Lin Chien-Yuan, Sun Yi, Song Jina, Chen Hsi-Chuan, Shi Rui, Yang Chenmin, Liu Jie, Tunlaya-Anukit Sermsawat, Liu Baoguang, Loziuk Philip L, Williams Cranos M, Muddiman David C, Lin Ying-Chung Jimmy, Sederoff Ronald R, Wang Jack P, Chiang Vincent L
Forest Biotechnology Group, Department of Forestry and Environmental Resources, North Carolina State University, Raleigh, NC, United States.
Joint BioEnergy Institute, Lawrence Berkeley National Laboratory, Berkeley, CA, United States.
Front Plant Sci. 2021 Oct 6;12:727932. doi: 10.3389/fpls.2021.727932. eCollection 2021.
Co-enzyme A (CoA) ligation of hydroxycinnamic acids by 4-coumaric acid:CoA ligase (4CL) is a critical step in the biosynthesis of monolignols. Perturbation of 4CL activity significantly impacts the lignin content of diverse plant species. In , two well-studied xylem-specific Ptr4CLs (Ptr4CL3 and Ptr4CL5) catalyze the CoA ligation of 4-coumaric acid to 4-coumaroyl-CoA and caffeic acid to caffeoyl-CoA. Subsequently, two 4-hydroxycinnamoyl-CoA:shikimic acid hydroxycinnamoyl transferases (PtrHCT1 and PtrHCT6) mediate the conversion of 4-coumaroyl-CoA to caffeoyl-CoA. Here, we show that the CoA ligation of 4-coumaric and caffeic acids is modulated by Ptr4CL/PtrHCT protein complexes. Downregulation of reduced Ptr4CL activities in the stem-differentiating xylem (SDX) of transgenic . The Ptr4CL/PtrHCT interactions were then validated using biomolecular fluorescence complementation (BiFC) and protein pull-down assays in SDX extracts. Enzyme activity assays using recombinant proteins of Ptr4CL and PtrHCT showed elevated CoA ligation activity for Ptr4CL when supplemented with PtrHCT. Numerical analyses based on an evolutionary computation of the CoA ligation activity estimated the stoichiometry of the protein complex to consist of one Ptr4CL and two PtrHCTs, which was experimentally confirmed by chemical cross-linking using SDX plant protein extracts and recombinant proteins. Based on these results, we propose that Ptr4CL/PtrHCT complexes modulate the metabolic flux of CoA ligation for monolignol biosynthesis during wood formation in .
4-香豆酸：辅酶A连接酶（4CL）将羟基肉桂酸与辅酶A（CoA）连接是单木质醇生物合成中的关键步骤。4CL活性的扰动会显著影响多种植物物种的木质素含量。在[研究对象]中，两个经过充分研究的木质部特异性Ptr4CL（Ptr4CL3和Ptr4CL5）催化4-香豆酸与4-香豆酰辅酶A的CoA连接以及咖啡酸与咖啡酰辅酶A的CoA连接。随后，两种4-羟基肉桂酰辅酶A：莽草酸羟基肉桂酰转移酶（PtrHCT1和PtrHCT6）介导4-香豆酰辅酶A向咖啡酰辅酶A的转化。在此，我们表明4-香豆酸和咖啡酸的CoA连接受到Ptr4CL/PtrHCT蛋白复合物的调节。[基因名称]下调会降低转基因[植物名称]茎分化木质部（SDX）中的Ptr4CL活性。然后在SDX提取物中使用生物分子荧光互补（BiFC）和蛋白质下拉试验验证了Ptr4CL/PtrHCT相互作用。使用Ptr4CL和PtrHCT重组蛋白进行的酶活性测定表明，添加PtrHCT时Ptr4CL的CoA连接活性升高。基于CoA连接活性进化计算的数值分析估计该蛋白复合物的化学计量比为一个Ptr4CL和两个PtrHCT，这通过使用SDX植物蛋白提取物和重组蛋白的化学交联实验得到了证实。基于这些结果，我们提出Ptr4CL/PtrHCT复合物在[植物名称]木材形成过程中调节单木质醇生物合成中CoA连接的代谢通量。
