Introduction

ATP Binding Cassette G (ABCG) transporters are associated with plant male reproduction, while their regulatory mechanisms underlying anther and pollen development remain largely unknown.

Objectives

Identify and characterize a male-sterility gene ZmMs13 encoding an ABCG transporter in modulating anther and pollen development in maize.

Methods

Phenotypic, cytological observations, and histochemistry staining were performed to characterize the ms13-6060 mutant. Map-based cloning and CRISPR/Cas9 gene editing were used to identify ZmMs13 gene. RNA-seq data and qPCR analyses, phylogenetic and microsynteny analyses, transient dual-luciferase reporter and EMSA assays, subcellular localization, and ATPase activity and lipidomic analyses were carried out to determine the regulatory mechanisms of ZmMs13 gene.

Results

Maize ms13-6060 mutant displays complete male sterility with delayed callose degradation, premature tapetal programmed cell death (PCD), and defective pollen exine and anther cuticle formation. ZmMs13 encodes a plasm membrane (PM)- and endoplasmic reticulum (ER)-localized half-size ABCG transporter (ZmABCG2a). The allele of ZmMs13 in ms13-6060 mutant has one amino acid (I311) deletion due to a 3-bp deletion in its fourth exon. The I311 and other conserved amino acid K99 are essential for the ATPase and lipid binding activities of ZmMS13. ZmMs13 is specifically expressed in anthers with three peaks at stages S5, S8b, and S10, which are successively regulated by transcription factors ZmbHLH122, ZmMYB84, and ZmMYB33-1/-2 at these three stages. The triphasic regulation of ZmMs13 is sequentially required for callose dissolution, tapetal PCD and pollen exine development, and anther cuticle formation, corresponding to transcription alterations of callose-, ROS-, PCD-, sporopollenin-, and anther cuticle-related genes in ms13-6060 anthers.

Conclusion

ms13-6060 mutation with one key amino acid (I311) deletion greatly reduces ZmMS13 ATPase and lipid binding activities and displays multiple effects during maize male reproduction. Our findings provide new insights into molecular mechanisms of ABCG transporters controlling anther and pollen development and male fertility in plants.

中文翻译：

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玉米中胼胝质溶解、花粉外壁和花药角质层的形成依次需要编码 ABCG 转运蛋白的 ZmMs13 的三相调节

介绍

ATP 结合盒 G (ABCG) 转运蛋白与植物雄性繁殖相关，但其花药和花粉发育的调控机制仍然很大程度上未知。

目标

鉴定并表征编码 ABCG 转运蛋白的雄性不育基因ZmMs13，该基因可调节玉米花药和花粉发育。

方法

通过表型、细胞学观察和组织化学染色来表征ms13-6060突变体。基于图位的克隆和CRISPR/Cas9基因编辑用于鉴定ZmMs13基因。进行RNA-seq数据和qPCR分析、系统发育和微系统分析、瞬时双荧光素酶报告基因和EMSA测定、亚细胞定位、ATP酶活性和脂质组学分析，以确定ZmMs13基因的调控机制。

结果

玉米ms13-6060突变体表现出完全雄性不育，伴有胼胝质降解延迟、绒毡层程序性细胞死亡 (PCD) 过早以及花粉外壁和花药角质层形成缺陷。ZmMs13编码质膜 (PM) 和内质网 (ER) 定位的半大小 ABCG 转运蛋白 (ZmABCG2a)。ms13-6060突变体中的ZmMs13等位基因由于其第四个外显子中的 3 bp 缺失而有一个氨基酸 (I311) 缺失。I311 和其他保守氨基酸 K99 对于 ZmMS13 的 ATP 酶和脂质结合活性至关重要。ZmMs13在花药中特异表达，在S5、S8b和S10阶段有3个峰，在这3个阶段依次受到转录因子ZmbHLH122、ZmMYB84和ZmMYB33-1/-2的调控。ZmMs13的三相调节依次是胼胝质溶解、绒毡层 PCD 和花粉外壁发育以及花药角质层形成所必需的，对应于 ms13 中胼胝质、ROS、PCD、孢粉质和花药角质层相关基因的转录改变6060 个花药。

结论

ms13-6060突变导致一个关键氨基酸 (I311) 缺失，大大降低了 ZmMS13 ATP 酶和脂质结合活性，并在玉米雄性繁殖过程中表现出多种影响。我们的研究结果为控制植物花药和花粉发育以及雄性育性的 ABCG 转运蛋白的分子机制提供了新的见解。