Table_2_Tackling functional redundancy of Arabidopsis fatty acid elongase complexes.xlsx

Very-long-chain fatty acids (VLCFA) are precursors for various lipids playing important physiological and structural roles in plants. Throughout plant tissues, VLCFA are present in multiple lipid classes essential for membrane homeostasis, and also stored in triacylglycerols. VLCFA and their derivatives are also highly abundant in lipid barriers, such as cuticular waxes in aerial epidermal cells and suberin monomers in roots. VLCFA are produced by the fatty acid elongase (FAE), which is an integral endoplasmic reticulum membrane multi-enzymatic complex consisting of four core enzymes. The 3-ketoacyl-CoA synthase (KCS) catalyzes the first reaction of the elongation and determines the chain-length substrate specificity of each elongation cycle, whereas the other three enzymes have broad substrate specificities and are shared by all FAE complexes. Consistent with the co-existence of multiple FAE complexes, performing sequential and/or parallel reactions to produce the broad chain-length-range of VLCFA found in plants, twenty-one KCS genes have been identified in the genome of Arabidopsis thaliana. Using CRISPR-Cas9 technology, we established an expression platform to reconstitute the different Arabidopsis FAE complexes in yeast. The VLCFA produced in these yeast strains were analyzed in detail to characterize the substrate specificity of all KCS candidates. Additionally, Arabidopsis candidate proteins were transiently expressed in Nicotiana benthamiana leaves to explore their activity and localization in planta. This work sheds light on the genetic and biochemical redundancy of fatty acid elongation in plants.

超长链脂肪酸（Very-long-chain fatty acids, VLCFA）是多种脂质的前体，在植物中发挥重要的生理与结构功能。VLCFA广泛分布于植物各组织，不仅存在于维持膜稳态所必需的多种脂质类别中，还储存在三酰甘油内。此外，VLCFA及其衍生物在脂质屏障中含量极高，例如地上表皮细胞的表皮蜡质以及根部的木栓质单体。VLCFA由脂肪酸延长酶（fatty acid elongase, FAE）合成，该酶是定位于内质网膜的整合型多酶复合体系，包含4个核心亚基。3-酮脂酰-CoA合酶（3-ketoacyl-CoA synthase, KCS）催化延长反应的第一步，并决定每个延长循环的底物链长特异性；而其余三种酶的底物特异性宽泛，为所有FAE复合体系所共有。鉴于存在多种可通过连续或并行反应合成植物中各类链长VLCFA的FAE复合体系，研究人员已在拟南芥基因组中鉴定出21个KCS基因。本研究借助CRISPR-Cas9技术构建了表达平台，可在酵母中重构不同的拟南芥FAE复合体系。随后对这些酵母菌株中合成的VLCFA进行详细分析，以表征所有候选KCS的底物特异性。此外，本研究将拟南芥候选蛋白在本氏烟草（Nicotiana benthamiana）叶片中进行瞬时表达，以探究其在植物体内的活性与定位。本研究阐明了植物脂肪酸延长过程的遗传与生化冗余性。