Studies on sterol biosynthesis mutants of Arabidopsis

This thesis examines the gene promoter activity and morphological characteristics of mutants of HYDRA 1 (HYD 1) and HYDRA2 I FACKEL (HYD2 I FK) (Mayer et al. 1991, Topping et al. 1997) from Arabidopsis. These loci are unique, and encode components of the sterol biosynthesis pathway (Schrick et al. 2000, Souter et at. 2002). Various patterning processes are disrupted in hydra mutants (Topping et at. 1997), and bulk sterol profiles are altered (Schrick et at. 2000, Souter et al. 2002). The mutants show heightened responses to auxin, and their phenotype is partly ameliorated by inhibition of ethylene signalling (Souter et al. 2002, 2004, He et al. 2003). Although much previous attention has been given to the analysis of their phenotype, the precise basis of the pleiotropic defects seen in the hydra mutants have not been attributed to any single phenomenon.This thesis examines the hydra mutants' body patterning and morphology, and aims to test the hypothesis that hydra mutants are defective in pattern coordination across the radial axis. The basis of phenotypic rescue through reduced ethylene perception, as conveyed by the ethylene insensitive2 (ein2) mutation (Alonso et al. 1999), is also examined using anatomical and transgenic markers of pattern definition and phytohormone signalling response. Mutants at the hydra loci have a substantial inter-sibling variability, including duplication or dissociation of the longitudinal axis. Reporter activities of the HYDRA 1 (HYD1) promoter implies an association of gene activity with stipules, and functional epidermal cells and ground tissue in both root and shoot tissues at the point of cell differentiation. Reporter expression defines a radial gradient across the root longitudinal axis which is maximal in thedifferentiation zone. All cell types highlighted by pHYD1 GUS activity sow anomalous cellular patterning in the root and rosette of hydra mutant seedlings, although pattern definition in lateral organs of the inflorescence stem appear relatively normal. Tissues of the hydra embryo and vegetative rosette have ectopic cell division activity; this persists in cotyledons beyond the point where wild-type cotyledon development has ceased Reduced ethylene perception via ein2 appears to confer a partial rescue of the hydra phenotype by facilitating an earlier transition from cell division to cell fate commitment, thus allowing greater coordination between cells in longitudinal cell files. This phenomenon may be attributable to enhanced auxin transport. In contrast, shoot dorsiventral cues are variably skewed or reversed, correlating with a loss of stipular function, in a manner independent of ethylene signalling. Other phytohormone signalling systems, as revealed by reporter constructs for auxin, cytokinin and gibberellin responsive genes, show a varied activity between seedlings. All of these responses appear anomalous in hydra single mutants, some with distinct differences between the two mutant sibling populations. These responses are partly modulated by ein2 in the hydra-ein2 double mutants, although ein2 itself has little or no effect on reporter activity. In particular the distinctive differences in cytokinin positional response between the two hydra mutants are abolished by the presence of ein2. HYDRA gene activity appears to modulate radial patterning and differentiation associated processes. The mutant shoot phenotypes suggest a role for sterolsin the definition of organ lateral boundaries and coordinated centrolateral expansion in flattened organs. In the mutant root, the control of the transition from division to differentiation in cortex cells is disrupted in hydra and may reflect a disrupted phosphate perception. As HYDRA gene activity is associated specifically with functional cells in the epidermis, this suggests that sterols may activate a mechanism for the timed differentiation of 'target cells'. Models are proposed to integrate the HYDRA gene expression data and the hydra mutant phenotype into a functional scheme of plant development.