MYC2‐SUMO protease feedback loops boost salt tolerance in wheat

Salt tolerance enables plants to withstand the toxicity of high concentrations of soluble salts, particularly NaCl. Increasing soil salinity slows plant growth and ultimately affects productivity with varying levels of impact depending on the plant species, duration of exposure, and stage of development. Therefore, engineering plant salt tolerance, defined as maintaining superior growth performance under high soil salinity, would be a valuable trait in response to the global salinisation of arable land (Munns et al., 2020). In a study recently published in New Phytologist, Xiao et al. (2024; doi: 10.1111/nph.20171) developed stable salt-tolerant wheat lines (Triticum aestivum L.) by overexpressing the small ubiquitin-like modifier (SUMO) protease-encoding gene TaDSU. '… transgenic lines had more spikes per plant than the untransformed background, with significant increases in yield under salt stress condition.' The ability to quickly reprogram protein functions in response to salt stress becomes crucial to activate cellular protection mechanisms. The covalent attachment of SUMO on target proteins is a well-recognised post-translational modification that can change the localisation, stability and activity of target proteins. In brief, SUMO is ligated by a specialised set of enzymes to lysine residues usually embedded within a canonical consensus of amino acids (Benlloch & Lois, 2018). After SUMO conjugation, SUMO deconjugating enzymes (belonging to different SUMO protease families, each encoded by multiple genes in plants) quickly remove SUMO from their targets to maintain a dynamic equilibrium between SUMOylated and nonSUMOylated target levels (Ghosh et al., 2024). The study by Xiao et al. contributes to the expanding list of abiotic stress responses mediated by SUMO proteases by highlighting the role of TaDSU. The authors observed increased shoot and root growth in wheat lines overexpressing TaDSU (TaDSU OX) compared with the wild-type (WT), specifically under saline soil conditions. High salt concentrations in the soil also cause osmotic stress, which reduces water uptake and impairs growth. Supporting the role of TaDSU in protecting plant cells from the osmotic aspect of salt stress, TaDSU OX lines showed reduced growth inhibition when cultivated in a medium containing mannitol, a nonmetabolisable sugar that induces osmotic stress. Metabolic and cation profiling of plants subject to salt stress revealed that TaDSU OX lines had reduced levels of Na+ content in the shoot (accompanied by increased K+), increased contents of soluble sugar and proline, and reduced ROS accumulation, which can be regarded as metabolic hallmarks for augmented salt tolerance. TaDSU, a homologue of OVERLY TOLERANT TO SALT1/2 (OTS1/2) from Arabidopsis and OsOTS1 from rice (Conti et al., 2008; Srivastava et al., 2016), has SUMO protease activity in vitro. Its overexpression in Arabidopsis leads to a global reduction in the SUMOylated proteome under salt stress, indicating deSUMOylation activity also in vivo. However, an important question raised by Xiao et al. concerns the specific target(s) of TaDSU-mediated deSUMOylation, which are responsible for the increased salt tolerance. A yeast two-hybrid screen followed by independent pairwise interaction assays identified the transcription factor TaMYC2 as a bona fide substrate for TaDSU SUMO protease activity. This was shown in transient assays and by using stable Arabidopsis lines, since Arabidopsis MYC2 also appears to interact with TaDSU and TaDSU overexpression confers increased salt tolerance to Arabidopsis. This suggests a conserved mechanism of salt tolerance mediated by TaDSU through MYC2 deSUMOylation. Exposure of WT Arabidopsis seedlings to high salt conditions resulted in increased levels of MYC2 accumulation accompanied by a corresponding increase of MYC2 SUMOylation. Conversely, when TaDSU is overexpressed in Arabidopsis, the MYC2 SUMOylated pool was reduced specifically under salt conditions, with no corresponding reduction in the nonSUMOylated pool of MYC2 relative to the WT. These data support a correlation between the reduced accumulation of SUMOylated MYC2 and the acquisition of salt tolerance traits. TaDSU OX lines (in wheat and Arabidopsis) also displayed increased levels of MYC2 transcripts but limited to salinity conditions. Supporting the role of TaMYC2 in conferring salt tolerance downstream of TaDSU, virus-induced gene-silencing (VIGS) of TaMYC2 significantly weakened the salt-tolerant phenotype of TaDSU OX lines in wheat. Therefore, the enhanced salt tolerance conferred by TaDSU overexpression can be attributed to an increase in TaMYC2 transcript accumulation and a reduced SUMOylation level of TaMYC2. The study of Xiao et al. also describes a direct transcriptional regulation of TaDSU mediated by MYC2. TaDSU promoter contains multiple MYC2 binding motifs and TaMYC2 can directly bind to these regions to activate TaDSU. The TaMYC2-dependent regulation of TaDSU expression was further investigated in wheat plants in which TaMYC2 was knocked down through VIGS. As expected, VIGS-treated plants had reduced levels of TaMYC2 and correspondingly decreased levels of TaDSU transcript accumulation, specifically under salt stress conditions. Interestingly, a similar MYC2-OTS1/2 direct regulation is conserved in Arabidopsis because OTS1/2 transcripts were upregulated in MYC2 overexpression lines. Additionally, a chromatin immunoprecipitation assay supports the direct binding of MYC2 to the OTS1/2 promoters. Therefore, under salt stress conditions, TaMYC2 accumulation feeds back into the transcriptional activation of TaDSU, which in turn promotes deSUMOylation of TaMYC2 (Fig. 1). Gains in stress tolerance measured under controlled environments are often less clear under field trials due to the higher complexity of natural stressors. Xiao et al. verified the performance of TaDSU wheat overexpression lines under saline soils through multiyear experiments. Overall, transgenic lines had more spikes per plant than the untransformed background, with significant increases in yield under salt stress conditions. TaDSU OX lines also performed better in another location characterised by saline–alkaline soil, whereas no yield penalty was observed in the presence of low salt. The study by Xiao et al. thus provides a path to an effective genetic strategy for wheat improvement under an agriculturally relevant scenario. It also raises intriguing questions across three interconnected areas, ranging from broader biological and physiological aspects of hormone signalling to more detailed molecular insights. First, it highlights the utility of SUMO modifications and, more generally, post-translational modification to modify key traits in crops. It also reveals important connections between TaDSU-MYC2 and other hormonal gene networks, mainly abscisic acid (ABA), since TaDSU OX lines had reduced ABA sensitivity. ABA and other phytohormones are known to play important roles in regulating ionic homeostasis and plant growth under salt stress conditions (Achard et al., 2006). Second, the relative paucity of the SUMO conjugation; encoding genes suggests a major role for SUMO deconjugating enzymes in providing specificity to adaptive responses in plants (Ghosh et al., 2024). TaDSU, like OTS1/2, falls in a subclade of cysteine proteases, which evolved mostly in angiosperms, perhaps in conjunction with the expansion and adaptation of flowering plants to an increasingly challenging environment or the evolution of more complex plant structures and functions. However, questions remain about how these proteases are post-translationally activated, particularly in response to salt stress. Third, SUMOylation is a fast and dynamic mode of influencing protein function. Here, the discovery of the TaDSU-TaMYC2 regulation opens new questions regarding the direct role of SUMOylation on MYC2 function. The recruitment of MYC2 to chromatin appears to be reduced in TaDSU OX lines. How does SUMOylation regulate MYC2 DNA binding? Does SUMOylation also influence MYC2-dependent transcriptional activation at its regulated promoters? Future studies will be required to better understand the TaDSU-TaMYC2 interplay and the broader role of SUMOylation in plant stress responses. Nevertheless, from an agriculture perspective, insights from this study, combined with advancements in detecting SUMOylation targets in vivo (Sang et al., 2024), enhance our ability to engineer SUMOylation for boosting salinity tolerance in crop species. Lucio Conti's Lab has been supported by a grant from the Italian Ministerial of Research PRIN 2022 'Light and drought signals integration driving development transitions and adaptations in plants – LIDS' Ref: 2022T2737Y. Giorgio Perrella is funded by the University Research Grants PSR2022 and My First Seed PSRL324.