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. 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. 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. 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. 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. 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.

中文翻译：

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BnaABF3 和 BnaMYB44 调节类胡萝卜素和脱落酸生物合成中玉米黄质环氧酶基因的转录

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化为紫黄质的关键酶。油菜籽 (Brassica napus) 基因组有 4 个 ZEP (BnaZEP) 拷贝，疑似经历了亚功能化，但这 4 个基因的潜在调控机制仍然未知。在这里，我们从遗传学上证实了基因对 BnaA09.ZEP/BnaC09.ZEP 和 BnaA07.ZEP/BnaC07.ZEP 的功能差异，它们分别编码在花和叶的类胡萝卜素和 ABA 生物合成中具有组织特异性作用的酶。分子和转基因实验表明，每个 BnaZEP 对分别通过 ABA 响应元件结合因子 3 (BnaABF3) 和 BnaMYB44 作为常见和特异性调节因子进行转录调节。 BnaABF3 直接与所有 4 个 BnaZEP 的启动子结合并激活它们的转录，在干旱胁迫下，单个 BnaABF3 的过度表达诱导 BnaZEP 表达和 ABA 积累。相反，BnaABF3s 功能的丧失导致类胡萝卜素和 ABA 代谢中起作用的几个基因的表达降低，并损害耐旱性。 BnaMYB44 特异性靶向并抑制 BnaA09.ZEP/BnaC09.ZEP 的表达，但不抑制 BnaA07.ZEP/BnaC07.ZEP 的表达。 BnaA07.MYB44 的过度表达导致花瓣中类胡萝卜素含量增加和类胡萝卜素谱改变。此外，RNA-seq 分析表明 BnaMYB44s 在苯丙素和类黄酮生物合成中起到阻遏物的作用。这些发现为重复基因的亚功能化提供了明确的证据，并有助于我们了解甘蓝型油菜中类胡萝卜素和 ABA 生物合成所涉及的复杂调控网络。