This project examines how the plant hormone gibberellin (GA) regulates seed germination, dormancy, and after-ripening. Besides being a major food source, seeds propagate, store, and disperse plants. Seeds are like lifeboats for embryonic plants. It is essential that seeds germinate only under conditions that will allow the seedlings to thrive and the species to survive. Thus seed germination is exquisitely regulated by the balance between two plant hormones, abscisic acid (ABA) and GA. During embryo maturation ABA triggers a condition called seed dormancy which prevents seed germination, even under favorable conditions. Dormancy is relieved by a period of dry storage or afterripening. This requirement for after-ripening assures seed dispersal over time as well as space. GA stimulates seed germination and has been implicated in after ripening. Plants such as Arabidopsis and tomato have an absolute requirement for GA synthesis to germinate. This project uses Arabidopsis to determine how the GA signal is perceived leading to germination. Germination is repressed by DELLA transcription factors. In Arabidopsis, GA stimulates germination by triggering destruction of the DELLA RGL2 protein. This destruction requires the GA receptor GID1 and the F-box protein SLY1. Interestingly, germination can occur in sly1 mutants that produce high levels of DELLA protein when seeds have been after-ripened a long time or when the GID1 receptor is over-produced. This suggests that a new mechanism controls seed germination and implicates the GA receptor. Three mechanisms will be tested: 1) DELLA-repression is bypassed by a parallel pathway; 2) DELLA is inactivated by interaction with the GID1 receptor; and 3) DELLA is inactivated by phosphorylation or ubiquitination. Elucidating these mechanisms will benefit society by providing molecular genetic methods to improve crop seedling establishment, yield, and resistance to preharvest sprouting in cereals like wheat. Knowledge about seed biology will be disseminated through annual presentations to Washington wheat growers and elementary schools. The project will promote education through training a postdoctoral fellow, an undergraduate and graduate student.
Seeds are critical to life on earth because they are the main means of propagating land plant species, and allow the convenient planting and harvesting of crops. Seeds of many species are dormant (unable to germinate under favorable conditions) at maturity, and acquire the ability to germinate through the process of dry after-ripening. It is still a mystery, how dry seeds can acquire the ability to germinate at 5-12% moisture. This project used hormone signaling mutants in the model plant Arabidopsis to examine the mechanisms that allow a seed to attain the ability to germinate. The sly1-2 mutant is insensitive to the germination-stimulating hormone gibberellin (GA); and needs much longer after-ripening time to acquire the ability to germinate - 2 years versus 2 weeks in wild type seeds. This after-ripening occurs even though sly1-2 F-box mutants cannot destroy DELLA repressors of seed germination. This study used sly1-2 as a tool to uncover mechanisms for dormancy loss that are independent of DELLA repressor destruction. The research answered many questions raised in the original proposal, and raised new questions for future research. 1) Do after-ripening and GID1 overexpression rescue sly1-2 via the same mechanism? The germination of sly1-2 seeds can be rescued overexpression of the GA hormone receptor, GID1, suggesting that GID1 triggers signaling events that can break dormancy without DELLA destruction. Our hypothesis was that these mechanisms overlap with those occurring during dry after-ripening. Based on transcriptome analysis, many more genes change expression due to after-ripening than due to GID1 overexpression (GID1-OE) (Nelson and Steber, in preparation). This tells us that GA/GID1 is only one of multiple mechanisms relieving seed dormancy during dry after-ripening. 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? When changes in gene transcription were examined, all of the genes whose expression was altered by GID1-OE were also found to be GA and DELLA regulated genes by comparison to previous microarray studies (Nelson and Steber, in prep.). Thus, there does not appear to be a bypass of DELLA repression that involves changes in gene transcription. Yeast 2-hybrid analysis identified new GID1-interacting proteins that might be involved bypass of DELLA destruction (Hauvermale and Steber, unpublished). The plant hormone ABA induces and maintains seed dormancy, whereas GA stimulates seed germination. Hormone measurements revealed that both after-ripening and GID1-OE resulted in decreased ABA hormone levels, suggesting that control of ABA levels is one shared way in which after-ripening and GID1-OE lift seed dormancy (Ariizumi et al., 2013; Nelson and Steber, unpublished). After-ripening also resulted in increased accumulation of GA in imbibing sly1-2 seeds. This together with increasing levels of GID1 mRNA and protein with after-ripening suggest that dormancy is lost via increased GA signaling (Hauvermale & Steber, in prep). 3) Is DELLA repression of seed germination blocked by direct protein-proten interaction between GID1 and DELLA? Our results suggest that the GID1-DELLA protein interaction rescues sly1-2 seed germination via proteolysis-independent down-regulation of DELLA repression. Transcripts showing altered expression with sly1-2 rescue by GID1-OE were previously shown to be GA/DELLA regulated genes, and many after-ripening-regulated genes were also GA/DELLA regulated. Thus, it appears that GID1 can down-regulate DELLA even though the sly1 mutation prevents DELLA destruction. After-ripening of sly1-2 seeds relied, at least in part, on DELLA inactivation by formation of the GID1-DELLA protein complex. Loss of function mutations in gid1a, gid1b, and gid1c prevented sly1-2 after-ripening. Rescue of sly1-2 seed germination required both GA and the presence of the DELLA domain required for the GID1-DELLA protein-protein interaction. Finally, higher GID1 protein levels correlated with better rescue of sly1-2 seed germination and with increasing protein-protein interaction between DELLA and GID1 proteins. This suggests that the GA-dependent GID1-DELLA protein-protein interaction is one mechanism rescuing sly1 seed germination (Ariizumi et al., 2013; Hauvermale et al., 2014). The Broader Impacts of the project: The research benefited society because improved understanding of the mechanisms controlling seed germiantion can be applied to improve crop stand establishment, yield, and seed quality. The research performed raises the possibility that the GA receptor GID1 may be a major regulator of seed germination in crop plants, and could be a candidate to prevent preharvest germination of cereals when rain occurs before harvest. The activity disseminates knowledge about seed biology and agriculture through annual presentations to farmers and to elementary schools. The activity promoted education by training one undergraduate student, three graduate students and one postdoctoral fellow. The activity broadened the participation of under-represented groups by training two female graduate students, one female postdoctoral fellow, as well as minority high school and undergraduate interns recruited through the Upward Bound program.
