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.     

Dynamic and compensatory responses of Arabidopsis shoot and floral meristems to CLV3 signaling

In Arabidopsis thaliana, the stem cell population of the shoot system is controlled by regulatory circuitry involving the WUSCHEL (WUS) and CLAVATA (CLV1-3) genes. WUS signals from the organizing center (OC) to promote stem cell fate at the meristem apex. Stem cells express the secreted peptide CLV3 that activates a signal transduction cascade to restrict WUS expression, thus providing a feedback mechanism. Stem cell homeostasis is proposed to be achieved by balancing these signals. We tested the dynamics of CLV3 signaling using an inducible gene expression system. We show here that increasing the CLV3 signal can very rapidly repress WUS expression during development, which in turn causes a fast reduction of CLV3 expression. We demonstrate that increased CLV3 signaling restricts meristem growth and promotes allocation of peripheral meristem cells into organ primordia. In addition, we extend the current model for stem cell control by showing that meristem homeostasis tolerates variation in CLV3 levels over a 10-fold range and that high-level CLV3 signaling can be partially compensated with time, indicating that the level of CLV3 expression communicates only limited information on stem cell number to the underlying OC cells.     

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.     

RPK2 is an essential receptor-like kinase that transmits the CLV3 signal in Arabidopsis

The shoot apical meristem (SAM) is the fundamental structure that is located at the growing tip and gives rise to all aerial parts of plant tissues and organs, such as leaves, stems and flowers. In Arabidopsis thaliana, the CLAVATA3 (CLV3) pathway regulates the stem cell pool in the SAM, in which a small peptide ligand derived from CLV3 is perceived by two major receptor complexes, CLV1 and CLV2-CORYNE (CRN)/SUPPRESSOR OF LLP1 2 (SOL2), to restrict WUSCHEL (WUS) expression. In this study, we used the functional, synthetic CLV3 peptide (MCLV3) to isolate CLV3-insensitive mutants and revealed that a receptor-like kinase, RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2), also known as TOADSTOOL 2 (TOAD2), is another key regulator of meristem maintenance. Mutations in the RPK2 gene result in stem cell expansion and increased number of floral organs, as seen in the other clv mutants. These phenotypes are additive with both clv1 and clv2 mutations. Moreover, our biochemical analyses using Nicotiana benthamiana revealed that RPK2 forms homo-oligomers but does not associate with CLV1 or CLV2. These genetic and biochemical findings suggest that three major receptor complexes, RPK2 homomers, CLV1 homomers and CLV2-CRN/SOL2 heteromers, are likely to mediate three signalling pathways, mainly in parallel but with potential crosstalk, to regulate the SAM homeostasis.     

Reaction-diffusion pattern in shoot apical meristem of plants

A fundamental question in developmental biology is how spatial patterns are self-organized from homogeneous structures. In 1952, Turing proposed the reaction-diffusion model in order to explain this issue. Experimental evidence of reaction-diffusion patterns in living organisms was first provided by the pigmentation pattern on the skin of fishes in 1995. However, whether or not this mechanism plays an essential role in developmental events of living organisms remains elusive. Here we show that a reaction-diffusion model can successfully explain the shoot apical meristem (SAM) development of plants. SAM of plants resides in the top of each shoot and consists of a central zone (CZ) and a surrounding peripheral zone (PZ). SAM contains stem cells and continuously produces new organs throughout the lifespan. Molecular genetic studies using Arabidopsis thaliana revealed that the formation and maintenance of the SAM are essentially regulated by the feedback interaction between WUSHCEL (WUS) and CLAVATA (CLV). We developed a mathematical model of the SAM based on a reaction-diffusion dynamics of the WUS-CLV interaction, incorporating cell division and the spatial restriction of the dynamics. Our model explains the various SAM patterns observed in plants, for example, homeostatic control of SAM size in the wild type, enlarged or fasciated SAM in clv mutants, and initiation of ectopic secondary meristems from an initial flattened SAM in wus mutant. In addition, the model is supported by comparing its prediction with the expression pattern of WUS in the wus mutant. Furthermore, the model can account for many experimental results including reorganization processes caused by the CZ ablation and by incision through the meristem center. We thus conclude that the reaction-diffusion dynamics is probably indispensable for the SAM development of plants.     

Centering the Organizing Center in the Arabidopsis thaliana Shoot Apical Meristem by a Combination of Cytokinin Signaling and Self-Organization

Plants have the ability to continously generate new organs by maintaining populations of stem cells throught their lives. The shoot apical meristem (SAM) provides a stable environment for the maintenance of stem cells. All cells inside the SAM divide, yet boundaries and patterns are maintained. Experimental evidence indicates that patterning is independent of cell lineage, thus a dynamic self-regulatory mechanism is required. A pivotal role in the organization of the SAM is played by the WUSCHEL gene (WUS). An important question in this regard is that how WUS expression is positioned in the SAM via a cell-lineage independent signaling mechanism. In this study we demonstrate via mathematical modeling that a combination of an inhibitor of the Cytokinin (CK) receptor, Arabidopsis histidine kinase 4 (AHK4) and two morphogens originating from the top cell layer, can plausibly account for the cell lineage-independent centering of WUS expression within SAM. Furthermore, our laser ablation and microsurgical experiments support the hypothesis that patterning in SAM occurs at the level of CK reception and signaling. The model suggests that the interplay between CK signaling, WUS/CLV feedback loop and boundary signals can account for positioning of the WUS expression, and provides directions for further experimental investigation.     

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.     

Isolation and characterization of a rice WUSCHEL-type homeobox gene that is specifically expressed in the central cells of a quiescent center in the root apical meristem

The Arabidopsis WUSCHEL (WUS) protein, which plays an important role in the specification of the stem cells in the shoot apical meristem (SAM), contains an 'atypical' homeodomain (HD) with extra residues in its loop and turn regions. We speculated that a WUS-type atypical HD protein might also be involved in the specification and maintenance of the root apical meristem (RAM) stem cells of rice. To investigate this possibility, we isolated and characterized a rice WUS-type homeobox gene designated quiescent-center-specific homeobox (QHB) gene. Using transformants carrying the QHB promoter-GUS and in situ hybridization, we found that QHB was specifically expressed in the central cells of a quiescent center (QC) of the root. During embryogenesis and crown root formation, QHB expression was observed prior to the morphological differentiation of the root. However, we detected different QHB expression patterns in the process of the RAM development, specifically between radicle and crown root formation, suggesting that the cell-fate determination of the QC may be controlled by different mechanisms. We also produced transformants that overexpress QHB or Arabidopsis WUS. These transformants did not form crown roots, but developed multiple shoots from ectopic SAMs with malformed leaves. On the basis of these observations, we propose that the WUS-type homeobox gene is involved in the specification and maintenance of the stem cells (QC cells) in the RAM, by a mechanism similar to that for WUS in the SAM.     

The CLAVATA signaling pathway mediating stem cell fate in shoot meristems requires Ca2+ as a secondary cytosolic messenger

CLAVATA1 (CLV1) is a receptor protein expressed in the shoot apical meristem (SAM) that translates perception of a non‐cell‐autonomous CLAVATA3 (CLV3) peptide signal into altered stem cell fate. CLV3 reduces expression of WUSCHEL (WUS) and FANTASTIC FOUR 2 (FAF2) in the SAM. Expression of WUS and FAF2 leads to maintenance of undifferentiated stem cells in the SAM. CLV3 binding to CLV1 inhibits expression of these genes and controls stem cell fate in the SAM through an unidentified signaling pathway. Cytosolic Ca²⁺ elevations, cyclic nucleotide (cGMP)‐activated Ca²⁺ channels, and cGMP have been linked to signaling downstream of receptors similar to CLV1. Hence, we hypothesized that cytosolic Ca²⁺ elevation mediates the CLV3 ligand/CLV1 receptor signaling that controls meristem stem cell fate. CLV3 application to Arabidopsis seedlings results in elevation of cytosolic Ca²⁺ and cGMP. CLV3 control of WUS was prevented in a genotype lacking a functional cGMP‐activated Ca²⁺ channel. In wild‐type plants, CLV3 inhibition of WUS and FAF2 expression was impaired by treatment with either a Ca²⁺ channel blocker or a guanylyl cyclase inhibitor. When CLV3‐dependent repression of WUS is blocked, altered control of stem cell fate leads to an increase in SAM size; we observed a larger SAM size in seedlings treated with the Ca²⁺ channel blocker. These results suggest that the CLV3 ligand/CLV1 receptor system initiates a signaling cascade that elevates cytosolic Ca²⁺, and that this cytosolic secondary messenger is involved in the signal transduction cascade linking CLV3/CLV1 to control of gene expression and stem cell fate in the SAM.     

Developmental events and shoot apical meristem gene expression patterns during shoot development in Arabidopsis thaliana

Critical developmental and gene expression profiles were charted during the formation of shoots from root explants in Arabidopsis tissue culture. Shoot organogenesis is a two-step process involving pre-incubation on an auxin-rich callus induction medium (CIM) during which time root explants acquire competence to form shoots during subsequent incubation on a cytokinin-rich shoot induction medium (SIM). At a histological level, the organization of shoot apical meristems (SAMs) appears to occur during incubation on SIM about the time of shoot commitment, i.e. the transition from hormone-dependent to hormone-independent shoot development. Genes involved in SAM formation, such as SHOOTMERISTEMLESS (STM) and CLAVATA1 (CLV1), were upregulated at about the time of shoot commitment, while WUSCHEL (WUS) was upregulated somewhat earlier. Genes required for STM expression, such as CUP-SHAPED COTYLEDON 1 and 2 (CUC1 and 2) were upregulated prior to shoot commitment. Gene expression patterns were determined for two GFP enhancer trap lines with tissue-specific expression in the SAM, including one line reporting on CUC1 expression. CUC1 was generally expressed in callus tissue during early incubation on SIM, but later CUC1 was expressed more locally in presumptive sites of shoot formation. In contrast, the expression pattern of the enhancer trap lines during zygotic embryogenesis was more localized to the presumptive SAM even in early stages of embryogenesis.     

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.     

Contributions of individual amino acid residues to the endogenous CLV3 function in shoot apical meristem maintenance in Arabidopsis

As a peptide hormone, CLV3 restricts the stem cell number in shoot apical meristem (SAM) by interacting with CLV1/CLV2/CRN/RPK2 receptor complexes. To elucidate how the function of the CLV3 peptide in SAM maintenance is established at the amino acid (AA) level, alanine substitutions were performed by introducing point mutations to individual residues in the peptide-coding region of CLV3 and its flanking sequences. Constructs carrying such substitutions, expressed under the control of CLV3 regulatory elements, were transformed to the clv3-2 null mutant to evaluate their efficiencies in complementing its defects in SAMs in vivo. These studies showed that aspartate-8, histidine-11, glycine-6, proline-4, arginine-1, and proline-9, arranged in an order of importance, were critical, while threonine-2, valine-3, serine-5, and the previously assigned hydroxylation and arabinosylation residue proline-7 were trivial for the endogenous CLV3 function in SAM maintenance. In contrast, substitutions of flanking residues did not impose much damage on CLV3. Complementation of different alanine-substituted constructs was confirmed by measurements of the sizes of SAMs and the WUS expression levels in transgenic plants. These studies established a complete contribution map of individual residues in the peptide-coding region of CLV3 for its function in SAM, which may help to understand peptide hormones in general.