Studies presented in this thesis combine both physiological and molecular approaches to investigate the genetic and hormonal regulation of shoot branching in the model plant species Pisum sativum L. (garden pea). This study employs the well characterised series of ramosus increased branching mutants (rms1 to rms5), that appear to specifically regulate shoot branching. Almost a decade of research with the rms mutants has led to the development of the RMS model of branching control that includes roles for the classic plant hormones and genes that regulate potentially novel branching signals. In this thesis, experiments were designed to critically test predictions of this model to expand our understanding of shoot branching control.
RMS1, a gene believed to regulate a potentially novel branching signal, was isolated with the assistance of work presented in this thesis. The RMS1 gene shares homology with a large family of polyene chain dioxygenases from plants, animals and bacteria and an enzymatic function for the RMS1 protein is consistent with a role in a biosynthetic pathway. Putative RMS1 orthologues have been identified in a range of plant species, including monocots and dicots, and may be potential tools for future crop improvement.
Analysis of the expression of the RMS1 gene has been a powerful tool to delineate the interactions between the RMS genes and the plant hormones, auxin and cytokinin. Previous physiological studies have indicated that auxin requires the mobile signal regulated by RMS1 to inhibit branching in decapitated plants. In a range of experiments, RMS1 expression levels were correlated with shoot auxin levels. In addition, protein synthesis in not required for auxin to modulate RMS1 expression. This data provides additional evidence that auxin may regulate shoot branching, in part, by modulating RMS1 activity.
Like rms1, decapitated rms2 to rms5 mutant plants also have reduced response to auxin. Preliminary expression studies with auxin-treated rms2, rms3 and rms4 plants have indicated that this is unlikely to be due to reduced ability to regulate RMS1 transcript level.
The RMS1 gene is differentially expressed in the range of rms mutants, indicating that the RMS genes may interact, at least in part, by regulating RMS1 transcript level. RMS1 expression is elevated in rms3 to rms5 mutant plants but not in rms2 mutant plants. This is consistent with physiological studies that indicate RMS2 may regulate RMS1 activity. Grafting studies with the rms mutants have revealed this regulation of RMS1 expression is mediated in part by a graft-transmissible signal. Elevated RMS1 expression is also associated with branching processes in wild type plants. As RMS1 acts to inhibit shoot branching, feedback regulation of RMS1 expression may be a mechanism to maintain branching homeostasis.
Previous grafting studies with the rms mutants have indicated that the shoot may regulate xylem sap cytokinin export from the roots. To investigate the nature of this feedback process, xylem sap cytokinin levels were monitored in Y-grafted plants, which have one branching mutant shoot and one WT shoot grafted to the same rootstock. Plants with at least one branching shoot exhibited low sap cytokinin levels, evidence that branching shoots may generate a negative regulator of sap cytokinin export.
RMS2 has been proposed to regulate a branching signal in addition to the plant hormones auxin, cytokinin and the signal regulated by RMS1. Grafting studies undertaken in this thesis critically tested this hypothesis. The phenotype of Y-grafted plants indicates RMS2 may regulate a signal that moves in the direction of shoot to root. Epicotyl interstock grafts (a small interstock is inserted between the scion and rootstock) between rms1, rms2 and rms5 plants provides additional evidence that the RMS1, RMS2 and RMS5 genes interact and indicate these genes may act in different tissues to regulate shoot branching.