The genetic control of vegetative phase change in pea

Plants exhibit numerous changes as they develop from a germinating seed through vegetative and reproductive stages to maturity. A seedling begins in a juvenile vegetative phase, and grows in size through the addition of new vegetative organs. As the plant acquires reproductive competence it enters an "adult" vegetative phase where flowering can occur in response to favourable environmental conditions. The initiation of reproductive structures signifies transition to the adult reproductive phase, in which gametes can eventually be produced and the formation of new seeds can occur, followed by senescence and death or a period of dormancy that may involve reversion to the adult vegetative phase. In order to maximise reproductive outputs, the life cycle of the plant and timing of development needs to be finely tuned and flexible enough to take advantage of favourable environmental conditions. The phases of plant development are thus tightly controlled genetically, but also have the ability to respond to the environment to some extent. This study used the model legume pea (Pisum sativum L.) to investigate genes with a possible role in developmental timing, in order to improve our understanding of the genetic control of developmental timing in pea and temperate legumes generally. It will address the following questions: What might the vegetative phase transition look like in pea and how is it related to the timing of flowering in the adult reproductive phase? Can mutants affecting vegetative phase change be identified in pea and if so, how do these mutants interact and what is their molecular nature? Finally, what are the composition and phylogenetic structure in pea of gene families for key Arabidopsis phase change genes, and is there any evidence that any of these genes could participate in phase change in pea? This research has made use of previously identified mutants, aeromaculata 1 (aero1) and aero2 and describes two new mutants, accelerated phase change 1 (apc1) and apc2, which all exhibit pleiotropic defects in various aspects of plant development. However, their most interesting common feature is an acceleration in the normal progression of compound leaves from simpler to more complex structure, such that mutants display more complex leaf structure earlier in development than the isogenic wild-type plants. This may indicate a shift in the timing of vegetative phase change in these mutants. The largely additive, but in some cases synergistic effects of these mutants in combination suggest that these genes have overlapping roles in control of several processes in plant development, including not only timing of vegetative development/compound leaf morphology, but also flower development and fertility, pod development, and phyllotaxy. Efforts were also made to improve or establish map positions for three of the four loci; AERO1, AERO2 and APC1. The AERO1 locus mapped close to the pea ortholog of CURLY LEAF (CLF), a gene with known roles in flowering time control in Arabidopsis. Sequencing revealed mutations in the CLF coding sequence in two independent aero1 mutant alleles, indicating CLF and AERO1 are likely to be the same gene. The known role of Arabidopsis CLF in epigenetic regulation of gene expression and the pleiotropic effects of AERO1 are consistent with the idea that AERO1/CLFmay operate as a master regulator of all aspects of plant developmental timing and might do this by modifying the activity of specific genes involved in phase change, since aero1 showed an acceleration of the timing of all phases of development, including vegetative development and flowering. For apc1, fine mapping and an RNA-sequencing approach were used to identify a potentially causal mutation in the ortholog of Arabidopsis FTSH11. This gene is not previously known to play a role in developmental timing in other species, so may prove to have a novel role in legume development. Genetic control of phase change in plants is achieved through the highly conserved miRNA156-SPL module. The final part of this study isolated, annotated and characterised the miR156 and SPL gene families in pea using newly-available genomic resources. This resulted in the identification of most but not all of the family members predicted from comparative analyses with Medicago truncatula. Of the pea miR156 precursor sequences isolated, none appeared to be obviously involved in vegetative phase change based on their expression patterns. However, investigation of expression patterns for SPL genes revealed several that are developmentally regulated in wild-type pea in a manner consistent with a possible role in regulation of phase change in pea. Overall, these findings represent an important contribution to the knowledge of developmental timing in pea and related legumes, the relationship between compound leaf development and vegetative phase change, and the genes that may be crucial for the control of these processes