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

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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.