Intra- and epicuticular-waxes primarily comprising of very long chain aliphatic lipid (VLCFA), terpenoids and secondary metabolites such as sterol and flavonoids played a major role in successful colonization of terrestrial ecosystem by aquatic plants and are thus considered as a key evolutionary innovation. The key rate limiting step of Fatty Acid (FA) biosynthesis of condensation/elongation are catalyzed by the enzyme, β-ketoacyl coenzyme A synthase (KCS), part of FAE (Fatty Acid Elongase) complex. KCS6 has been shown to be responsible for elongation using C22 fatty acid as substrate and is considered essential for synthesis of VLCFA for cuticular waxes. Earlier studies have established KCS5 as a close paralog of KCS6 in Arabidopsis thaliana, albeit with non-redundant function. We subsequently established segmental duplication responsible for origin of KCS6-KCS5 paralogy which is exclusive to Brassicaceae. In the present study, we aim to understand impact of duplication on regulatory diversification and evolution, through sequence and functional analysis of cis-regulatory element of KCS5 and KCS6. High level of sequence variation leading to conservation of only the proximal end of the promoter corresponding to the core promoter was observed among Brassicaceae members; such high diversity was also revealed when sliding window analysis revealed only two to three phylogenetic footprints. Profiling of transcription factor binding sites (TFBS) across Brassicaceae shows presence of light, hormone and stress responsive motifs; a few motifs involved in tissue specific expression (Skn-1; endosperm) were also detected. Functional characterization using transcriptional fusion constructs revealed regulatory diversification when promoter activity of homologs from A. thaliana and Brassica juncea were compared. When subjected to 5-Azacytidine, altered promoter activity was observed, implying role of DNA methylation in transcriptional regulation. Finally, investigation of the role of an 87 bp fragment from first intron that is retained in a splice variant, revealed it to be a transcriptional repressor. This is a first report on comparative sequence and functional analysis of transcriptional regulation of KCS5 and KCS6; further studies are required before manipulation of cuticular waxes as a strategy for mitigating stress.

主要由超长链脂肪族脂质 (VLCFA)、萜类和次生代谢物（如甾醇和类黄酮）组成的内蜡和表皮蜡在水生植物成功殖民陆地生态系统中发挥了重要作用，因此被认为是一项关键的进化创新。脂肪酸 (FA) 缩合/延伸生物合成的关键限速步骤由酶、β-酮酰基辅酶 A 合酶( KCS )催化，该酶是 FAE（脂肪酸延伸酶）复合物的一部分。KCS6已被证明负责使用 C22 脂肪酸作为底物进行伸长，并且被认为是合成表皮蜡的 VLCFA 所必需的。早期的研究已经将KCS5 确定为KCS6 的近旁系在拟南芥中，尽管具有非冗余功能。我们随后建立了负责KCS6-KCS5旁系同源的节段重复，这是十字花科独有的。在本研究中，我们旨在通过对KCS5和KCS6顺式调控元件的序列和功能分析，了解重复对调控多样化和进化的影响. 在十字花科成员中观察到导致仅与核心启动子相对应的启动子近端保守的高水平序列变异；当滑动窗口分析仅显示两到三个系统发育足迹时，也揭示了如此高的多样性。十字花科转录因子结合位点 (TFBS) 的分析表明存在光、激素和应激反应基序；还检测到一些参与组织特异性表达的基序（Skn-1；胚乳）。使用转录融合构建体的功能表征揭示了来自拟南芥和芥菜的同源物的启动子活性的调控多样化进行了比较。当受到 5-氮杂胞苷时，观察到启动子活性改变，这意味着 DNA 甲基化在转录调控中的作用。最后，对保留在剪接变体中的第一个内含子的 87 bp 片段的作用的研究表明，它是一个转录阻遏物。这是关于KCS5和KCS6转录调控的比较序列和功能分析的第一份报告；在将角质层蜡作为减轻压力的策略之前，需要进一步研究。