elongation zone in OsOTS1-RNAi transgenic lines was 40% less than the vector control (t-test, P < 0.05). OsOTS1 overexpression resulted in an increased expansion of the root elongation zone cell size (36% increase compared to vector only control, t-test P < 0.01). In conclusion, our discoveries reveal a regulatory mechanism underlying rice root development, which acts through SUMO proteases. Thus our work provides a foundation for further understanding the role of SUMO proteases in growth and development of the crop plants such as rice.

Figure 2.

SUMO protease OsOTS1 overexpression increase the cell size of the root elongation zone but not the cell number in presence of 150 mM of NaCl salt. (A) cell size of the vecot only plant, Os-OTS1-OX plants and OsOTS-RNAo plants and (B) cell number int respective transgenics lines with the vector only control. Error bars indicate SD. P values for differences between transgenic lines compared to empty cevot only. *P < 0.05 and **P < 0.01 respecticely.(Two way Anova Test).

Disclosure of potential conflicts of interest

No potential conflicts of interest were disclosed.

Funding

This work was supported by European Research Council (ERC) grant to AS.