Identification of signalling components and regulatory pathways determining development of the barley endosperm
Final Report Abstract
A new seg8 interval was mapped in the centromeric region of chromosome 7H flanked by the markers G7C2 and Bmag341. This interval is larger than published before (Röder et al. 2006). From this interval, one candidate (morex_contig_95668) shows expression below the detection limit in the seg8 NP in all analyzed developmental stages but is expressed in the Bowman NP. This might be a candidate for the mutated seg8 gene. Further analyses are necessary to confirm this suggestion. The maternal affected endosperm mutant seg8 is defective in cellularization and differentiation of endosperm transfer cells (ETCs) and starchy endosperm. Histological analyses in frame of an associated project showed impaired differentiation also of the nucellar projection (NP). We decided not to perform comparative molecular analysis of ETCs and prismatic endosperm as proposed but to concentrate our work on differentiation of NP and ETCs and interactions between the two tissues in seg8 and wild type (WT) Bowman. Expression of ABA biosynthesis und –signaling genes was visualized in distinct tissues of developing WT grains and compared to the patterns of ABA distribution as estimated by immune-localization. We suggest that ABA is imported from vascular tissues between anthesis and 3 days after flowering (DAF). Thus, early grain development is influenced by maternally supplied ABA. ABA import declines at 5 DAF. Instead, ABA biosynthesis starts in the maternal chlorenchyma, and ABA localizes at 5 DAF in NP and endosperm. Thus, ABA produced in the chlorenchyma and transported to NP and endosperm might trigger grain development at the beginning of transition from prestorage to storage phase. 7 DAF, ABA biosynthesis starts in ETCs. 10 DAF, ABA was detected in NP and ETCs. Between 10 and 14 DAF, ABA signaling correlates with ABA biosynthesis in the accumulating endosperm. We consider that between 7 and 10 DAF filial grain parts take over ABA-dependent regulation. The ETC transcriptome showed high abundance of elements of the two component signaling (TCS) system suggesting an outstanding role in ETCs differentiation. Transcript profiling of TCS elements by qRT-PCR suggested pivotal roles for specific phosphorelays activated in a coordinated time flow during ETC cellularization and differentiation. Features of candidate genes of distinct phosphorelays and transcriptional activation of genes putatively implicated in hormone signaling hint to a crosstalk of hormone influences and TCS signaling. Our findings suggest an integral function for the TCS in ETC differentiation possibly coupled to sequent hormonal regulation by ABA and ethylene. Transcript profiling of developing NP and ETCs of seg8 and Bowman and subsequent PCA analysis showed remarkably low variance of the transcriptome of seg8 and Bowman NPs in all analyzed developmental stages. This is also true for seg8 and Bowman ETCs at 3 and 5 DAF. At 7 DAF, the transcriptome of seg8 ETCs is remarkably different from that of Bowman. Furthermore, the transcriptome of Bowman ETCs at 7 DAF and that of seg8 ETCs at 10 DAF cluster together indicating high similarity (low variance). We propose disturbances in transcriptional regulation of seg8 ETCs between 5 and 7 DAF resulting in delayed development. From the transcriptome data, differentially expressed genes related to ABA biosynthesis and signaling as well as differentially expressed TCS genes were depicted. The data hint to general ABA shortage in developing NP and ETCs of seg8 in comparison to the WT, furthermore to early (3 DAF) disturbance of ethylene signaling mediated by TCS in the two seg8 tissues. These speculations are preliminary and have to be confirmed by comprehensive analysis of the Omics data. Unfortunately, Dynamic Bayesian Network (DBN) modeling as planned for reconstruction of regulatory networks had to be cancelled because the responsible expert (Swetlana Friedel) left the IPK at project half-time. Instead, visualization of differences in metabolism is planned together with Uwe Scholz. Data established in tight cooperation with the associated project gave strong hints that deregulated ABA to GA balances impair NP differentiation in seg8 and potentially compromise transfer of signals and assimilates to the endosperm. Additional work: TCS components were suppressed by RNAi approaches in developing barley grains. The transgenic grains show some similarity to the seg8 phenotype pointing to relationships between the seg8 gene and TCS-determined regulation. Furthermore, protein-protein interactions between TCS components were verified by Bimolecular Fluorescence Complementation. Together with the project-specific results these data were prerequisite for a new proposal.
Publications
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Laser-capture microdissection of developing barley seeds and cDNA array analysis of selected tissues. Methods Mol Biol 755: 461-475
Thiel, J., Weier, D., Weschke, W.
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454 transcriptome sequencing suggests a role for two-component signalling in cellularization and differentiation of barley endosperm transfer cells. PLoS One 7 (2012) e41867
Thiel, J., J. Hollmann, T. Rutten, H. Weber, U. Scholz & W. Weschke
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Differentiation of endosperm transfer cells of barley - a comprehensive analysis at the micro-scale. Plant J. 71 (2012) 639-655
Thiel, J., D. Riewe, T. Rutten, M. Melzer, S. Friedel, F. Bollenbeck, W. Weschke & H. Weber
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(2014) Development of endosperm transfer cells in barley (review article). Frontiers Plant Science Front. Plant Sci. 5:108, 26 March 2014
Thiel, J.
