The Rice Cysteine Protease OsEP3A Promotes Seedling Growth and Seed Development and Contains a Nitrogen Starvation Responsive Sequence

Nitrogen (N) is a critical macronutrient that influences plant growth, development, and productivity. This study characterizes the rice cysteine protease gene OsEP3A and its promoter to elucidate its role in N-mediated developmental and transcriptional regulation. Transgenic rice lines overexpressing ( OsEP3A-Ox ) or silenced ( OsEP3A-Ri ) for OsEP3A were generated to assess its physiological functions. Overexpression of OsEP3A significantly enhanced shoot and root growth, whereas RNAi -silenced plants exhibited reduced height, shorter roots, and smaller seeds compared to wild type, indicating that OsEP3A positively regulates seedling and seed development. Expression analyses revealed that OsEP3A transcription was strongly induced under N deficient conditions and repressed by both inorganic (NH₄NO₃) and organic (glutamine, asparagine) N sources. Under N-limited hydroponic culture, OsEP3A-RNAi seedlings showed severely impaired growth, underscoring the gene’s essential role in internal N remobilization during deficiency. Promoter-reporter analyses using OsEP3A::GUS lines demonstrated strong activation of the OsEP3A promoter under N starvation and repression upon N resupply, suggesting N dependent transcriptional control. Deletion and insertion analyses of the OsEP3A promoter identified a 43 bp N starvation responsive sequence ( NSRS ; -278 to -236 bp) as necessary and sufficient for starvation-induced transcriptional activation. This NSRS represents a novel cis-regulatory element responsive to N deprivation. Overall, OsEP3A acts as a N starvation-activated cysteine protease that facilitates N recycling and seedling vigor, providing new insight into N responsive regulatory mechanisms in rice and offering a potential molecular target for improving N use efficiency in cereal crops.