Regulation of stem cell characters in the haploid generation of the moss Physcomitrella patens ヒメツリガネゴケ配偶体世代における幹細胞の性質の制御機構
Regulation of stem cell characters in the haploid generation of the moss Physcomitrella patens
Stem cells are characterized by their ability to self-renew and give rise to differentiated cells and formed at particular times and positions during the development of multicellular organisms. Land plants have a haplodiplontic life cycle, in which gametophyte and sporophyte generations alternate. Whereas flowering plants form stem cells only in the sporophyte generation, non-seed plants form stem cells in both the sporophyte and gametophyte generations. Since the closest relatives of land plants, the charophytes, have a haplontic life cycle and retain stem cells only in the gametophyte generation, it is hypothesized that the molecular mechanisms underlying stem cell regulation in sporophytes were co-opted from pre-existing mechanisms in gametophytes. For stem cell regulation in sporophyte generation, various research were extensively performed using Arabidopsis thaliana as a model organism. In A. thaliana, stem cells exist at the growing tips of shoot and root as shoot apical meristem (SAM) and root apical meristem (RAM). Class1 KNOX genes play a key role to maintain pluripotent stem cells in SAM. Orthologous genes of Class1 KNOX genes in P. patens were found, and it is shown that these genes did function not in stem cells of gametophytes but in development of sporophytes. WUSCHEL (WUS) and CLAVATA (CLV) genes are also important regulators of stem cells in SAM. WUS gene is a homeobox transcription factor and positively regulates the expression of CLV3 gene. In contrast, CLV3 gene, which encode precursor of glycopeptide, negatively regulates the expression of WUS gene via CLV1, which encode receptor-like kinase. This negative feedback loop between WUS and CLV3 genes is necessary to maintain a stem cell population in SAM. It is reported that genes, which belong to WOX family, exist in P. patens, although functions of these genes are unknown. Therefore, I generated the disruption lines of WOX genes in P. patens. However, these disruption lines showed normal growth and no morphological difference compared with wild type in the gametophyte generation. Recently, it is shown that loss-of-function of WOX genes caused defects in regeneration from leaf cell and sporophyte development in P. patens. Although these results suggest that there are different mechanisms for stem cell regulation between gametophyte and sporophyte generations, relevance of the “co-option” hypothesis is still unclear because there is a little information for stem cell regulation in the gametophyte generation. It is important to identify the genes that regulate gametophyte stem cell formation in order to elucidate the general principles of stem cell formation in plants.The moss Physcomitrella patens forms a hypha-like body (protonema) and a shoot-like body (gametophore) from a protonema apical cell and a gametophore apical cell, respectively, in gametophyte generation. These apical cells have stem cell characteristics and are formed as side branches of differentiated protonema cells. Differentiation of two different types of stem cells from side branch initial cells is controlled by two phytohormones, auxin and cytokinin. However, molecular mechanisms for such a stem cell formation has been largely unknown. In addition to simple stem cell system in P. patens, availability of whole genome sequence and the feasibility of gene targeting allow us to investigate developmental mechanisms in detail. The AP2-type transcription factors, which are characterized by the AP2/ERF DNA-binding domain, form a plant-specific protein family. The AINTEGUMENTA (ANT) subfamily consists of eight genes: ANT, AINTEGUMENTA-LIKE (AIL) 1, PLETHORA (PLT) 5/AIL5, PLT1, PLT2, PLT3/AIL6, PLT7/AIL7 and BABY BOOM (BBM) that are involved in the development of flowering plants. In A. thaliana, PLT genes are required for stem cell niche formation in root apical meristems, and loss-of-function of these genes causes a defect in stem cell maintenance. In addition, it is shown that ANT, AIL6, and AIL7 regulate shoot apical meristem function. Since homologs of these genes exist in P. patens, these genes are good candidate to analyze the molecular mechanisms for stem cell regulation in gametophyte generation.In this study, I show that four AP2-type transcription factors orthologous to Arabidopsis thaliana eight genes are indispensable for the formation of gametophore apical cells from protonema cells. I named the four P. patens genes APB1, APB2, APB3, and APB4, based on the initials of their orthologous genes ANT, PLT, and BBM in A. thaliana. Quadruple disruption of all APB genes blocked gametophore formation, even in the presence of cytokinin, which enhances gametophore apical cell formation in the wild type. Time-lapse observation showed that side branch initial cells do not acquire gametophore apical cell identity and directly differentiate into protonema apical cells even in the conditions of gametophore apical cell induction, at least based on their morphology. All APB-reporter fusion proteins were detected in side branch initial cells just after side branch formation. These signals were also detected in emerging gametophore apical cells and continuously detected during gametophore apical cell formation, whereas the signals disappeared during protonema apical cell formation. Heat-shock induction of a APB4 transgene driven by a heat-shock promoter increased the number of gametophores. Furthermore, constitutive induction of a APB4 transgene driven by EF1-α promoter increased the proportion of gametophore apical cells to protonema apical cells in the presence of cytokinin. Expression of all APB genes was induced by auxin but not by cytokinin, although ectopic expression of APB-reporter fusion proteins was not observed even in the presence of these phytohormones. Thus, the APB genes work under auxin signaling and function synergistically with cytokinin signaling to determine the identity of the two types of stem cells.This study provides some new insights into regulation of stem cells in gametophyte and sporophyte generations. Our research suggests that APBs control the fate of gametophore apical stem cells in gametophyte generation of P. patens. Orthologous genes of APBs are involved in the regulation of shoot and root apical meristem in sporophyte generation of A. thaliana. Therefore, this is the first report to show that orthologous genes function in the regulation of stem cells in both gametophyte and sporophyte generation.