ABSTRACT OF THE DISSERTATION
Characterization of LATERAL ORGAN BOUNDARIES (LOB) and leaflet inclination in Vigna unguiculata (L.) Walp (Cowpea)
Michael Frederick Schwartz
Plants undergo organogenesis throughout their lifetime by maintaining populations of pluripotent cells at their apices. The shoot apical meristem (SAM) produces lateral organs, such as leaves, which form at the periphery of the SAM. The SAM and the lateral organs are separated by the boundary region. The boundary region contains a population of cells which are smaller and divide infrequently and is a region of reduced growth. The boundary region plays an important role in regulating shoot architecture as it is the region of the plant that influences leaf inclination. The Arabidopsis transcription factor LATERAL ORGAN BOUNDARIES (LOB) is expressed in plant organ boundaries and functions to limit growth and separate the organs. This dissertation focuses on characterizing an ortholog of LOB, VuLOB, in cowpea, an economically important legume, and its role in regulating leaf inclination.
In chapter 1, I identify the LOB ortholog in cowpea, termed VuLOB, through the identification of the LOB-domain genes in cowpea and phylogenetic analysis. Characterization of the expression pattern of VuLOB shows transcript accumulation in the pulvini, structures at the base of the leaves and leaflets which incline the leaves under water-deficit. VuLOB is positively regulated by the phytohormone brassinosteroid (BR) which mimics LOB’s regulation, but functional analysis using Arabidopsis suggests that VuLOB may not form the boundary by restricting growth. VuLOB transcripts are not significantly changed in inclined pulvini. BRs have been shown to regulate leaf inclination in the grasses. When BRs are removed from cowpea using propiconazole they fail to incline. Propiconazole treatment changes the cellular morphology of the epidermis of the pulvinus which correlates with the failure to incline. This suggests that BRs are required to promote leaf inclination in cowpea
In chapter 2, I use genome-wide association studies (GWAS) to identify regions of the cowpea genome which correlate with the variation in the size of the pulvinus and leaflet inclination after water-deficit. Several significant peaks were identified which correlate to the variation in pulvinus size, but no peaks were identified with regards to leaflet angle. From the significant peaks a candidate gene, VuLBD41, was identified. VuLBD41 is a LOB-domain transcription factor that is orthologous to the Arabidopsis transcription factors LBD40 and LBD41. Transcripts of VuLBD41 accumulate in the pulvini of cowpea. However, functional analysis using Arabidopsis did not reveal any function in regard to development. Transgenic plants overexpressing VuLBD41 appear morphologically normal. This suggests that VuLBD41 has an unknown role in boundary formation.
In chapter 3, I characterize the genetic relationship between LOB and 2 putative targets, TREHALOSE-6-PHOSPHATE PHOSPHATASE I/J (TPPI and TPPJ). LOB directly regulates TPPJ, but not TPPI in yeast. However, higher order mutants don’t have an enhancement of the organ fusions found in lob mutant plants, but they do have a significant increase in the number of branches, which suggests that they have some role in the boundary. Further, I show that ectopic TPPJ accumulation in the boundary is sufficient to rescue the lob mutant phenotype. This suggests that LOB requires TPPJ to restrict growth at the boundary.