Vegetative shoot branching in pea (Pisum sativum) is regulated, in part, by a network of five Ramosus (RMS) genes, in combination with the plant hormones auxin and cytokinin and other unidentified long-distance signals. The unidentified signals include one named Shoot Multiplication Signal (SMS) and a feedback signal(s), which operate in a root-shoot-root regulatory loop. As is commonly the case for many biological systems, research with the rms mutants of pea has reached a point where the number of variables and components that need to be considered renders the integration of new and existing hypotheses both complex and cumbersome. In this thesis, the interactions between the RMS gene products, cytokinin, and the other long-distance signals are examined with the aid of a rule-based, algebraic approach to model the system. Additionally, the function of auxin, and its transport, in regulating axillary bud outgrowth is investigated experimentally in pea.
The modelling process shows feedback regulation is an essential element of the RMS branching regulatory network. The model predicts an unexpected physiological function for RMS3 and RMS4 in the rootstock that is subsequently verified using grafting and gene expression studies. Subtle but significant differences in the interactions between RMS3 and RMS4 function, and RMS1 and RMS5 transcription in the shoot and rootstock are added to the model based on rootstock xylem-sap cytokinin and RMS1 gene expression analyses. The model predicts and accounts for spatial differences observed for RMS1 and RMS5 gene expression in root tissue and different shoot internodes of rms2 rms4 plants relative to wild¬type plants. This research indicates that a branch-derived signal, possibly auxin, up-regulates RMS1 transcript abundance independently of the RMS2-mediated feedback signal. Furthermore, xylem-sap cytokinin from the rootstock is proposed to promote sustained bud outgrowth when SMS levels or perception are low. These advances in understanding of the regulation of branching in pea emphasize the power of computational modelling as a research tool in understanding complex biological systems.
A hypothesis is developed to account for the vastly different ideas recently proposed in literature about how auxin is involved in branching regulation; the bud transition hypothesis. The bud transition hypothesis proposes that there are multiple stages of bud development at which an axillary bud might reside: a stage of dormancy, a stage of transition, or a stage of sustained growth. It is suggested that many factors, such as photoperiod, decapitation, bud age and the node at which the bud resides can contribute to the determination of whether an axillary bud is in a stage of dormancy or transition. When in a stage of transition, the bud is suggested to be receptive to other signals, such as cytokinin, auxin or SMS that can trigger or inhibit, respectively, the progression of the bud from a stage of transition to that of sustained growth. Thus, different experimental systems, such as intact, detached or decapitated, could influence the stage at which an axillary bud resides. It is therefore proposed that different species do not necessarily have divergent mechanisms for branching control but rather that the architecture of diverse species has led to studies using plants of various developmental stages, grown under different conditions being assessed using contrasting techniques resulting in altered outcomes. Auxin transport experiments and PsPIN1 gene expression analyses suggest that auxin transport capacity may be increased as a consequence, rather than cause, of bud outgrowth in the rms branching mutants of pea. Bud outgrowth experiments following the chemical inhibition of auxin transport by application of 1-N-naphthylphthalamic acid suggest that apically derived auxin travelling in the polar auxin transport stream also inhibits the progression of buds to sustained growth independently of SMS. It is suggested that auxin might affect shoot branching in a multitude of ways.