1a. Objectives (from AD-416):
The objective of this cooperative research project is to elucidate the mechanisms by which the plant hormone gibberellin (GA) stimulates seed germination. Specifically, it will examine whether the non-proteolytic release from DELLA repression by after-ripening and by GID1 overexpression result from similar mechanisms, and test potential mechanisms for nonproteolytic release from DELLA repression. Three alternative mechanisms for DELLA RGL2 inactivation will be investigated: 1) bypass of DELLA RGL2 repression via a parallel pathway, 2) DELLA RGL2 inactivation via direct protein-protein interaction with GID1, and 3) changes in RGL2 protein ubiquitination and phosphorylation. These objectives are all part of a funded NSF proposal submitted through Washington State University (WSU). This research will be performed by WSU personnel under the guidance of ARS Principal Investigator(adjunct faculty, WSU).
1b. Approach (from AD-416):
The approaches that will be used to examine each research objective are described briefly below. If an increase in GID1 protein accumulation or GID1-DELLA interaction correlates with after-ripening, this will open up a new area for investigation of the role of GA signaling genes in the breaking of seed dormancy. These approaches are described in greater detail in the NSF proposal that will fund the project, Washington State University Budget No. 1N-3019-5447. Objective 1. Do after-ripening and GID1 overexpression rescue sly1-2 germination via the same mechanism? a. Determine whether GID1 mRNA and protein show differential expression in dormant and after-ripened using RT-PCR and western analysis of imbibing sly1 seeds. It will be necessary to raise an antibody to GID1 protein. (Year 1, 2) b. Determine whether after-ripening and GID1 overexpression in sly1-2 seeds cause similar changes in the global pattern of gene expression using microarray analysis. (Year 1, 2) Objective 2. Does rescue of sly1 germination by after-ripening and GID1 overexpression result from a bypass of the requirement for DELLA destruction or DELLA inactivation? a. Perform a yeast 2-hybrid screen to determine if GID1b interacts with as yet unidentified seed proteins expressed in a prey cDNA library derived from seeds. (Year 2, 3) b. Determine whether GID1 overexpression and after-ripening in sly1 result in altered expression of GA-regulated transcripts in a DELLA-independent manner using RT-PCR analysis of imbibing seeds. (Year 1, 2) Objective 3. Is DELLA RGL2 repression of seed germination blocked by direct protein-protein interaction between GID1 and RGL2? a. Determine if rescue of sly1 germination requires GA and the presence of the RGL2 DELLA domain required for interaction with GID1. This will be done through the construction and phenotypic analysis of double mutants. (Year 1, 2) b. Determine if differences in DELLA RGL2 activity are associated with differences in GA levels in dormant and after-ripened sly1-2 seeds. This will be done by measuring GA hormone levels in imbibing seeds. (Year 2) c. Determine whether RGL2 interacts with other transcription factors expressed in seeds by yeast 2-hybrid assay. (Year 2, 3) d. Determine whether GID1-OE results in an increased ratio of RGL2-GID1 heterodimer to RGL2 using co-immunoprecipitation assays. (Year 3) Documents Non-Funded cooperative agreement with Washington State University.
3. Progress Report:
This study examines the mechanisms underlying control of seed germination by the plant hormone gibberellin (GA). GA stimulates seed germination in dicots, and also stimulates the seed germination of dormancy cereal grains. Thus, understanding the genetic mechanisms by which GA stimulates germination can provide gene targets for increasing seed dormancy through reduced function and improving emergence through gain of gene function. Over-production of the GA hormone receptor GID1 (GA-insensitive dwarf) was shown to stimulate seed germination, even in lines with extremely low GA hormone production. GID1 stimulates germination in part by lifting repression of germination by DELLA genes. GID1 can downregulate DELLA repressors either by DELLA destruction or competition for DELLA binding. An ARS Researcher and WSU graduate student used microarray analysis to identify multiple GA-GID1-DELLA regulated transcripts including the wheat dormancy-associated gene Mother of FT. ARS Researcher and WSU graduate student found that the hormone GA is needed not only for seed germination, but for the transition to vigorous seedling growth. The progress of two Washington State University graduate students on this project was monitored through weekly laboratory meetings, biweekly individual meetings, and monthly project reports. This supports parent project 5348-21000-023-00D, Objective 1, Sub-objective C.