Identification of signalling components and regulatory pathways determining development of the barley endosperm

在大麦籽粒中，胚乳传递细胞(ETCs)的协调发育负责胚乳的正常分化，产生糊粉和淀粉胚乳两种主要细胞类型，包括棱柱状胚乳(面向ETCs)和翅区的淀粉胚乳。大麦胚乳突变体Seg8在ETC的细胞化和分化过程中存在缺陷，表现为棱柱状胚乳减少或缺失，种子重量减少70%。因此，SEG8是鉴定决定胚乳发育的成分的合适模型。该提案的一个目标是利用大麦基因组序列信息来鉴定突变的Seg8基因。此外，还将对SEG8和野生型(WT)籽粒的显微切割的ETCs和棱形胚乳细胞进行转录本、代谢物和激素的比较分析。有强有力的证据表明，Seg8颗粒的变化与ABA功能的变化有关。因此，另一个目标是研究ABA的生物合成、信号传递和分布与SEG8和WT籽粒的胚乳细胞化和分化有关。ABA生物合成基因和信号成分的表达将通过mRNA原位杂交和qRT-PCR进行研究。与WT相比，那些在SEG8中显示ABA生物合成模式改变的区域将在显微解剖后通过转录本和代谢物图谱进行分析。生物信息学网络分析将被用来识别调节胚乳发育的信号级联。

In barley grains the coordinated development of endosperm transfer cells (ETCs) is responsible for the regular differentiation of the endosperm resulting in two major cell types: aleurone and starchy endosperm, which includes the prismatic endosperm (facing ETCs) and the starchy endosperm of the wing regions. The barley endosperm mutant seg8, which is defective in cellularisation and differentiation of ETCs, reveals reduced or missing prismatic endosperm and 70% reduction of seed weight. Thus, seg8 is a suitable model for the identification of components determining endosperm development. One objective of the proposal is to identify the mutated seg8 gene by using genomic barley sequence information. In addition, comparative transcript, metabolite and hormone profiling will be performed on microdissected ETCs and prismatic endosperm cells of seg8 and wild type (WT) grains. There is strong evidence that changes in seg8 grains are related to altered ABA functions. Thus, another objective is to study ABA biosynthesis, signalling and distribution as related to endosperm cellularisation and differentiation in seg8 and WT grains. Expression of ABA biosynthesis genes and signalling components will be studied by mRNA in situ hybridisation and qRT-PCR. Those regions showing altered patterns of ABA biosynthesis in seg8 will be analysed by transcript and metabolite profiling after microdissection in comparison to the WT. Bioinformatics network analysis will be used to identify signalling cascades regulating endosperm development.