Final Report Summary - PHYLLORHIZOTAXIS (A conserved mechanism regulating shoot and root lateral organ placement) Executive Summary:Phyllotaxis, the pattern of leaf initiation, and rhizotaxis, the pattern of lateral root initiation, are controlled by a common genetic element. In the model plant Arabidopsis, these processes are controlled by members of the PLETHORA plade of transcription factors, namely PLT3, PLT5 and PLT7. Triple plt3, plt5, plt7 mutants display a switch from the normal spiral phyllotaxis to a metastable distichous pattern, and lateral roots show clustering that is not seen in wild-type plants. In lateral roots, expression of the PLT genes depends on activity of AUXIN RESPONSE FACTOR (ARF) proteins, which control the responses to concentrations of the phytohormone auxin in many contexts throughout the plant body. auxin. The lateral root-expressed ARF7, ARF19, as well as ARF10, ARF16, ARF5/MP which are all expressed in the shoot, were therefore selected as candidate genes for controlling expression of PLT genes in the shoot. Based on PLT expression and phyllotaxis in the corresponding mutants, MP emerged as the most promising candidate. MP appears to regulate expression of all shoot-expressed PLT genes but the manner of this regulation is complex and may change throughout development. An additional candidate regulator of PLTs came from yeast 1-hybrid screening in the Scheres Lab. JLO was found to bind to the promoters of all three PLT genes in this assay. A JLO artificial microRNA gene-knockdown strategy was developed, and reduction of JLO resulted in shoot termination, consistent with a critical role of this gene in shoot meristem patterning. To analyze the dynamics of gene expression in phyllotaxis control, reporter genes were constructed and/or crossed into the plt3, plt5, plt7 mutant background. Analysis of the auxin transporter PIN1 and auxin response reporter DR5 together showed for the first time that auxin transport and response markers can be uncoupled in plt3,plt5,plt7 mutants. In wild-type plants auxin up-regulates both at sites of organ inception whereas in triple mutants one or the other could be absent. Confocal imaging was used to quantify how many cells in the peripheral zone were responding to auxin. Further, by using a KAN1 reporter to mark the peripheral zone, the exact expression domain of PLT3 was more clearly defined. As KAN1 expression was expanded into the central zone in plt3, plt5, plt7 triple mutants despite central zone markers such as WUS, CLV3 being expressed normally, the identity of these cells is mixed in these mutants. Finally, PLT3, having the most uneven expression pattern of the three genes, was shown to respond to auxin such that expression increased but the domain of expression remained unchanged. Therefore all domains of PLT3 expression are competent to respond to auxin but regions with no expression do not respond under these experimental conditions. Taken together, and in addition to results recently published from the group (Pinon et al. 2013), these results show that PLT genes exert relatively minor influence on both meristem size and zonal identity, but greatly influence both auxin transport and response to auxin, and their activity itself is influenced by the auxin response machinery. This complex looped network shows similarities and differences in topology to the known looped network of auxin transport and PLT expression in the primary root meristem. The control of plant architecture is of great importance in crop plant selection. Agriculturally important traits such as inflorescence morphology and apical dominance are controlled at least in part by phyllotactic patterning, which determines the position of leaves, flowers and lateral branches. Decisions in root architecture play a large part in effective nutrient acquisition. Therefore dissecting the genetic networks controlling phyllotaxis serves as a cornerstone to understanding the evolution and development of plant architecture. The research carried out in this project illuminates parts of the network surrounding PLT gene activity in both shoot and root lateral organ positioning decisions, and how these decisions tie in with regulation of and response to auxin maxima and homeostasis. The PLT gene family is conserved throughout seed plants, but information on function of PLT-like genes outside of Arabidopis remains scarce. Thus, these genes and the networks they act in may serve as candidates for genetic manipulation and breeding for altering plant root and shoot body plan in diverse crop plants.
