Molecular Physiology of Endosperm-limited Seed Germination - Gene Function and Regulation in the Endosperm of Brassicaceae Species
Final Report Abstract
The mature seeds of most angiosperm species are endospermic and our work shows that micropylar endosperm (CAP) weakening is an important process preceeding the completion of seed germination by radicle protrusion. The completion of germination by endosperm rupture is requires the interaction of the key seed compartments CAP and RAD (lower hypocotyl/radicle axis, embryo growth zone), and as the CAP must weaken the RAD . Using a cross-species approach with the Lepidium sativum (big seeds) and Arabidopsis thaliana (tiny seeds) we provide compelling direct and indirect evidence that endosperm weakening is an important trait of these Brassicaceae species. Using direct biomechanical quantification by the puncture force method we demonstrate that L. sativum CAP weakening is promoted by gibberellins (GA) and ethylene, and inhibited by abscisic acid (ABA), jasmonates and the allelochemical myrigalone A (MyA). We provide evidence that the molecular downstream mechanisms underlying endosperm weakening and rupture are mediated by cell-wall remodelling proteins expressed in the CAP, including expansins, XTHs, pectin-related enzymes and proteins which generate apoplastic reactive oxygen species (aROS). Their expression is regulated, at least in part, by GA signalling via the GID1-type GA receptors. Molecular phylogenetic analysis of angiosperm GID1 proteins reveald that they cluster into two eudicot (GID1ac, GID1b) groups and one monocot group. Eudicots have at least one gene from each of these two groups and our cross-species work with L. sativum and A. thaliana mutants demonstrates that the GID1ac and GID1b pathways fulfil distinct roles during Brassicaceae seed germination. We identified and characterised seed-tissue specific gene expression in L. sativum seeds but the analysis of their promoters will be conducted when the genome sequence is completed. Myrigalone A (MyA), a rare flavonoid, is secreted by Myrica gale fruits and is an allelochemical and phytotoxin which inhibits seed germination and seedling growth of target species including L. sativum. We investigated its mode of action and found that it interferes with RAD elongation by inhibiting the production of aROS and endoduplication required for cell expansion growth and embryo growth. It enhances testa permeability and water uptake by imbibition, but inhibits GA3 oxidase and thereby the production of bioactive GAs, and it inhibits CAP weakening and endosperm rupture required for seed germination. Our work shows that transcription and translation is required for CAP weakening and CAP hole formation by autolysis. We published "A Guideline to Family-wide Comparative State-of-the-art qRT-PCR Analysis Exemplified with a Brassicaceae Crossspecies Seed Germination Case Study" as a Plant Cell Front Section Feature which is a milestone in seed biology also because it identified superior cross-species reference genes may be used for family-wide comparative qRT-PCR analysis of seed germination for wide use. We generated transgenic L. sativum seeds to further directly test the importance of CAP weakening and RAD growth as key processes in seed endosperm rupture. Taken together, the works conducted in DFG project has established L. sativum as the rosid model system for the interdisciplinary investigation of CAP weakening.
Publications
- (2010). The evolution of seeds. Tansley review: New Phytologist 186: 817-831
Linkies A, Graeber K, Knight C, Leubner-Metzger G
- (2011). A guideline to family-wide comparative state-of-the-art qRT-PCR analysis exemplified with a Brassicaceae cross-species seed germination case study. The Plant Cell 23: 2045-2063
Graeber K, Linkies A, Wood ATA, Leubner-Metzger G
- (2011). Members of the gibberellin receptor gene family (GIBBERELLIN INSENSITIVE DWARF1) play distinct roles during Lepidium sativum and Arabidopsis thaliana seed germination. Journal of Experimental Botany 62: 5131–5147
Voegele A, Linkies A, Müller K, Leubner-Metzger G
- (2011). Regulation of seed germination in the close Arabidopsis relative Lepidium sativum: A global tissue specific transcript analysis. Plant Physiology 155: 1851-1870
Morris K, Linkies A, Müller K, Oracz K, Wang X, Lynn JR, Leubner-Metzger G, Finch-Savage WE
- (2012). Embryo growth, testa permeability, and endosperm weakening are major targets for the environmentally regulated inhibition of Lepidium sativum seed germination by myrigalone A. Journal of Experimental Botany 63: 5337-5350
Voegele A, Graeber K, Oracz K, Tarkowská D, Jacquemoud D, Turecková V, Urbanová D, Strnad M, Leubner-Metzger G
(See online at https://doi.org/10.1093/jxb/ers197) - (2012). Myrigalone A inhibits Lepidium sativum seed germination by interference with gibberellin metabolism and apoplastic superoxide production required for embryo extension growth and endosperm rupture. Plant and Cell Physiology 53: 81-95
Oracz K, Voegele A, Tarkowská D, Jacquemoud D, Turečková V, Urbanová T, Strnad M, Sliwinska E, Leubner-Metzger G
(See online at https://doi.org/10.1093/pcp/pcr124)
