The Role and Regulation of Ethylene in the Cell Death Program of Maize Endosperm

在谷物种子中，含淀粉的胚乳发育为储存组织，注定在成熟之前死亡，但持续到成熟的种子中。玉米籽粒发育过程中胚乳细胞死亡以高度组织化的方式进行，同时伴随着核糖核酸酶活性的适度增强和核DNA的核小体间碎裂。小麦和玉米的胚乳细胞死亡程序部分受乙烯的调节。因此，对负责乙烯生物合成和感知的基因以及负责DNA片段化的核酸酶的分析将扩大我们对谷类胚乳细胞死亡程序调控的理解。本项目的具体目标是：(1)研究玉米器官发育过程中乙烯生物合成和感知机制的表达，并确定每个基因家族中胚乳特异的成员；(2)确定在胚乳发育过程中细胞死亡过程中乙烯生物合成和感知机制的时空调节；(3)分离ACC合成酶和乙烯受体敲除突变体，以从遗传学角度描述玉米胚乳细胞死亡对乙烯生物合成和感知机制的需求；(4)鉴定胚乳表达的核酸酶，并研究其在指导核体内DNA碎裂中的作用。在玉米胚乳发育的整个过程中，将使用RT-PCR和RNA原位定位技术进行表达研究。转座子插入突变技术已被用来产生影响乙烯生物合成的突变体，这些突变体将用于玉米发育过程中乙烯的表型分析。拟议的实验将在理解谷物籽粒发育的一个基本但鲜为人知的方面取得重大进展，该方面将通过提供一种新的方法来通过延长谷物种子发育期间的储藏储存窗口来提高产量，从而有助于改善谷类作物。

In cereal seeds, the starchy endosperm develops as a storage tissue that is fated to die prior to maturation but persists into the mature seed. The progression of cell death in the endosperm of developing maize kernels occurs in a highly organized manner which is accompanied by a moderate increase in nuclease activity and internucleosomal fragmentation on the nuclear DNA. The endosperm cell death program of wheat and maize is regulated, in part, by ethylene. Consequently, the analysis of the genes responsible for the biosynthesis and perception of ethylene, as well as the nuclease responsible for DNA fragmentation will extend our understanding of the regulation of the cell death program in cereal endosperm. The specific aims of this project are: (1) to investigate the expression of the ethylene biosynthetic and perception machinery during maize organ development and identify the endosperm-specific members of each gene family; (2) to determine the spatial and temporal regulation of the ethylene biosynthetic and perception machinery during the initiation and progression of the cell death program during endosperm development; (3) to isolate ACC synthase and ethylene receptor knock out mutants to genetically characterize the requirement for ethylene biosynthesis and perception for endosperm cell death; and (4) to characterize an endosperm-expressed nuclease and investigate its role in directing internucleosomal DNA fragmentation. The expression studies will be carried out using RT-PCR and in situ RNA localization throughout the development of the maize endosperm. Transposon-insertion mutagenesis has been used to generate mutants affecting ethylene biosynthesis which will be used for phenotypic analysis of ethylene during maize development. The proposed experiments will represent a significant advancement in understanding a basic yet poorly understood aspect of cereal kernel development that will contribute to the improvement of cereal crops by providing a novel means to increase yield through extending the window of storage reserve deposition during cereal seed development.