Combined SHOOT MERISTEMLESS and WUSCHEL trigger ectopic organogenesis in Arabidopsis

Almost all aerial parts of plants are continuously generated at the shoot apical meristem (SAM). To maintain a steady pool of undifferentiated cells in the SAM while continuously generating new organs, it is necessary to balance the rate of cell division with the rate of entrance into differentiation pathways. In the Arabidopsis meristem, SHOOT MERISTEMLESS (STM) and WUSCHEL (WUS) are necessary to keep cells undifferentiated and dividing. Here, we tested whether ectopic STM and WUS functions are sufficient to revert differentiation and activate cell division in differentiating tissues. Ectopic STM and WUS functions interacted non-additively and activated a subset of meristem functions, including cell division, CLAVATA1 expression and organogenesis, but not correct phyllotaxy or meristem self-maintenance. Our results suggest that WUS produces a non-cell autonomous signal that activates cell division in combination with STM and that combined WUS/STM functions can initiate the progression from stem cells to organ initiation.     

Regulation of CLV3 expression by two homeobox genes in Arabidopsis

The ability of meristems to continuously produce new organs depends on the activity of their stem cell populations, which are located at the meristem tip. In Arabidopsis, the size of the stem cell domain is regulated by two antagonistic activities. The WUS (WUSCHEL) gene, encoding a homeodomain protein, promotes the formation and maintenance of stem cells. These stem cells express CLV3 (CLAVATA3), and signaling of CLV3 through the CLV1/CLV2 receptor complex restricts WUS activity. Homeostasis of the stem cell population may be achieved through feedback regulation, whereby changes in stem cell number result in corresponding changes in CLV3 expression levels, and adjustment of WUS expression via the CLV signal transduction pathway. We have analyzed whether expression of CLV3 is controlled by the activity of WUS or another homeobox gene, STM (SHOOT MERISTEMLESS), which is required for stem cell maintenance. We found that expression of CLV3 depends on WUS function only in the embryonic shoot meristem. At later developmental stages, WUS promotes the level of CLV3 expression, together with STM. Within a meristem, competence to respond to WUS activity by expressing CLV3 is restricted to the meristem apex.     

A quantitative and dynamic model for plant stem cell regulation

Plants maintain pools of totipotent stem cells throughout their entire life. These stem cells are embedded within specialized tissues called meristems, which form the growing points of the organism. The shoot apical meristem of the reference plant Arabidopsis thaliana is subdivided into several distinct domains, which execute diverse biological functions, such as tissue organization, cell-proliferation and differentiation. The number of cells required for growth and organ formation changes over the course of a plants life, while the structure of the meristem remains remarkably constant. Thus, regulatory systems must be in place, which allow for an adaptation of cell proliferation within the shoot apical meristem, while maintaining the organization at the tissue level. To advance our understanding of this dynamic tissue behavior, we measured domain sizes as well as cell division rates of the shoot apical meristem under various environmental conditions, which cause adaptations in meristem size. Based on our results we developed a mathematical model to explain the observed changes by a cell pool size dependent regulation of cell proliferation and differentiation, which is able to correctly predict CLV3 and WUS over-expression phenotypes. While the model shows stem cell homeostasis under constant growth conditions, it predicts a variation in stem cell number under changing conditions. Consistent with our experimental data this behavior is correlated with variations in cell proliferation. Therefore, we investigate different signaling mechanisms, which could stabilize stem cell number despite variations in cell proliferation. Our results shed light onto the dynamic constraints of stem cell pool maintenance in the shoot apical meristem of Arabidopsis in different environmental conditions and developmental states.     

植物干细胞决定基因WUS的研究进展

WUS(WUSCHEL)基因编码一转录因子,它的存在使周围细胞具有干细胞的特征,与之相关的信号系统近年逐步被阐明.在茎尖分生组织内WUS和CLV(CLAVATA)之间形成一个反馈调节环,使得干细胞保持自我更新,维持茎尖的顶端优势.在胚胎分生组织内,CLV3的表达只依赖于WUS的存在,然而在胚以后的发育中,CLV3的表达受到WUS和STM(SHOOTMERISTEMLESS)的双重调节,启动器官发生.在花分生组织中,WUS和LFY(LEAFY)共同激活AG(AGAMOUS)基因的表达,WUS受AG的反馈抑制.由WUS建立的信号体系还参与胚珠的发育.当WUS蛋白和生长素共存时,可以高效启动体细胞胚的发生.细胞对WUS信号的感应性与细胞所处的微环境有关,WUS在不同环境条件下可以启动不同的下游基因表达.     

APETALA2 regulates the stem cell niche in the Arabidopsis shoot meristem

Postembryonic organ formation in higher plants relies on the activity of stem cell niches in shoot and root meristems where differentiation of the resident cells is repressed by signals from surrounding cells. We searched for mutations affecting stem cell maintenance and isolated the semidominant l28 mutant, which displays premature termination of the shoot meristem and differentiation of the stem cells. Allele competition experiments suggest that l28 is a dominant-negative allele of the APETALA2 (AP2) gene, which previously has been implicated in floral patterning and seed development. Expression of both WUSCHEL (WUS) and CLAVATA3 (CLV3) genes, which regulate stem cell maintenance in the wild type, were disrupted in l28 shoot apices from early stages on. Unlike in floral patterning, AP2 mRNA is active in the center of the shoot meristem and acts via a mechanism independent of AGAMOUS, which is a repressor of WUS and stem cell maintenance in the floral meristem. Genetic analysis shows that termination of the primary shoot meristem in l28 mutants requires an active CLV signaling pathway, indicating that AP2 functions in stem cell maintenance by modifying the WUS-CLV3 feedback loop.     

SHOOT MERISTEMLESS trafficking controls axillary meristem formation,meristem size and organ boundaries in Arabidopsis

The shoot stem cell niche, contained within the shoot apical meristem (SAM) is maintained in Arabidopsis by the homeodomain protein SHOOT MERISTEMLESS (STM). STM is a mobile protein that traffics cell‐to‐cell, presumably through plasmodesmata. In maize, the STM homolog KNOTTED1 shows clear differences between mRNA and protein localization domains in the SAM. However, the STM mRNA and protein localization domains are not obviously different in Arabidopsis, and the functional relevance of STM mobility is unknown. Using a non‐mobile version of STM (2xNLS‐YFP‐STM), we show that STM mobility is required to suppress axillary meristem formation during embryogenesis, to maintain meristem size, and to precisely specify organ boundaries throughout development. STM and organ boundary genes CUP SHAPED COTYLEDON1 (CUC1), CUC2 and CUC3 regulate each other during embryogenesis to establish the embryonic SAM and to specify cotyledon boundaries, and STM controls CUC expression post‐embryonically at organ boundary domains. We show that organ boundary specification by correct spatial expression of CUC genes requires STM mobility in the meristem. Our data suggest that STM mobility is critical for its normal function in shoot stem cell control.     

The essential gene EMB1611 maintains shoot apical meristem function during Arabidopsis development

The Arabidopsis thaliana genome contains hundreds of genes essential for seed development. Because null mutations in these genes cause embryo lethality, their specific molecular and developmental functions are largely unknown. Here, we identify a role for EMB1611/MEE22 , an essential gene in Arabidopsis, in shoot apical meristem maintenance. EMB1611 encodes a large, novel protein with N-terminal coiled-coil regions and two putative transmembrane domains. We show that the partial loss-of-function emb1611-2 mutation causes a range of pleiotropic developmental phenotypes, most dramatically a progressive loss of shoot apical meristem function that causes premature meristem termination. emb1611-2 plants display disorganization of the shoot meristem cell layers early in development, and an associated stem cell fate change to an organogenic identity. Genetic and molecular analysis indicates that EMB1611 is required for maintenance of the CLV-WUS stem cell regulatory pathway in the shoot meristem, but also has WUS -independent activity. In addition, emb1611-2 plants have reduced shoot and root growth, and their rosette leaves form trichomes with extra branches, a defect we associate with an increase in endoreduplication. Our data indicate that EMB1611 functions to maintain cells, particularly those in the shoot meristem, roots and developing rosette leaves, in a proliferative or uncommitted state.     

Shoot apical meristem and cotyledon formation during Arabidopsis embryogenesis: interaction among the CUP-SHAPED COTYLEDON and SHOOT MERISTEMLESS genes

The shoot apical meristem and cotyledons of higher plants are established during embryogenesis in the apex. Redundant CUP-SHAPED COTYLEDON 1 (CUC1) and CUC2 as well as SHOOT MERISTEMLESS (STM) of Arabidopsis are required for shoot apical meristem formation and cotyledon separation. To elucidate how the apical region of the embryo is established, we investigated genetic interactions among CUC1, CUC2 and STM, as well as the expression patterns of CUC2 and STM mRNA. Expression of these genes marked the incipient shoot apical meristem as well as the boundaries of cotyledon primordia, consistent with their roles for shoot apical meristem formation and cotyledon separation. Genetic and expression analyses indicate that CUC1 and CUC2 are redundantly required for expression of STM to form the shoot apical meristem, and that STM is required for proper spatial expression of CUC2 to separate cotyledons. A model for pattern formation in the apical region of the Arabidopsis embryo is presented.     

Requirement of homeobox gene STIMPY/WOX9 for Arabidopsis meristem growth and maintenance

Most organs of flowering plants develop postembryonically from groups of pluripotent cells called meristems [1]. The shoot apical meristem (SAM) is specified by two complementary pathways [2-4]. SHOOT MERISTEMLESS (STM; [5]) defines the entire SAM region [6]. WUSCHEL (WUS), on the other hand, functions in a more restricted set of cells to promote stem-cell fate and is regulated by the CLAVATA genes in a negative feedback loop [7-10]. In contrast, little is known about how the growth of the SAM, which increases in size during vegetative development [11], is regulated. We have characterized STIMPY (STIP; also called WOX9 [12]), a homeobox gene required for the growth of the vegetative SAM, in part by positively regulating WUS expression. In addition, STIP is required in several other aerial organs and the root. What sets STIP apart from STM and WUS is that stip mutants can be fully rescued by stimulating the entry into the cell cycle with sucrose. Therefore, STIP is likely to act in all these tissues by maintaining cell division and preventing premature differentiation. Taken together, our findings suggest that STIP identifies a new genetic pathway integrating developmental signals with cell-cycle control.     

Pattern formation during de novo assembly of the Arabidopsis shoot meristem

Most multicellular organisms have a capacity to regenerate tissue after wounding. Few, however, have the ability to regenerate an entire new body from adult tissue. Induction of new shoot meristems from cultured root explants is a widely used, but poorly understood, process in which apical plant tissues are regenerated from adult somatic tissue through the de novo formation of shoot meristems. We characterize early patterning during de novo development of the Arabidopsis shoot meristem using fluorescent reporters of known gene and protein activities required for shoot meristem development and maintenance. We find that a small number of progenitor cells initiate development of new shoot meristems through stereotypical stages of reporter expression and activity of CUP-SHAPED COTYLEDON 2 (CUC2), WUSCHEL (WUS), PIN-FORMED 1 (PIN1), SHOOT-MERISTEMLESS (STM), FILAMENTOUS FLOWER (FIL, also known as AFO), REVOLUTA (REV), ARABIDOPSIS THALIANA MERISTEM L1 LAYER (ATML1) and CLAVATA 3 (CLV3). Furthermore, we demonstrate a functional requirement for WUS activity during de novo shoot meristem initiation. We propose that de novo shoot meristem induction is an easily accessible system for the study of patterning and self-organization in the well-studied model organism Arabidopsis.     

The Arabidopsis OBERON1 and OBERON2 genes encode plant homeodomain finger proteins and are required for apical meristem maintenance

Maintenance of the stem cell population located at the apical meristems is essential for repetitive organ initiation during the development of higher plants. Here, we have characterized the roles of OBERON1 (OBE1) and its paralog OBERON2 (OBE2), which encode plant homeodomain finger proteins, in the maintenance and/or establishment of the meristems in Arabidopsis. Although the obe1 and obe2 single mutants were indistinguishable from wild-type plants, the obe1 obe2 double mutant displayed premature termination of the shoot meristem, suggesting that OBE1 and OBE2 function redundantly. Further analyses revealed that OBE1 and OBE2 allow the plant cells to acquire meristematic activity via the WUSCHEL-CLAVATA pathway, which is required for the maintenance of the stem cell population, and they function parallel to the SHOOT MERISTEMLESS gene, which is required for preventing cell differentiation in the shoot meristem. In addition, obe1 obe2 mutants failed to establish the root apical meristem, lacking both the initial cells and the quiescent center. In situ hybridization revealed that expression of PLETHORA and SCARECROW, which are required for stem cell specification and maintenance in the root meristem, was lost from obe1 obe2 mutant embryos. Taken together, these data suggest that the OBE1 and OBE2 genes are functionally redundant and crucial for the maintenance and/or establishment of both the shoot and root meristems.     

CORONA, a member of the class III homeodomain leucine zipper gene family in Arabidopsis, regulates stem cell specification and organogenesis

Organogenesis at the shoot meristem requires a delicate balance between stem cell specification and differentiation. In Arabidopsis thaliana, WUSCHEL (WUS) is a key factor promoting stem cell identity, whereas the CLAVATA (CLV1, CLV2, and CLV3) loci appear to promote differentiation by repressing WUS expression. In a screen for mutations modifying clv1 mutants, we have identified a novel regulator of meristem development we term CORONA (CNA). Whereas cna single mutant plants exhibit subtle defects in meristem development, clv cna double mutants develop massively enlarged apices that display early loss of organogenesis, misexpression of WUS and CLV3, and eventual differentiation of the entire apex. The CNA gene was isolated by positional cloning and found to encode a class III homeodomain Leu zipper protein. A missense mutation resulting in the dominant-negative cna-1 allele was identified in a conserved domain of unknown function, and a likely null allele was shown to display a similar but weaker phenotype. CNA is expressed in developing vascular tissue, diffusely through shoot and flower meristems, and within developing stamens and carpels. Our analysis of WUS expression in wild-type, clv, and clv cna plants revealed that, contrary to current models, WUS is neither necessary nor sufficient for stem cell specification and that neither WUS nor CLV3 is a marker for stem cell identity. We propose that CNA functions in parallel to the CLV loci to promote organ formation.