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Abstract
背景+问题：Zeaxanthin epoxidase (ZEP) is a key enzyme that catalyzes the conversion of zeaxanthin to violaxanthin in the carotenoid and abscisic acid (ABA) biosynthesis pathways. The rapeseed (Brassica napus) genome has 4 ZEP (BnaZEP) copies that are suspected to have undergone subfunctionalization, yet the 4 genes’ underlying regulatory mechanisms remain unknown.
结果1-BnaZEPs功能分化：Here, we genetically confirmed the functional divergence of the gene pairs BnaA09.ZEP/BnaC09.ZEP and BnaA07.ZEP/BnaC07.ZEP, which encode enzymes with tissue-specific roles in carotenoid and ABA biosynthesis in flowers and leaves, respectively.
结果2-调控机制：Molecular and transgenic experiments demonstrated that each BnaZEP pair is transcriptionally regulated via ABA-responsive element–binding factor 3 s (BnaABF3s) and BnaMYB44s as common and specific regulators, respectively.
结果2-ABF3s通用型调控：BnaABF3s directly bound to the promoters of all 4 BnaZEPs and activated their transcription, with overexpression of individual BnaABF3s inducing BnaZEP expression and ABA accumulation under drought stress. Conversely, loss of BnaABF3s function resulted in lower expression of several genes functioning in carotenoid and ABA metabolism and compromised drought tolerance.
结果3-MYB44特异性调控：BnaMYB44s specifically targeted and repressed the expression of BnaA09.ZEP/BnaC09.ZEP but not BnaA07.ZEP/BnaC07.ZEP. Overexpression of BnaA07.MYB44 resulted in increased carotenoid content and an altered carotenoid profile in petals. Additionally, RNA-seq analysis indicated that BnaMYB44s functions as a repressor in phenylpropanoid and flavonoid biosynthesis.
结论：These findings provide clear evidence for the subfunctionalization of duplicated genes and contribute to our understanding of the complex regulatory network involved in carotenoid and ABA biosynthesis in B. napus.
摘 要
玉米黄质环氧化酶ZEP是类胡萝卜素和脱落酸ABA生物合成途径中催化玉米黄质转变为紫黄质的关键酶。油菜基因组编码4个BnaZEP基因拷贝，被认为经历了亚功能化，但其潜在的调控机制仍不清楚。本文中，作者通过遗传学方法确定了BnaA09.ZEP/BnaC09.ZEP在花中发挥特异性功能，而BnaA07.ZEP/BnaC07.ZEP在叶中发挥特异性功能。进一步的分子和转基因试验显示，ABA响应元件结合因子BnaABF3s对4个BnaZEP基因均能发挥转录调控作用，而BnaMYB44s存在特异性的转录调控。其中，BnaABF3s能够直接集合到4个BnaZEP基因的启动子上，激活这些基因的转录，而单个BnaABF3基因的过表达均会诱导干旱条件下BnaZEP的表达以及ABA的积累。相反，BnaABF3s功能缺失会导致类胡萝卜素和ABA代谢途径中相关基因的表达量降低，并且降低干旱耐受性。BnaMYB44s能够特异性靶向并抑制BnaA09.ZEP/BnaC09.ZEP的表达，但对BnaA07.ZEP/BnaC07.ZEP的表达并无影响。BnaA07.MYB44的过表达会导致类胡萝卜素含量的增加，并且导致花瓣中的类胡萝卜素谱发生变化。另外，RNA-seq分析显示BnaMYB44s作为苯丙酸和类黄酮生物合成的抑制子发挥功能。本文的研究揭示了重复基因的亚功能化，并且有助于加深我们对于油菜类胡萝卜素和ABA生物合成的复杂调控网络的理解。
华中农业大学文静副教授为本文通讯作者，叶沈华博士为本文第一作者。该研究得到了国家科技创新2030-重大专项（2022ZD04010）、国家现代农业产业技术体系（CARS-12）和中央高校基本科研业务费专项资金（2662023PY004）的联合资助。
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