Biosynthesis and distribution of ABA in relation to cellularization, differentiation and storage product accumulation in the barley endosperm
Plant Physiology
Final Report Abstract
Maternally effected mutants, such as "seg8" showing strongly affected endosperm phenotype and no alterations in maternal tissues and embryo, are good models to study hormonal regulation of barley grain development and hormone functions in maternal/filial signal transduction during endosperm cellularization and differentiation. The phytohormone abscisic acid (ABA) was described to influence cellularization and storage product accumulation of the developing barley endosperm, since obvious differences in the endogenous ABA levels of developing caryopses were found between wild type and "seg8". In this project, we aimed to identify gradients of ABA distribution in developing seeds based on visualization of regions that differ in ABA distribution between wild type and "seg8" seeds, but also on laser microdissection of specific tissues followed by transcript and metabolite (ABA) quantification. ABA quantification was optimized using an LC-MS based approach. Although an important objective of our work, ABA quantification in micro-dissected cells or tissues of barley seeds was not possible. Therefore, monitoring the accumulation of ABA at cellular level was done by an antibody-based immuno-labeling approach. Highly specific antibodies raised against ABA were generated by immunization of rabbits with ABA conjugated to BSA. Resulting IgG fractions were characterized by ELISA and showed high specificity for ABA. Concomitantly, fixation and embedding of developing barley caryopses were optimized using a carbodiimide as fixative and polyethylene glycol 1500 (PEG1500) as embedding medium. Pre-incubation of antibodies with 25 mM ABA before applying them to the section was used as negative control. Combining ABA localization data with transcriptome data obtained from laser-micro-dissected tissues from developing wild type caryopses, two phases in the seed development were proposed which are characterized by an import of ABA from vascular tissues into the middle part of the seed. Mainly the communication between chlorenchyma and endosperm transfer cells might regulate the transition of the endosperm into the storage phase, a process in which ABA might have a major role. To visualize the occurrence of ABA in developing seeds of the mutant "seg8" and to correlate it to the mutant phenotype, a detailed characterization of seed morphology was done. Seeds of "seg8" exhibited not only a shrunken endosperm, but also an altered anatomy of the nucellar projection characterized by a diminished cell elongation, enhanced cell wall thickening and reduced number of cells undergoing programmed cell death or autolysis. These structural alterations were accompanied by enhanced levels of ABA in these cells leading to the assumption that deregulated and thereby enhanced ABA levels might inhibit cell elongation and cell death. These results will help to better understand the mechanisms underlying the regulation of ABA levels/signaling in seeds and can be used later on to improve seed growth and maturation.
Publications
- 2011. Laser-capture microdissection of developing barley seeds and cDNA array analysis of selected tissues. In: Murray GI, ed. Laser Capture Microdissection: Methods and Protocols. Methods in Molecular Biology, Vol. 755.2011, pp 461-475.
Thiel J, Weier D, Weschke W.
(See online at https://dx.doi.org/10.1007/978-1-61779-163-5_39) - 2012. An UPLC-MS/MS method for highly sensitive highthroughput
analysis of phytohormones in plant tissues. Plant Methods, Vol. 8. 2012: 47.
Balcke G, Handrick V, Bergau N, Fichtner M, Henning A, Stellmach H, Tissier A, Hause B, Frolov A.
(See online at https://doi.org/10.1186/1746-4811-8-47)
