NpSCI2A, a potato inhibitor I protein from a wild sugarcane relative, negatively regulates drought tolerance by targeting subtilases

Drought severely limits sugarcane productivity. The wild relative of sugarcane, Narenga porphyrocoma , is highly drought-tolerant and represents a valuable source of stress-resistance genes. We identified NpSCI2A , encoding a s potato inhibitor I that is significantly downregulated under prolonged drought. Overexpression of NpSCI2A in rice markedly increased drought sensitivity, accompanied by severe leaf wilting, reduced antioxidant enzyme activities, and elevated hydrogen peroxide and malondialdehyde (MDA) levels, leading to reduced survival rates. Physiological analyses revealed that NpSCI2A-overexpressing plants exhibited significantly decreased chlorophyll content, maximum photochemical efficiency of PSII (Fv/Fm), and net photosynthetic rate (Pn), coupled with increased intercellular CO₂ concentration (Ci) and disrupted chloroplast ultrastructure under drought stress, indicating severe impairment of photosynthetic capacity. Transcriptome analysis revealed that NpSCI2A overexpression together with drought additively suppressed chloroplast- and photosynthesis-associated genes. Through co-expression profiling and protein–protein interaction assays, two subtilases, SBT1.2 and SBT1.8, were identified as direct physical interactors of NpSCI2A. In vitro enzyme activity assays further demonstrated that NpSCI2A directly suppressed the protease activities of SBT1.2 and SBT1.8. Notably, their transcript levels were paradoxically upregulated in NpSCI2A -overexpressing lines under drought, suggesting a compensatory transcriptional response to impaired protease activity. These findings establish NpSCI2A as a negative regulator of drought tolerance that acts by inhibiting SBT proteases, thereby disrupting chloroplast protein homeostasis and exacerbating oxidative damage. Our study uncovers a functional link between protease inhibitor-mediated proteolytic regulation and chloroplast function, offering new insights into plant drought adaptation and a potential target for the molecular breeding of stress-tolerant sugarcane.