The naked endosperm1 (nkd1), naked endosperm2 (nkd2), and thick aleurone1 (thk1) genes are important regulators of maize (Zea mays L.) endosperm development. Double mutants of nkd1 and nkd2 (nkd1,2) show multiple aleurone (AL) cell layers with disrupted AL cell differentiation, whereas mutants of thk1 cause multiple cell layers of fully differentiated AL cells. Here, we conducted a comparative analysis of nkd1,2 and thk1 mutant endosperm transcriptomes to study how these factors regulate gene networks to control AL layer specification and cell differentiation. Weighted gene coexpression network analysis was incorporated with published laser capture microdissected transcriptome datasets to identify a coexpression module associated with AL development. In this module, both Nkd1,2+ and Thk1+ appear to regulate cell cycle and division, whereas Nkd1,2+, but not Thk1+, regulate auxin signaling. Further investigation of nkd1,2 differentially expressed genes combined with published putative targets of auxin response factors (ARFs) identified 61 AL-preferential genes that may be directly activated by NKD-modulated ARFs. All 61 genes were upregulated in nkd1,2 mutant and the enriched Gene Ontology terms suggested that they are associated with hormone crosstalk, lipid metabolism, and developmental growth. Expression of a transgenic DR5–red fluorescent protein auxin reporter was significantly higher in nkd1,2 mutant endosperm than in wild type, supporting the prediction that Nkd1,2+ negatively regulate auxin signaling in developing AL. Overall, these results suggest that Nkd1,2+ and Thk1+ may normally restrict AL development to a single cell layer by limiting cell division, and that Nkd1,2+ restrict auxin signaling in the AL to maintain normal cell patterning and differentiation processes.

中文翻译：

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比较转录组学和网络分析定义了控制玉米糊粉发育和生长素信号传导的基因共表达模块

裸胚乳1 ( nkd1 )、裸胚乳2 ( nkd2 )和厚糊粉层1 ( thk1 )基因是玉米( Zea mays L.)胚乳发育的重要调控因子。nkd1和nkd2 ( nkd1,2 )的双突变体显示多个糊粉层 (AL) 细胞层破坏了 AL 细胞分化，而thk1突变体导致完全分化的 AL 细胞的多个细胞层。在这里，我们对nkd1,2和thk1进行了对比分析突变胚乳转录组研究这些因子如何调节基因网络以控制 AL 层规范和细胞分化。加权基因共表达网络分析与已发表的激光捕获显微切割转录组数据集相结合，以识别与 AL 发展相关的共表达模块。在该模块中，Nkd1,2+和Thk1+似乎都调节细胞周期和分裂，而Nkd1,2+而非Thk1+调节生长素信号传导。进一步调查nkd1,2差异表达基因与已发表的生长素反应因子 (ARF) 的推定靶点相结合，确定了 61 个可能被 NKD 调节的 ARF 直接激活的 AL 优先基因。所有 61 个基因在nkd1,2突变体中上调，并且丰富的基因本体术语表明它们与激素串扰、脂质代谢和发育生长有关。nkd1,2突变胚乳中转基因 DR5-红色荧光蛋白生长素报告基因的表达显着高于野生型，这支持了Nkd1,2+在发育中的 AL 中负调节生长素信号传导的预测。总的来说，这些结果表明Nkd1,2+和Thk1+通常可以通过限制细胞分裂将 AL 发育限制在单个细胞层，并且Nkd1,2+限制 AL 中的生长素信号传导以维持正常的细胞模式和分化过程。