Identification and functional analyses of genes regulating seed dormancy, longevity and germination

Abstract

Fundamental knowledge about the processes affecting seed performance, including the regulation of germination, dormancy and longevity can provide insight to improve these traits, which is of economic importance for agricultural use and storage of seed crops. Accordingly, the objective of this study was to reveal genetic regulators for seed dormancy and possibly other traits related to seed performance. For this purpose the study started with the transcriptome profiling of freshly harvested (dormant) and after-ripened (AR; non-dormant) 24-hour imbibed seeds of four different DELAY OF GERMINATION near isogenic lines (DOGNILs) and the Landsberg erecta (Ler) wild type, which have different levels of primary dormancy. This comparative transcriptomics approach lead to the identification of genes that were either related to seed dormancy or to after-ripening in all of these genotypes (46 and 25 genes for the dormancy-up and after-ripened-up set, respectively). The study was followed by reverse genetic analysis in the model plant Arabidopsis thaliana and resulted in several dormancy and other germination-related phenotypes (Chapter 2). Three mutants displaying the most interesting phenotypes were selected for in depth studies of their corresponding genes. In Chapter 3 we studied FAST GERMINATING SEEDS (FGS), a novel gene whose lack of function mutant seeds displayed reduced seed dormancy, faster and more uniform germination and lower sensitivity to abiotic stress, while seed longevity was not affected. The germination phenotypes represented by the fgs mutant resemble the requirements for vigorous seeds in an agricultural context and make FGS an interesting candidate gene for the seed industry. By genetic complementation of the fgs mutant we showed that the mutant phenotype is indeed due to disruption of the FGS gene. The expression of FGS mRNA is induced during seed maturation and decreases upon seed imbibition. The fgs mutant was further investigated using metabolomics and proteomics analysis which revealed a reduced abundance of seed storage related proteins in dormant dry seeds and a quicker release of amino acids in germinating seeds in comparison to wild type Columbia. Furthermore, reciprocal crosses indicated that FGS controls seed germination through the maternal tissue. These findings together with localization of FGS in protein storage vacuoles suggests a role for FGS in seed storage protein mobilization. In Chapter 4, I studied the role of the NADP-MALIC ENZYME 1 (NADP-ME1) gene in seed longevity. Loss-of-function of NADP-ME1 resulted in a reduced seed viability relative to wild type. NADP-ME1 is one of four Arabidopsis NADP-ME isoforms that catalyses the oxidative decarboxylation of malate to yield pyruvate, CO2, and NADPH. The effect on seed longevity is specific for NADP-ME1 as was revealed by mutant analyses and complementation cloning. Furthermore, dry nadp-me1 mutant seeds display higher levels of protein carbonylation than wild type and upon seed imbibition malate and amino acids accumulate in the embryos of aged mutant seeds compared to wild type. NADP-ME1 expression is increased in imbibed aged as compared to non-aged seeds and NADP-ME1 activity at testa rupture promotes normal germination of aged seeds. Moreover, in seedlings of aged seeds NADP-ME1 is specifically active in the root meristematic zone. We propose a role for NADP-ME1 in the protection of seed proteins against oxidative damage. The last gene that I studied is ALLANTOATE AMIDOHYDROLASE (AtAAH) that encodes an enzyme in the uric acid catabolic pathway. Seeds carrying mutations in this gene display increased seed dormancy. Since AtAAH is a key enzyme in the pathway that produces usable nitrogen for subsequent anabolic reactions, I hypothesized that higher dormancy of the Ataah mutant seeds is the consequence of a defective nitrogen production. To test this hypothesis, exogenous nitrate was applied during seed maturation and seed germination to see whether it can rescue the germination behaviour of the mutant. Results showed that applying exogenous nitrate during both seed maturation and germination partially complement the higher dormancy phenotype of the mutant seeds. Based on these findings we conclude that a defective AtAAH leads to a reduction in nitrogen (ammonia) release in freshly harvested seeds. This causes a block of germination which can be overcome by seed after-ripening or by the application of exogenous nitrate (Chapter 5). Finally, I integrated and discussed the work presented in this thesis in Chapter 6. The power of our approaches (multi-NIL transcriptomic analysis) in search for candidate genes was emphasized, as knock-out mutants in several genes showed dormancy and germination-related phenotypes. I also discussed the potential of this knowledge for improving traits that contribute to seed performance including seed dormancy, seed longevity, germination rate, pre-harvest sprouting and germination under stress condition. In addition I described the processes and strategies that can be used for translating the knowledge gained form this thesis to improve crops species.