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 scientist Dr. Camille M. Steber (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
The germination of dormant seeds is repressed by a gene family that makes DELLA protein. The block of seed germination by DELLA is lifted by the plant hormone GA in part by causing DELLA protein destruction. However, our recent data indicates that there is a destruction-free mechanism for lifting DELLA repression of seed germination. This project is investigating whether this works through, DELLA protein binding by the GA receptor GID1, but DELLA modification, or through a DELLA bypass by the ABA hormone signaling pathway. Initial work has: 1) determined that DELLA protein repression of seed germination can be lifted by binding of the GA receptor GID1; 2) has identified candidate proteins in this process using the yeast 2-hybrid method; 3) and has examined whether the lifting of DELLA repression of seed germination is associated with changes in GA and ABA hormone levels. These hormone measurements were performed at the RIKEN institute in Japan. This project is monitored through weekly laboratory meeting and through individual meeting with the Washington State University graduate students performing the research.