Tingchao Yin, Z K Zhang, Huanhuan Hao, Yanrong Yang, L Wang, Qi ZHOU, Aying Zhang, Yingjun Chi, Jie Zhang, Bin Xu
Leaf senescence and heat tolerance are critical determinants of productivity and quality in perennial ryegrass (Lolium perenne). Although the chlorophyll (Chl) degradation gene LpNOL negatively regulates both processes, its upstream regulatory mechanism remains elusive. In this study, a bHLH transcription factor LpCbDR2 was identified as a direct activator of LpNOL through yeast one-hybrid screening. LpCbDR2 binds the G-box cis-element within the proximal LpNOL promoter, as demonstrated by EMSA, in planta transactivation, and CUT&Tag-qPCR assays. Expression analysis reveals that LpCbDR2 transcript levels positively correlate with LpNOL expression during leaf development and in response to heat stress. Functional characterization using overexpression and RNAi transgenic lines demonstrates that LpCbDR2 negatively regulates both heat tolerance and leaf senescence. Overexpression lines show severer degradation of photosystem complex proteins, increased reactive oxygen species (ROS) accumulation, reduced antioxidant enzyme activities, and lower survival rates under heat stress, whereas RNAi lines exhibit opposite phenotypes. Epistatic analysis by suppressing both LpCbDR2 and LpNOL confirmed that LpCbDR2 acts upstream of LpNOL. Importantly, RNAi-LpCbDR2 lines accumulate 35.2-54.4% higher crude protein contents without yield penalty. These findings establish LpCbDR2 as a key regulator of LpNOL that links heat tolerance and senescence pathways, making it a potential target for enhancing stress tolerance and feedstock quality in perennial ryegrass and other forage species.