Yujie Fan, Chunyan Mo, Chuang Wang, Jianfeng Xing, Yunlong Zhang, Nan Sun, Qingbiao Xie, Yuan Yao, Kaiye Liu, Xue Yang, Xiaodong Liu, Jun Tao, Yongjun Fang, Xiaohu Xiao, Zhendong Zhao, Xueqing Zhou, Xiaolin Tian, Haiteng Deng, Jianquan Liu, Zhenhua Ming, Jiang Wang, Chaorong Tang
Cytosolic invertases (CINVs) are essential for plant sugar homeostasis, growth and development, yet their enzymatic regulation remains unclear. Here we show that land plant CINV enzymes are tightly autoinhibited via synergistic actions of their N- and C-termini on the central catalytic GH100 domain. A conserved C-terminal hexapeptide functions as a dual switch: an intrinsic 'brake' that inhibits GH100 and an 'accelerator' that recruits 14-3-3 activators upon phosphorylation. During plant terrestrialization, this motif evolved from 'TRSXpSW' to '[K/R]RSXpSW', enhancing both autoinhibition and 14-3-3 binding. In Hevea brasiliensis, 14-3-3-dependent CINV activation is the primary source of endogenous CINV enzyme activity in rubber-producing laticifers. In Arabidopsis thaliana, replacing its hexapeptide with ancestral variant relieves autoinhibition, elevates CINV activity and promotes growth. Our study unveils an evolutionarily refined, hexapeptide-mediated mechanism that controls CINV autoinhibition with potential for biotechnological crop improvement.