The transcription factor TGA2 orchestrates salicylic acid signal to regulate cold-induced proline accumulation in Citrus

Plants subjected to cold stress have been observed to accumulate proline, but the underlying regulatory mechanism remains to be elucidated. In this study, we identified a pyrroline-5-carboxylate synthetase (P5CS)-encoding gene (CtrP5CS1) from trifoliate orange (Citrus trifoliata L.), a cold-hardy citrus species, as a critical gene for cold-induced proline accumulation. CtrTGA2 bound directly to the TGACG motif of the CtrP5CS1 promoter and activated its expression. Moreover, CtrTGA2 functioned positively in cold tolerance via modulation of proline synthesis by regulating CtrP5CS1 expression. Up-regulation of CtrP5CS1 and CtrTGA2 under cold stress was dependent on salicylic acid (SA) biosynthesis. CtrTGA2 directly regulated the expression of CtrICS1, a gene encoding isochorismate synthase (ICS) involved in SA biosynthesis, forming a positive feedback loop to intensify the CtrTGA2-mediated transcriptional activation of CtrP5CS1. The cold-induced SA receptor NONEXPRESSOR OF PATHOGENESIS-RELATED GENES3 (CtrNPR3) interacted with CtrTGA2 to inhibit its transcriptional activation activity; however, the inhibition was released by SA. Our results uncover the CtrTGA2-CtrP5CS1/CtrICS1 regulatory module that orchestrates the SA signal to regulate proline synthesis, giving important insights into the transcriptional mechanism underlying proline accumulation in plants under cold stress. Salicylic acid-dependent transcription factor TGA2 of Citrus trifoliata, a cold-hardy species, positively regulates P5CS1-mediated proline accumulation for enhanced cold tolerance.