Biosynthesis and distribution of ABA in relation to cellularization, differentiation and storage product accumulation in the barley endosperm

在以前的项目中，我们表明阿坝影响发育中的大麦胚乳的细胞化和贮藏产物的积累。在本申请中，我们计划鉴定（1）产生阿坝以调节种子发育的组织，（2）发育种子中阿坝分布的梯度和（3）与阿坝相关的种子发育的调节剂，基于野生型和突变体/转基因种子之间阿坝分布不同的区域的可视化，以及随后通过显微方法对相应组织的分析。为了鉴定ABA产生组织，将定位阿坝生物合成的转录物。不同种子部分中的阿坝量将通过冷冻显微切片的GC分析来定量。免疫定位和MALDI成像质谱将用于显示ABA的空间分布。发育中的谷粒中的pH梯度将被可视化并与阿坝分布相关。将进一步开发生物信息学工具，并用于可视化野生型和突变体/转基因种子之间阿坝分布和pH梯度的差异。为了鉴定与阿坝分布和胚乳分化相关的新序列，计划对幼嫩胚乳表皮的转录组进行454测序。使用这些序列，将生产44 K AGILENT芯片。将显微解剖WT和突变体/转基因种子之间具有不同阿坝分布的区域，并通过转录物和代谢物谱分析进行分析，以鉴定ABA相关的调控组分和过程。

In the previous project, we showed that ABA influences both, cellularization and storage product accumulation of the developing barley endosperm. In this application, we plan to identify (1) tissues producing ABA to regulate seed development, (2) gradients of ABA distribution in developing seeds and (3) regulators of seed development as related to ABA, based on visualization of regions that differ in ABA distribution between wild type and mutant/transgenic seeds, and on subsequent analysis of the respective tissues by micro-methods. To identify ABA-producing tissues, transcripts of ABA biosynthesis will be localized. ABA amounts in different seed parts will be quantified by GC analysis of cryo-microdissectates. Immuno-localization and MALDI-imaging mass spectrometry will be used to show spatial ABA distribution. pH gradients in developing grains will be visualized and correlated to ABA distribution. Bioinformatics tools will be further developed and used to visualize differences in ABA distribution and pH gradients between wild type and mutant/transgenic seeds. To identify new sequences related to ABA distribution and endosperm differentiation, 454 sequencing of the transcriptome of the young endosperm epidermis is planned. Using the sequences, a 44K AGILENT chip will be produced. Regions with different ABA distribution between WT and mutant/transgenic seeds will be micro-dissected and analyzed by transcript and metabolite profiling to identify ABA-related regulatory components and processes.