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)
3. Progress Report:
This project supports the parent project Objective 3 by determining how hormones control preharvest sprouting of seeds on the mother plant and seedling emergence. This study examined the mechanisms underlying control of seed germination by the plant hormone gibberellin (GA). GA stimulates seed germination in dicots, and stimulates the seed germination of dormant cereal grains. Thus, future work will use new understanding the genetic mechanisms by which GA stimulates germination to provide gene targets for increasing seed dormancy through reduced function and improving emergence through gain of gene function. The GA hormone receptor GID1 (GA-insensitive dwarf) stimulates 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 down regulate DELLA repressors either by DELLA destruction or competition for DELLA binding. This year,the ARS Researcher, WSU postdoctoral fellow, and WSU graduate student found that increased expression of GID1 is correlated with higher germination potential and loss of dormancy through after-ripening. This suggests that you may be able to maintain higher dormancy and sprouting tolerance if you interfere with GID1 gene action.