Seed germination is a critical phase in the plant life cycle, both ecologically and agriculturally, and is sensitive to both endogenous hormones and environmental factors. Seeds integrate physiological and environmental information to determine whether to germinate or remain dormant. Two important physiological regulators of germination are gibberellin (GA), which generally promotes germination, and abscisic acid (ABA), an inhibitor of germination. A key environmental determination of germination behavior is the availability of water, since radicle emergence requires water uptake and cell expansion. Single-gene mutations in tomato which block synthesis of either GA or ABA result in contrasting effects on dormancy, germination rates, and sensitivity to reduced water availability. These mutants have been used in combination with hormonal application to demonstrate that the regulation of germination occurs via alterations in the water potential threshold, allowing radicle emergence. A quantitative mathematical model has been developed that can describe the responses of seed germination to the plant growth regulators GA and ABA and to the seed water potential. A key feature of this is the recognition that individual seeds vary in their threshold responses to GA, ABA, and water potential. The sensitivity thresholds for tomato seed germination are determined by the mechanical resistance of a thin endosperm layer to penetration by the radicle. When stimulated to germinate, the walls of these endosperm cap cells are degraded and weakened, allowing radicle emergence. Multiple factors are likely to be required for successful completion of germination, with their combined influence setting the sensitivity threshold for a given seed. The goal of this project is to determine whether the transition from quiescence or dormancy to germination is associated with specific changes in gene expression. The effects of GA and ABA on mRNA populations in mutant tomato endosper m caps will be visualized by a differential display reverse transcription PCR technique. Imbibed wild type seeds will also be sorted into dormant and nondormant categories prior to visible germination using a single seed assay. The mRNAs expressed in the endosperm caps of the two categories will be similarly displayed. Messages that are consistently associated with either dormancy or germination in all comparisons will be evaluated by RNA blot and in situ hybridization to determine whether their abundance, timing and tissue location are regulated by GA, ABA, and water potential as expected. Expression of selected genes under a range of hormonal and environmental conditions will be investigated in single seeds using tissue printing to determine whether they are regulated among seeds within a population in keeping with the predictions of the threshold models. This approach will distinguish genes involved in the regulation of radicle emergence from those that are associated with post-germinative events such as reserve mobilization. Characterization of the function of these genes and their promoters will provide insight into how plants integrate multiple internal and environmental signals to control a critical developmental process.
