The biosynthesis of storage reserves and auxin is coordinated by a hierarchical regulatory network in maize endosperm [RNA-Seq]

Grain filling in maize (Zea mays L.) is intricately linked to cell development, involving the regulation of genes responsible for the biosynthesis of storage reserves (starch, proteins, and lipids) and phytohormones. However, the regulatory network coordinating these biological functions remains unclear. In this study, we identified 1,744 high-confidence target genes co-regulated by the transcription factors (TFs) ZmNAC128 and ZmNAC130 (ZmNAC128/130) through chromatin immunoprecipitation sequencing coupled with RNA-seq analysis in the zmnac128/130 loss-of-function mutants. We further constructed a hierarchical regulatory network using DNA affinity purification sequencing analysis of downstream TFs regulated by ZmNAC128/130. In addition to target genes involved in the biosynthesis of starch and zeins, we discovered novel target genes of ZmNAC128/130 involved in the biosynthesis of lipids and indole-3-acetic acid (IAA). Consistently, the number of oil bodies, as well as the contents of triacylglycerol and IAA were significantly reduced in zmnac128/130. The hierarchical regulatory network centered by ZmNAC128/130 revealed a significant overlap between the direct target genes of ZmNAC128/130 and their downstream TFs, particularly in regulating the biosynthesis of storage reserves and IAA. Our results indicated that the biosynthesis of storage reserves and IAA is coordinated by a multi-TFs hierarchical regulatory network in maize endosperm. Overall design: Identification of differentially expressed genes of zmnac128, zmnac130 and zmnac128/130 during maize endosperm

玉米（Zea mays L.）籽粒灌浆过程与细胞发育紧密相关，涉及贮藏储备物质（淀粉、蛋白质与脂质）及植物激素生物合成相关基因的调控。然而，协调这些生物学功能的调控网络仍未明确。本研究通过在zmnac128/130功能缺失突变体中开展染色质免疫共沉淀测序（chromatin immunoprecipitation sequencing）与RNA测序（RNA-seq）联合分析，鉴定出1744个由转录因子（transcription factors, TFs）ZmNAC128与ZmNAC130（ZmNAC128/130）共同调控的高可信度靶基因。我们进一步针对ZmNAC128/130调控的下游转录因子开展DNA亲和纯化测序（DNA affinity purification sequencing）分析，构建了层级调控网络。除了参与淀粉与玉米醇溶蛋白（zeins）生物合成的靶基因之外，本研究还发现了ZmNAC128/130的新型靶基因，这些基因参与脂质与吲哚-3-乙酸（indole-3-acetic acid, IAA）的生物合成。实验结果一致表明，zmnac128/130突变体中的油体数量、三酰甘油与IAA含量均显著降低。以ZmNAC128/130为核心的层级调控网络显示，ZmNAC128/130的直接靶基因与其下游转录因子存在显著重叠，尤其在调控贮藏储备物质与IAA的生物合成方面。我们的研究结果表明，玉米胚乳中贮藏储备物质与IAA的生物合成由多转录因子层级调控网络协同完成。总体实验设计：鉴定玉米胚乳发育过程中zmnac128、zmnac130及zmnac128/130的差异表达基因。