Nutrient release by cellular disintegration in developing barley seeds
Final Report Abstract
TUNEL assay was used to visualize PCD events in the developing barley grain. Degenerating nuclei were detected in cells of the nucellar projection (NP) adjacent to the endosperm transfer cells from 6 days after flowering (DAF) till grain maturation. Nuclei degradation was further visualized in the embryo-surrounding region (ESR) and especially in the endosperm-near nucellus at 6 DAF. Also cells of the embryo-near endosperm were TUNEL labelled at 8 and 10 DAF. A collection of cDNA libraries prepared from micro-dissected grain tissues, among them nucellus, NP and ESR was used to quantify Jekyll transcript amounts by qRT-PCR. Jekyll transcripts were detected in nucellus and NP but not in ESR. Thus, PCD in ESR is obviously not related to Jekyll activity and presumably follows Jekyll-independent regulatory mechanisms. The transcriptome of the Jekyll-suppressed NP of transgenic line N91 was analyzed in comparison to the nontransgenic control. Analysis of differential gene expression in ESR was proposed but deleted from the work plan because of missing Jekyll expression. Instead, transcriptome analysis of transgenic and control endosperm was performed. Combined transcript and metabolite profiling showed that dysfunction in differentiation of the Jekyll-suppressed NP of line 91 results in interrupted assimilate flow to the endosperm and abnormal storage product accumulation in the transgenic NP. Reduced starch accumulation and transcriptional activation of starch degrading enzymes indicate assimilate starvation of the developing transgenic endosperm being responsible for the strongly reduced N91 thousand grain weight. Usage of a newly designed rabbit polyclonal anti-Jekyll antibodies failed to identify members of the Jekyll protein complex. Six different scFv specifically binding to Jekyll were identified by screening of a scFv phage display library. The scFv are ready for use in immunoprecipitation experiments. Expression pattern of the transcription factor gene HvMADS29 coincides temporally and spatially with Jekyll expression. The recombinant HvMADS29 protein binds specifically to CArG motifs in the Jekyll promoter indicating that HvMADS29 is a direct regulator of Jekyll activity.
Publications
- (2011) Dynamic 13C/1H NMR imaging uncovers sugar allocation in the living seed. Plant Biotechnol. J. 9: 1022-1037
Melkus G., Rolletschek H., Fuchs J., Radchuk V., Grafahrend-Belau E., Sreenivasulu N., Rutten T., Weier D., Heinzel N., Schreiber F., Altmann T., Jakob P., Borisjuk L.
- (2012) Fertility in barley flowers depends on Jekyll functions in male and female sporophytes. New Phytol. 194: 142-157
Radchuk V., Kumlehn J., Rutten T., Sreenivasulu N., Radchuk R., Rolletschek H., Herrfurth C., Feussner I., Borisjuk L.
(See online at https://doi.org/10.1111/j.1469-8137.2011.04032.x) - (2014) Physical, metabolic and developmental functions of the seed coat. Front. Plant Sci., 5:510
Borisjuk L., Radchuk V.
(See online at https://doi.org/10.3389/fpls.2014.00510)
