INTRODUCTION

Sugarcane (Saccharum spp. hybrids) is a perennial grass characterised by tall fibrous stalks that accumulate high amounts of sucrose and lignocellulosic biomass for sugar and bioethanol production, respectively (Waclawovsky et al. 2010). This crop is especially sensitive to drought as a consequence of climate change due to its long cropping cycle, which spans all seasons of the year. Among abiotic stressors, water deficit is considered the most deleterious to crop yield and productivity, accounting for yield losses of up to 60% (Ferreira et al. 2017). Adequate water supply, in the form of rainfall or irrigation, is especially important during the earlier stages of growth, such as the tillering phase where the stalk population is determined, and the grand growth phase, during which exponential vegetative crop growth occurs (Inman-Bamber & Smith 2005).

Post-translational modifications (PTMs) of proteins by Small Ubiquitin-like Modifiers (SUMO) regulate plant development and stress responses. The process of SUMOylation has demonstrated distinctive signalling roles in the regulation of salt and drought stress (Conti et al. 2008; Castro 2013; Castro et al. 2016; Srivastava et al. 2016a,b; Srivastava et al. 2017; Rosa et al. 2018; Le Roux et al. 2019; Ibrahim et al. 2022). Its mode of operation resonates with that of ubiquitination, where it shares the same substrate on target proteins and employs an enzymatic cascade for activity. However, while ubiquitinated proteins are mainly signalled for degradation, SUMOylated proteins are modified, consequently influencing their activity, intracellular distribution, interactions with other proteins and longevity (Han et al. 2018; Srivastava & Sadanandom 2020).

SUMOylation is a highly dynamic and cyclic process where, during stress signal perception, an ~11 kDa SUMO protein is interchanged between its free, inactive immature form, and its active mature form through the peptidase activity of SUMO proteases (Benlloch & Lois 2018). The SUMO proteases cleave 10 amino acids (~1.1 kDa) from the free immature SUMO protein, exposing a diglycine motif on its carboxyl terminal. This renders its maturity to enter the cyclic process that oversees its activation, conjugation to the target protein, ligation to the protein and deconjugation from the protein into a new cycle (Morrell & Sadanandom 2019). Under prolonged stress conditions, SUMO proteins are mainly deconjugated from target substrates to enable them to enter another cycle or, less likely, to be restored to their free, immature state (Park & Yun 2013). For deSUMOylation, SUMO isopeptidases catalyse the cleavage of the isopeptide bond connecting SUMO proteins to their target proteins (Colby et al. 2006; Yates et al. 2016; Benlloch & Lois 2018).

SUMO proteases are characterised as cysteine proteases as they possess a cysteine thiol in their catalytic site for a nucleophilic attack that breaks thioester bonds between SUMO proteins and target proteins and/or within the SUMO proteins (Hickey et al. 2012). The ubiquitin-like protein-specific protease 1 (ULP1; ~28 kDa) isolated from yeast was the first identified SUMO protease (Li & Hochstrasser 1999), while ESD4 (Early in Short Days 4) from Arabidopsis represented the first SUMO protease characterised from plants (Reeves et al. 2002; Murtas et al. 2003). Through sequence similarity, Kurepa et al. (2003) identified the ELS (ESD4-like SUMO protease) 1 and 2, as well as the OTS (Overly Tolerant to Salt) 1 and 2 SUMO proteases. The ESD4 and OTS1/2 proteases have been reported to display both peptidase (for maturity) and isopeptidase (for recycling) activity (Colby et al. 2006; Conti et al. 2008). Of these two, the OTS enzymes have been implicated as playing key roles in the regulation of salt and drought stress in crop species.

The simultaneous knockout of both proteases [depicted ots1-1 (for OTS1 knockout mutant), ots2-1 (for OTS2 knockout mutant)] was initially characterised as conferring hypersensitivity to salt in Arabidopsis (Conti et al. 2008). In terms of drought, Castro et al. (2016) functionally characterised these proteases through loss-of-function reverse genetics in Arabidopsis, where the double mutants displayed deregulation of genes controlled by various drought-associated transcriptional regulators, resulting in morphologically diminished root growth, higher stomatal conductivity, and no overall enhanced drought tolerance. In addition, Castro et al. (2016) introduced a knocked-out SUMO ligase (E3) SIZ1 gene to the OTS double mutant. From this, they demonstrated that the OTS1 and 2 SUMO proteases (in combination) operate primarily as isopeptidases in the SUMOylation process controlling water deficit responses.

The characteristic of salt stress hypersensitivity was also reported in rice under the overexpression or silencing of the OTS1 gene (Srivastava et al. 2016a). The OsOTS1-RNAi rice lines displayed high levels of SUMO-conjugates, which rendered them hypersensitive to salt stress and displayed no distinguishable characteristics compared to the wild type (WT) under normal conditions. Reversibly, the overexpression of OsOTS1 in rice displayed enhanced salt tolerance, with reduced SUMO conjugates during stress periods (Srivastava et al. 2016b). Interestingly, the OsOTS1-RNAi transgenic rice, which display hypersensitivity to salt stress, contrastingly displayed enhanced drought tolerance (Srivastava et al. 2017). The increased drought tolerance in these plants was linked to the OsbZIP transcription factor, where the absence of the OsOTS1 maintained the attachment of SUMO proteins to the transcription factor, and thus increased its stability, which led to the transcription of drought-responsive genes. Furthermore, Le Roux et al. (2019) demonstrated that wheat overexpressing the AtOTS1 gene displayed improved growth under water deficit stress. In the wheat plants, SUMOylation of total protein increased in untransformed plants but not in the AtOTS1 transformed plants, where it was suggested that SUMO-proteases may influence an array of mechanisms to ensure enhanced tolerance to water deficit stress. Unlike the OTS1, which has been shown to independently impact the response to drought stress, (Castro 2013) demonstrated that Atots2-1, in OTS2 knockout lines, displayed similar results as the WT in response to drought and/or salt stress in Arabidopsis.

So far limited research has been conducted on the SUMOylation system in plants, particularly in crop species. However, available research has shown the importance of SUMO proteases in the regulation of SUMOylation, particularly during plants' drought stress responses. This study serves as the first report on the effect of overexpressed cysteine protease OVERLY TOLERANT TO SALT-1 from A. thaliana (AtOTS1) in the polyploid crop sugarcane during drought stress. Transgenic plants were analysed in terms of plant growth, physiological and biochemical variables, and changes in SUMO-enriched protein expression, after exposure to water deficit stress when compared with untransformed WT plants.