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1.  Signaling in the Arabidopsis shoot meristem stem cell niche correlates with ligand-dependent trafficking of the CLV1 receptor kinase 
Current biology : CB  2011;21(5):345-352.
Summary
Cell numbers in above-ground meristem types of plants are thought to be maintained by a feedback loop driven by perception of the glycopeptide ligand CLAVATA3 (CLV3) by the CLAVATA1 (CLV1) receptor kinase and the CLV2/CORYNE (CRN) receptor-like complex [1]. CLV3 made in the stem cells at the meristem apex limits the expression level of the stem cell-promoting homeodomain protein WUSCHEL (WUS) in the cells beneath, where CLV1 as well as WUS RNA are localized. WUS downregulation nonautonomously reduces stem cell proliferation. High-level overexpression of CLV3 eliminates the stem cells and causes meristem termination [2], and loss of CLV3 function allows meristem overproliferation [3]. There are many open questions regarding the CLV3/CLV1 interaction, including where in the meristem it occurs, how it is regulated, and how it is that a large range of CLV3 concentrations gives no meristem size phenotype [4]. Here we use genetics and live imaging to examine the cell biology of CLV1 in Arabidopsis meristematic tissue. We demonstrate that plasma membrane-localized CLV1 is reduced in concentration by CLV3, which causes trafficking of CLV1 to lytic vacuoles. We find that changes in CLV2 activity have no detectable effects on CLV1 levels. We also find that CLV3 appears to diffuse broadly in meristems, contrary to a recent sequestration model which states that CLV3 is quantitatively bound by CLV1 in the apical regions of the meristem, allowing continued WUS activity in lower regions [5]. This study provides a new model for CLV1 function in plant stem cell maintenance and suggests that downregulation of plasma membrane-localized CLV1 by its CLV3 ligand can account for the buffering of CLV3 signaling in the maintenance of stem cell pools in plants.
doi:10.1016/j.cub.2011.01.039
PMCID: PMC3072602  PMID: 21333538
2.  FON2 SPARE1 Redundantly Regulates Floral Meristem Maintenance with FLORAL ORGAN NUMBER2 in Rice 
PLoS Genetics  2009;5(10):e1000693.
CLAVATA signaling restricts stem cell identity in the shoot apical meristem (SAM) in Arabidopsis thaliana. In rice (Oryza sativa), FLORAL ORGAN NUMBER2 (FON2), closely related to CLV3, is involved as a signaling molecule in a similar pathway to negatively regulate stem cell proliferation in the floral meristem (FM). Here we show that the FON2 SPARE1 (FOS1) gene encoding a CLE protein functions along with FON2 in maintenance of the FM. In addition, FOS1 appears to be involved in maintenance of the SAM in the vegetative phase, because constitutive expression of FOS1 caused termination of the vegetative SAM. Genetic analysis revealed that FOS1 does not need FON1, the putative receptor of FON2, for its action, suggesting that FOS1 and FON2 may function in meristem maintenance as signaling molecules in independent pathways. Initially, we identified FOS1 as a suppressor that originates from O. sativa indica and suppresses the fon2 mutation in O. sativa japonica. FOS1 function in japonica appears to be compromised by a functional nucleotide polymorphism (FNP) at the putative processing site of the signal peptide. Sequence comparison of FOS1 in about 150 domesticated rice and wild rice species indicates that this FNP is present only in japonica, suggesting that redundant regulation by FOS1 and FON2 is commonplace in species in the Oryza genus. Distribution of the FNP also suggests that this mutation may have occurred during the divergence of japonica from its wild ancestor. Stem cell maintenance may be regulated by at least three negative pathways in rice, and each pathway may contribute differently to this regulation depending on the type of the meristem. This situation contrasts with that in Arabidopsis, where CLV signaling is the major single pathway in all meristems.
Author Summary
The body plan of plants is regulated by the function of apical meristems that are generated in the embryo. Leaves and floral organs are derived from cells supplied by stem cells in the vegetative shoot apical meristem (SAM) and the floral meristem (FM), respectively. Thus, genetic regulation of stem cell maintenance is a central issue in plant development. In the model plant Arabidopsis thaliana, CLAVATA3 (CLV3) functions as a key signaling molecule to restrict the size of the stem cell population in both the SAM and the FM. In rice, however, we show here that two CLV3-like genes, FLORAL ORGAN NUMBER2 (FON2) and FON2 SPARE1 (FOS1), redundantly regulate maintenance of the FM. We also show that FOS1 is likely to be involved in maintenance of the vegetative SAM, whereas FON2 plays no role in regulation in this meristem. FOS1 appears to act via a putative receptor that differs from the FON2 receptor, suggesting that these two signaling molecules function in independent pathways to restrict stem cells in different ways depending on the type of meristem. In addition, we show that the FOS1 gene was compromised in the standard rice, Oryza sativa spp. japonica, during the evolution of rice.
doi:10.1371/journal.pgen.1000693
PMCID: PMC2752996  PMID: 19834537
3.  Translational Control of Arabidopsis Meristem Stability and Organogenesis by the Eukaryotic Translation Factor eIF3h 
PLoS ONE  2014;9(4):e95396.
Essentially all aboveground plant tissues develop from the stem cells in the primary shoot apical meristem. Proliferation of the stem cell population in the Arabidopsis shoot apical meristem is tightly controlled by a feedback loop formed primarily by the homeodomain transcription factor WUSCHEL (WUS) and the CLAVATA ligand-receptor system. In this study, it is shown that mutation of a translation initiation factor, eIF3h, causes a tendency to develop a strikingly enlarged shoot apical meristem with elevated and ectopic expression of WUS and CLAVATA3 (CLV3). Many of the mRNAs that function in apical meristem maintenance possess upstream open reading frames (uORFs), translational attenuators that render translation partially dependent on eIF3h. Specifically, the mRNA for the receptor kinase, CLV1, is undertranslated in the eif3h mutant as shown by transient and transgenic expression assays. Concordant phenotypic observations include defects in organ polarity and in translation of another uORF-containing mRNA, ASYMMETRIC LEAVES 1 (AS1), in eif3h. In summary, the expression of developmental regulatory mRNAs is attenuated by uORFs, and this attenuation is balanced in part by the translation initiation factor, eIF3h. Thus, translational control plays a key role in Arabidopsis stem cell regulation and organogenesis.
doi:10.1371/journal.pone.0095396
PMCID: PMC3988188  PMID: 24736281
4.  Multiple receptor complexes assembled for transmitting CLV3 signaling in Arabidopsis 
Plant Signaling & Behavior  2010;5(3):300-302.
In Arabidopsis, the feedback regulatory loop between CLAVATA3 (CLV3) signaling pathway and transcription factor, WUSCHEL (WUS) plays a significant role in shoot apical meristems (SAM) maintenance. Previously, CLV1/CLV2 heterodimers were supposed to perceive and transmit CLV3 signaling. Recent genetic analysis isolated a novel receptor kinase, CORYNE (CRN), which was found to be involved in the CLV3 pathway. Therefore, new hypothesis was put forward that CRN probably acts with CLV2 to transmit CLV3 in parallel with CLV1 based on genetic analysis. In our recent work, we took advantage of firefly luciferase complementation imaging (LCI) assay to analyze the interactions among CLV1, CLV2 and CRN in both Arabidopsis thaliana protoplasts and Nicotiana benthamiana leaves. We identified the physical interaction between CLV2 and CRN in the absence of CLV3 and found some interesting phenomenon such as CLV1, CLV2 and CRN may form a complex, and that CRN was able to form homodimers. These new observations make the relationships among these three proteins more complex than that indicated in two-parallel pathway model. Combining current genetic and our new biochemical evidence, a more possible and detailed model for CLV3 pathway was developed.
PMCID: PMC2881284  PMID: 20220313
CLAVATA3 (CLV3) signaling pathway; firefly luciferase complementation imaging (LCI) assay; Arabidopsis thaliana protoplasts; Nicotiana benthamiana leaves; homodimers
5.  The ERECTA receptor kinase regulates Arabidopsis shoot apical meristem size, phyllotaxy and floral meristem identity 
Development (Cambridge, England)  2014;141(4):830-841.
In plants, the shoot apical meristem (SAM) serves as a reservoir of pluripotent stem cells from which all above ground organs originate. To sustain proper growth, the SAM must maintain homeostasis between the self-renewal of pluripotent stem cells and cell recruitment for lateral organ formation. At the core of the network that regulates this homeostasis in Arabidopsis are the WUSCHEL (WUS) transcription factor specifying stem cell fate and the CLAVATA (CLV) ligand-receptor system limiting WUS expression. In this study, we identified the ERECTA (ER) pathway as a second receptor kinase signaling pathway that regulates WUS expression, and therefore shoot apical and floral meristem size, independently of the CLV pathway. We demonstrate that reduction in class III HD-ZIP and ER function together leads to a significant increase in WUS expression, resulting in extremely enlarged shoot meristems and a switch from spiral to whorled vegetative phyllotaxy. We further show that strong upregulation of WUS in the inflorescence meristem leads to ectopic expression of the AGAMOUS homeotic gene to a level that switches cell fate from floral meristem founder cell to carpel founder cell, suggesting an indirect role for ER in regulating floral meristem identity. This work illustrates the delicate balance between stem cell specification and differentiation in the meristem and shows that a shift in this balance leads to abnormal phyllotaxy and to altered reproductive cell fate.
doi:10.1242/dev.104687
PMCID: PMC3930468  PMID: 24496620
AGAMOUS (AG); Cell fate; WUSCHEL (WUS)
6.  The Function of the CLE Peptides in Plant Development and Plant-Microbe Interactions 
The CLAVATA3 (CLV3)/ENDOSPERM SURROUNDING REGION (ESR) (CLE) peptides consist of 12 or 13 amino acids, including hydroxylated proline residues that may or may not contain sugar modifications, and function in a non-cell-autonomous fashion. The CLE gene was first reported in Zea mays (maize) as an endosperm-specific gene, ESR, in 1997 (Opsahl-Ferstad et al., 1997). CLE genes encode secreted peptides that function in the extracellular space as intercellular signaling molecules and bind to cellular surface receptor-like proteins to transmit a signal. CLE peptides regulate various physiological and developmental processes and its signaling pathway are conserved in diverse land plants. Recent CLE functional studies have pointed to their significance in regulating meristematic activity in plant meristems, through the CLE-receptor kinase-WOX signaling node. CLV3 and CLE40 are responsible for maintenance of shoot apical meristem (SAM) and root apical meristem (RAM) function, regulating homeodomain transcription factors, WUSCHEL (WUS) and WUSCHEL-related homeobox 5 (WOX5), respectively. CLE and WOX form an interconnected and self-correcting feedback loop to provide robustness to stem cell homeostasis. CLE peptides are required for certain plant-microbe interactions, such as those that occur during legume symbiosis and phytopathogenic nematode infection. Understanding the molecular properties of CLE peptides may provide insight into plant cell-cell communication, and therefore also into plant-microbe interactions.
doi:10.1199/tab.0149
PMCID: PMC3268505  PMID: 22303273
7.  WUSCHEL protein movement and stem cell homeostasis 
Plant Signaling & Behavior  2012;7(5):592-594.
Stem cell maintenance is essential for growth and development of plants and animals. Similar to animal studies, transcription factors play a critical role in plant stem cell maintenance, however the regulatory logic is not well understood. Shoot apical meristems (SAMs) harbor a pool of pluoripotent stem cells and they provide cells for the development of all above-ground organs. Molecular genetic studies spanning more than a decade have revealed cell-cell communication logic underlying stem cell homeostasis. WUSCHEL (WUS), a homeodomain transcription factor expressed in cells of the organizing center specifies stem cells in overlying cells of the central zone (CZ) and also activates a negative regulator-CLAVATA3 (CLV3). CLV3, a small secreted peptide, binds to CLAVATA1 (CLV1) and also possibly to CLV1-related receptors to activate signaling which restricts WUS transcription. Though the CLV-WUS feedback network explains the cell-cell communication logic of stem cell maintenance, how WUS communicates with adjacent cells had remained elusive. In October 15 2011 issue of Genes and Development, we report that WUS protein synthesized in cells of organizing center migrates into adjacent cells via cell-cell movement and activates CLV3 transcription by directly binding to promoter elements.
doi:10.4161/psb.19793
PMCID: PMC3419026  PMID: 22516820
shoot apical meristem; CLAVATA3; CLAVATA1; central zone and peripheral zone
8.  CCS52A2/FZR1, a cell cycle regulator, is an essential factor for shoot apical meristem maintenance in Arabidopsis thaliana 
BMC Plant Biology  2012;12:135.
Background
Cell division and cell fate decisions regulate organ formation and function in plant growth and development. It is still unclear how specific meristematic regulatory networks operate with the cell cycle machinery to translate stem cell identity and maintenance into cellular behavior. In this study, we address these questions by analysis of a shoot apex defective mutant, namely xcm9.
Results
Phenotypic analysis of the xcm9 mutant reveals concomitant premature termination of floral shoots with frequent bifurcation of the shoot apices, stems, and flowers. Microscopic observations show irregular cell organization in shoot apical meristems of xcm9. Positional cloning revealed that xcm9 is a loss of function allele of the CCS52A2/FZR1 gene, which has previously been implicated in root development. Expression analysis demonstrated that CCS52A2 maintains a higher transcriptional expression level in actively dividing tissue. Genetic studies indicated that the CCS52A2 gene functions together with WUSCHEL (WUS) and CLAVATA3 (CLV3) in regulating the development of the shoot meristem, and also contributes to this regulation together with the chromatin remodeling pathway. In addition, fewer xcm9 cells express CYCLIN B1:1, showing that cell cycle progression is disrupted in the mutant.
Conclusion
We propose that the CCS52A2 gene is a mediator that functions together with meristematic genes to regulate meristem organization, and cross-functions with chromatin regulators in cell cycle progression during shoot apical meristem development.
doi:10.1186/1471-2229-12-135
PMCID: PMC3534500  PMID: 22873486
9.  CLAVATA2 forms a distinct CLE-binding receptor complex regulating Arabidopsis stem cell specification 
SUMMARY
CLAVATA1 (CLV1), CLV2, CLV3, CORYNE (CRN), BAM1 and BAM2 are key regulators that function at the shoot apical meristem (SAM) of plants to promote differentiation by limiting the size of the organizing center that maintains stem cell identity in neighboring cells. Previous results have indicated that the extracellular domain of the receptor-kinase CLV1 binds to the CLV3-derived CLE ligand. The biochemical role of receptor-like protein CLV2 has remained largely unknown. While genetic analysis suggested that CLV2, together with the membrane kinase CRN, act in parallel with CLV1, recent studies using transient expression indicated that CLV2 and CRN from a complex with CLV1. Here we report evidence for distinct CLV2/CRN heteromultimeric and CLV1/BAM multimeric complexes in transient expression and in Arabidopsis. Weaker interactions between the two complexes were detectable in transient expression. We also find that CLV2 alone generates a membrane-localized CLE binding activity independent of CLV1. CLV2, CLV1 and the CLV1 homologs BAM1 and BAM2 all bind to the CLV3-derived CLE peptide with similar kinetics, but BAM receptors show a broader range of interactions with different CLE peptides. Finally, we show that BAM and CLV1 over-expression can compensate for the loss of CLV2 function in vivo. These results suggest two parallel ligand-binding receptor complexes controlling stem cell specification in Arabidopsis.
doi:10.1111/j.1365-313X.2010.04295.x
PMCID: PMC2974754  PMID: 20626648
CLAVATA2; CLE peptide; CORYNE; CLAVATA1; receptor complex; meristem
10.  CLE genes may act in a variety of tissues/cells and involve other signaling cascades in addition to CLV3-WUS-like pathways 
Plant Signaling & Behavior  2011;6(1):105-108.
CLE, which is the term for the CLV3/ESR-related gene family, is thought to participate in CLAVATA3-WUSCHEL (CLV3-WUS) and CLV3-WUS-like signaling pathways to regulate meristem activity in plant. Although some CLE genes are expressed in meristems, many CLE genes appear to express in a variety of tissues/cells. Here we report that CLE14 and CLE20 express in various specific tissues/cells outside the shoot/root apical meristem (SAM/RAM), including in highly differentiated cells, and at different developmental stages. Overexpressing CLE14 or CLE20 also causes multiple phenotypes, which is consistent with its expression pattern in Arabidopsis. These results suggest that CLE genes may play multiple roles and involve other signaling cascades in addition to the CLV3-WUS and CLV3-WUS-like pathways.
doi:10.4161/psb.6.1.14186
PMCID: PMC3122018  PMID: 21270538
CLE; CLAVATA3-WUSCHEL; cell signaling and development; root apical meristem; arabidopsis
11.  The auxin signalling network translates dynamic input into robust patterning at the shoot apex 
We provide a comprehensive expression map of the different genes (TIR1/AFBs, ARFs and Aux/IAAs) involved in the signalling pathway regulating gene transcription in response to auxin in the shoot apical meristem (SAM).We demonstrate a relatively simple structure of this pathway using a high-throughput yeast two-hybrid approach to obtain the Aux/IAA-ARF full interactome.The topology of the signalling network was used to construct a model for auxin signalling and to predict a role for the spatial regulation of auxin signalling in patterning of the SAM.We used a new sensor to monitor the input in the auxin signalling pathway and to confirm the model prediction, thus demonstrating that auxin signalling is essential to create robust patterns at the SAM.
The plant hormone auxin is a key morphogenetic signal involved in the control of cell identity throughout development. A striking example of auxin action is at the shoot apical meristem (SAM), a population of stem cells generating the aerial parts of the plant. Organ positioning and patterning depends on local accumulations of auxin in the SAM, generated by polar transport of auxin (Vernoux et al, 2010). However, it is still unclear how auxin is distributed at cell resolution in tissues and how the hormone is sensed in space and time during development. A complex ensemble of 29 Aux/IAAs and 23 ARFs is central to the regulation of gene transcription in response to auxin (for review, see Leyser, 2006; Guilfoyle and Hagen, 2007; Chapman and Estelle, 2009). Protein–protein interactions govern the properties of this transduction pathway (Del Bianco and Kepinski, 2011). Limited interaction studies suggest that, in the absence of auxin, the Aux/IAA repressors form heterodimers with the ARF transcription factors, preventing them from regulating target genes. In the presence of auxin, the Aux/IAA proteins are targeted to the proteasome by an SCF E3 ubiquitin ligase complex (Chapman and Estelle, 2009; Leyser, 2006). In this process, auxin promotes the interaction between Aux/IAA proteins and the TIR1 F-box of the SCF complex (or its AFB homologues) that acts as an auxin co-receptor (Dharmasiri et al, 2005a, 2005b; Kepinski and Leyser, 2005; Tan et al, 2007). The auxin-induced degradation of Aux/IAAs would then release ARFs to regulate transcription of their target genes. This includes activation of most of the Aux/IAA genes themselves, thus establishing a negative feedback loop (Guilfoyle and Hagen, 2007). Although this general scenario provides a framework for understanding gene regulation by auxin, the underlying protein–protein network remains to be fully characterized.
In this paper, we combined experimental and theoretical analyses to understand how this pathway contributes to sensing auxin in space and time (Figure 1). We first analysed the expression patterns of the ARFs, Aux/IAAs and TIR1/AFBs genes in the SAM. Our results demonstrate a general tendency for most of the 25 ARFs and Aux/IAAs detected in the SAM: a differential expression with low levels at the centre of the meristem (where the stem cells are located) and high levels at the periphery of the meristem (where organ initiation takes place). We also observed a similar differential expression for TIR1/AFB co-receptors. To understand the functional significance of the distribution of ARFs and Aux/IAAs in the SAM, we next investigated the global structure of the Aux/IAA-ARF network using a high-throughput yeast two-hybrid approach and uncover a rather simple topology that relies on three basic generic features: (i) Aux/IAA proteins interact with themselves, (ii) Aux/IAA proteins interact with ARF activators and (iii) ARF repressors have no or very limited interactions with other proteins in the network.
The results of our interaction analysis suggest a model for the Aux/IAA-ARF signalling pathway in the SAM, where transcriptional activation by ARF activators would be negatively regulated by two independent systems, one involving the ARF repressors, the other the Aux/IAAs. The presence of auxin would remove the inhibitory action of Aux/IAAs, but leave the ARF repressors to compete with ARF activators for promoter-binding sites. To explore the regulatory properties of this signalling network, we developed a mathematical model to describe the transcriptional output as a function of the signalling input that is the combinatorial effect of auxin concentration and of its perception. We then used the model and a simplified view of the meristem (where the same population of Aux/IAAs and ARFs exhibit a low expression at the centre and a high expression in the peripheral zone) for investigating the role of auxin signalling in SAM function. We show that in the model, for a given ARF activator-to-repressor ratio, the gene induction capacity increases with the absolute levels of ARF proteins. We thus predict that the differential expression of the ARFs generates differences in auxin sensitivities between the centre (low sensitivity) and the periphery (high sensitivity), and that the expression of TIR1/AFB participates to this regulation (prediction 1). We also use the model to analyse the transcriptional response to rapidly changing auxin concentrations. By simulating situations equivalent either to the centre or the periphery of our simplified representation of the SAM, we predict that the signalling pathway buffers its response to the auxin input via the balance between ARF activators and repressors, in turn generated by their differential spatial distributions (prediction 2).
To test the predictions from the model experimentally, we needed to assess both the input (auxin level and/or perception) and the output (target gene induction) of the signalling cascade. For measuring the transcriptional output, the widely used DR5 reporter is perfectly adapted (Figure 5) (Ulmasov et al, 1997; Sabatini et al, 1999; Benkova et al, 2003; Heisler et al, 2005). For assaying pathway input, we designed DII-VENUS, a novel auxin signalling sensor that comprises a constitutively expressed fusion of the auxin-binding domain (termed domain II or DII) (Dreher et al, 2006; Tan et al, 2007) of an IAA to a fast-maturating variant of YFP, VENUS (Figure 5). The degradation patterns from DII-VENUS indicate a high auxin signalling input both in flower primordia and at the centre of the SAM. This is in contrast to the organ-specific expression pattern of DR5::VENUS (Figure 5). These results indicate that the signalling pathway limits gene activation in response to auxin at the meristem centre and confirm the differential sensitivity to auxin between the centre and the periphery (prediction 1). We further confirmed the buffering capacities of the signalling pathway (prediction 2) by carrying out live imaging experiments to monitor DII-VENUS and DR5::VENUS expression in real time (Figure 5). This analysis reveals the presence of important temporal variations of DII-VENUS fluorescence, while DR5::VENUS does not show such global variations. Our approach thus provides evidence that the Aux/IAA-ARF pathway has a key role in patterning in the SAM, alongside the auxin transport system. Our results illustrate how the tight spatio-temporal regulation of both the distribution of a morphogenetic signal and the activity of the downstream signalling pathway provides robustness to a dynamic developmental process.
A comprehensive expression and interaction map of auxin signalling factors in the Arabidopsis shoot apical meristem is constructed and used to derive a mathematical model of auxin signalling, from which key predictions are experimentally confirmed.
The plant hormone auxin is thought to provide positional information for patterning during development. It is still unclear, however, precisely how auxin is distributed across tissues and how the hormone is sensed in space and time. The control of gene expression in response to auxin involves a complex network of over 50 potentially interacting transcriptional activators and repressors, the auxin response factors (ARFs) and Aux/IAAs. Here, we perform a large-scale analysis of the Aux/IAA-ARF pathway in the shoot apex of Arabidopsis, where dynamic auxin-based patterning controls organogenesis. A comprehensive expression map and full interactome uncovered an unexpectedly simple distribution and structure of this pathway in the shoot apex. A mathematical model of the Aux/IAA-ARF network predicted a strong buffering capacity along with spatial differences in auxin sensitivity. We then tested and confirmed these predictions using a novel auxin signalling sensor that reports input into the signalling pathway, in conjunction with the published DR5 transcriptional output reporter. Our results provide evidence that the auxin signalling network is essential to create robust patterns at the shoot apex.
doi:10.1038/msb.2011.39
PMCID: PMC3167386  PMID: 21734647
auxin; biosensor; live imaging; ODE; signalling
12.  A Quantitative and Dynamic Model for Plant Stem Cell Regulation 
PLoS ONE  2008;3(10):e3553.
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.
doi:10.1371/journal.pone.0003553
PMCID: PMC2570333  PMID: 18958283
13.  Reaction-Diffusion Pattern in Shoot Apical Meristem of Plants 
PLoS ONE  2011;6(3):e18243.
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.
doi:10.1371/journal.pone.0018243
PMCID: PMC3066213  PMID: 21479227
14.  Spatial expression of CLAVATA3 in the shoot apical meristem suggests it is not a stem cell marker in soybean 
Journal of Experimental Botany  2013;64(18):5641-5649.
CLAVATA3 (CLV3), a stem cell marker in Arabidopsis thaliana, encodes a secreted peptide that maintains the stem cell population within the shoot apical meristem. This work investigated the CLV3 orthologue in a major legume crop, soybean (GmCLV3). Instead of being expressed in the three outermost layers of the meristem as in Arabidopsis, GmCLV3 was expressed deeper in the central zone beneath the fourth layer (L4) of the meristem, overlapping with the expression of soybean WUSCHEL. Subsequent investigation using an alternative stem cell marker (GmLOG1) revealed its expression within layers L2–L4, indicating that GmCLV3 is not a stem cell marker. Overexpression studies of GmCLV3 in Arabidopsis and complementation of clv3-2 mutant suggest similar functional capacity to that of Arabidopsis CLV3. The expression of soybean CLV1, which encodes a receptor for CLV3 in Arabidopsis, was not detectable in the central zone of the meristem via reverse-transcription PCR analysis of amplified RNA from laser-microdissected samples or in situ, implicating a diverged pathway in soybean. This study also reports the novel expression of GmLOG1 in initials of axillary meristem in the boundary region between the SAM and developing leaf primordia, before the expression of GmWUS or GmCLV3, indicating cytokinin as one of the earliest signals in initiating and specifying the stem cell population.
doi:10.1093/jxb/ert341
PMCID: PMC3871822  PMID: 24179098
CLAVATA3; cytokinin; shoot apical meristem; soybean; stem cell.
15.  Is the Arabidopsis root niche protected by sequestration of the CLE40 signal by its putative receptor ACR4? 
Plant Signaling & Behavior  2009;4(7):634-635.
A tight but also dynamic regulation is necessary to control the size of stem cell populations in response to internal and external cues. The stem cells of the Arabidopsis shoot and root meristems are governed by the niche cells of the organizing centre (OC) and the quiescent centre (QC), respectively. The well characterized CLV3/WUS negative feedback loop adjusts homeostasis of the stem cell population in the shoot. Here, the CLAVATA3 (CLV3) dodecapeptide, expressed by the stem cells, signals to repress WUSCHEL (WUS), which is expressed in the subjacent OC cells, and in turn activates CLV3 expression non-cell autonomously. However, a similar signaling module controlling the root stem cell population was as yet unknown. In the June issue of Current Biology we report on such a signaling module comprising CLE40 (a CLV3 homologue) that acts via the receptor kinase Arabidopsis Crinkly4 (ACR4) to repress the WUS homologue WOX5 which maintains distal root stem cells. Furthermore, we showed that CLE40 peptide (CLE40p) treatment upregulates ACR4 expression. In this Addendum, we are further elaborating our hypothesis in which the upregulation of ACR4 as a consequence of ectopic CLE40p builds a protective barrier for the QC niche cells.
PMCID: PMC2710560  PMID: 19820344
arabidopsis; root meristem; niche; stem cells; QC; CLE40; ACR4; WOX5
16.  Strigolactone Can Promote or Inhibit Shoot Branching by Triggering Rapid Depletion of the Auxin Efflux Protein PIN1 from the Plasma Membrane 
PLoS Biology  2013;11(1):e1001474.
Shoot branching is regulated by competition between branches to export the phytohormone auxin into the main stem. The phytohormone strigolactone balances shoot system growth by making auxin export harder to establish, thus modulating the auxin transport network.
Plants continuously extend their root and shoot systems through the action of meristems at their growing tips. By regulating which meristems are active, plants adjust their body plans to suit local environmental conditions. The transport network of the phytohormone auxin has been proposed to mediate this systemic growth coordination, due to its self-organising, environmentally sensitive properties. In particular, a positive feedback mechanism termed auxin transport canalization, which establishes auxin flow from active shoot meristems (auxin sources) to the roots (auxin sinks), has been proposed to mediate competition between shoot meristems and to balance shoot and root growth. Here we provide strong support for this hypothesis by demonstrating that a second hormone, strigolactone, regulates growth redistribution in the shoot by rapidly modulating auxin transport. A computational model in which strigolactone action is represented as an increase in the rate of removal of the auxin export protein, PIN1, from the plasma membrane can reproduce both the auxin transport and shoot branching phenotypes observed in various mutant combinations and strigolactone treatments, including the counterintuitive ability of strigolactones either to promote or inhibit shoot branching, depending on the auxin transport status of the plant. Consistent with this predicted mode of action, strigolactone signalling was found to trigger PIN1 depletion from the plasma membrane of xylem parenchyma cells in the stem. This effect could be detected within 10 minutes of strigolactone treatment and was independent of protein synthesis but dependent on clathrin-mediated membrane trafficking. Together these results support the hypothesis that growth across the plant shoot system is balanced by competition between shoot apices for a common auxin transport path to the root and that strigolactones regulate shoot branching by modulating this competition.
Author Summary
Plants can adapt their form to suit the environment in which they are growing. For example, genetically identical plants can develop as a single unbranched stem or as a highly ramified bush. This broad developmental potential is possible because the shoot system is produced continuously by growing tips, known as shoot meristems. Meristems produce the stem and leaves of a shoot, and at the base of each leaf, a new meristem is formed. This meristem can remain dormant as a small bud or activate to produce a branch. Thus, the shoot system is a community of shoot meristems, the combined activity and inactivity of which shape shoot form. Here we provide evidence that growth is balanced across the Arabidopsis shoot system by competition between the shoot meristems. This competition is likely mediated by the requirement of meristems to export the plant hormone auxin in order to activate bud outgrowth. In our model, auxin in the main stem, exported from active branches, can prevent auxin export by dormant buds, thus preventing their activation. Our findings show that a second hormone, strigolactone, increases the level of competition between branches by making auxin export harder to establish. Together, these hormones balance growth across the shoot system, adjusting it according to the environmental conditions in which a plant is growing.
doi:10.1371/journal.pbio.1001474
PMCID: PMC3558495  PMID: 23382651
17.  Influence of clavata3-2 mutation on early flower development in Arabidopsis thaliana: quantitative analysis of changing geometry 
Journal of Experimental Botany  2008;60(2):679-695.
Early development of the flower primordium has been studied in Arabidopsis thaliana clavata3-2 (clv3-2) plants with the aid of sequential in vivo replicas and longitudinal microtome sections. Sequential replicas show that, although there is no regular phyllotaxis in the clv3-2 inflorescence shoot apex, the sites of new primordium formation are, to a large extent, predictable. The primordium always appears in a wedge-like region of the meristem periphery flanked by two older primordia. In general, stages of primordium development in clv3-2 are similar to the wild type, but quantitative geometry analysis shows that the clv3-2 primordium shape is affected even before the CLAVATA/WUSCHEL regulatory network would start to operate in the wild-type primordium. The shape of the youngest primordium in the mutant is more variable than in the wild type. In particular, the shape of the adaxial primordium boundary varies and seems to be related to the shape of the space available for the given primordium formation, suggesting that physical constraints play a significant role in primordium shape determination. The role of physical constraints is also manifested in that the shape of the primordium in the later stages, as well as the number and position of sepals, are adjusted to the available space. Longitudinal sections of clv3-2 apices show that the shape of surface cells of the meristem and young primordium is different from the wild type. Moreover, there is only one tunica layer in both the meristem and in the primordium until it becomes a bulge that is distinctly separated from the meristem. Starting from this stage, the anticlinal divisions predominate in subprotodermal cells, suggesting that the distribution of periclinal and anticlinal cell divisions in the early development of the flower primordium is not directly affected by the clv3-2 mutation.
doi:10.1093/jxb/ern312
PMCID: PMC2651453  PMID: 19088334
Arabidopsis thaliana; clavata3-2; early flower development; shoot apex; primordium geometry
18.  Histone H4R3 Methylation Catalyzed by SKB1/PRMT5 Is Required for Maintaining Shoot Apical Meristem 
PLoS ONE  2013;8(12):e83258.
The shoot apical meristem (SAM) is the source of all of the above-ground tissues and organs in post-embryonic development in higher plants. Studies have proven that the expression of genes constituting the WUSCHEL (WUS)-CLAVATA (CLV) feedback loop is critical for the SAM maintenance. Several histone lysine acetylation and methylation markers have been proven to regulate the transcription level of WUS. However, little is known about how histone arginine methylation regulates the expression of WUS and other genes. Here, we report that H4R3 symmetric dimethylation (H4R3sme2) mediated by SKB1/PRMT5 represses the expression of CORYNE (CRN) to maintain normal SAM geometrics. SKB1 lesion results in small SAM size in Arabidopsis, as well as down-regulated expression of WUS and CLV3. Up-regulation of WUS expression enlarges SAM size in skb1 mutant plants. We find that SKB1 and H4R3sme2 associate with the chromatin of the CRN locus to down-regulate its transcription. Mutation of CRN rescues the expression of WUS and the small SAM size of skb1. Thus, SKB1 and SKB1-mediated H4R3sme2 are required for the maintenance of SAM in Arabidopsis seedlings.
doi:10.1371/journal.pone.0083258
PMCID: PMC3861506  PMID: 24349476
19.  Membrane distributions of two ligand-binding receptor complexes in the CLAVATA pathway 
Plant Signaling & Behavior  2010;5(11):1442-1445.
Genetic studies have suggested that transmembrane proteins CLAVATA1 (CLV1), CLV2, CORYNE (CRN), BAM1 and BAM2 all play a role in relaying the CLV3 signal and thus regulating stem cell homeostasis at the shoot meristem (SM). The extracellular domain of CLV1 was previously shown to bind the CLE peptide derived from CLV3, providing direct evidence that CLV3-CLV1 function as a ligand-receptor pair. How the other putative receptors function in the CLV pathway, however, remained unclear. We demonstrated in a recent Plant Journal article that the receptor-like protein CLV2 and the receptor-kinases BAM1 and BAM2 also bind to the CLV3 CLE peptide ligand with an affinity similar to that of CLV1. Critically, these ligand binding receptors form two distinct complexes in both transient expression in tobacco and in Arabidopsis meristem cells: a CLV2/CRN multimer and a CLV1/BAM multimer. Here we examine in detail the subcellular membrane partitioning for the receptor proteins in transient expression by two-phase partitioning and co-expression with known subcellular markers. All tested proteins measurably accumulate at the plasma membrane. While CLV1 primarily co-localizes with a plasma membrane marker, CLV2 shows greater co-localization with an endoplasmic reticulum (ER) marker.
doi:10.4161/psb.5.11.13359
PMCID: PMC3115250  PMID: 21051944
CLAVATA2; CLAVATA1; CORYNE; subcellular localization; plasma membrane; endoplasmic reticulum; receptor complex; meristem
20.  Mitogen-Activated Protein Kinase Regulated by the CLAVATA Receptors Contributes to Shoot Apical Meristem Homeostasis 
Plant and Cell Physiology  2010;52(1):14-29.
In Arabidopsis, the CLAVATA (CLV) pathway operates in the regulation of the size of the stem cell population in the shoot apical meristem (SAM). CLV3 functions as a small peptide ligand to negatively regulate the expression of the WUSCHEL (WUS) transcription factor through three major receptor kinase complexes of CLV1, CLV2-SUPPRESSOR OF LLP1-2 (SOL2)/CORYNE (CRN) and recently identified RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2)/TOADSTOOL 2 (TOAD2). Aiming to understand the precise molecular details of CLV3 signaling, we investigated the contribution of phospho-signaling, potentially regulated by these kinase complexes, to the CLV pathway. We detected CLV3-triggered CLV1 phosphorylation, which is also conditioned by the rest of the CLV receptors, presumably by their direct association. Our comprehensive analysis of the activities of the respective CLV receptors on mitogen-activated protein kinases (MAPKs) suggested that the precise balanced regulation of MAPK activity by the CLV receptors is likely to be key for SAM homeostasis.
doi:10.1093/pcp/pcq157
PMCID: PMC3023851  PMID: 20965998
Arabidopsis; CLAVATA; CLE; MAPK; Nicotiana benthamiana; Shoot apical meristem
21.  The CUC1 and CUC2 genes promote carpel margin meristem formation during Arabidopsis gynoecium development 
Carpel margin meristems (CMMs), a pair of meristematic tissues present along the margins of two fused carpel primordia of Arabidopsis thaliana, are essential for the formation of ovules and the septum, two major internal structures of the gynoecium. Although a number of regulatory factors involved in shoot meristem activity are known to be required for the formation of these gynoecial structures, their direct roles in CMM development have yet to be addressed. Here we show that the CUP-SHAPED COTYLEDON genes CUC1 and CUC2, which are essential for shoot meristem initiation, are also required for formation and stable positioning of the CMMs. Early in CMM formation, CUC1 and CUC2 are also required for expression of the SHOOT MERISTEMLESS gene, a central regulator for stem cell maintenance in the shoot meristem. Moreover, plants carrying miR164-resistant forms of CUC1 and CUC2 resulted in extra CMM activity with altered positioning. Our results thus demonstrate that the two regulatory proteins controlling shoot meristem activity also play critical roles in elaboration of the female reproductive organ through the control of meristematic activity.
doi:10.3389/fpls.2014.00165
PMCID: PMC4012194  PMID: 24817871
Arabidopsis thaliana; carpel margin meristem; shoot meristem; leaf development; MicroRNA (miRNA); fruit development
22.  The bZIP Transcription Factor PERIANTHIA: A Multifunctional Hub for Meristem Control 
As sessile organisms, plants are exposed to extreme variations in environmental conditions over the course of their lives. Since plants grow and initiate new organs continuously, they have to modulate the underlying developmental program accordingly to cope with this challenge. At the heart of this extraordinary developmental plasticity are pluripotent stem cells, which are maintained during the entire life-cycle of the plant and that are embedded within dynamic stem cell niches. While the complex regulatory principles of plant stem cell control under artificial constant growth conditions begin to emerge, virtually nothing is known about how this circuit adapts to variations in the environment. In addition to the local feedback system constituted by the homeodomain transcription factor WUSCHEL (WUS) and the CLAVATA signaling cascade in the center of the shoot apical meristem (SAM), the bZIP transcription factor PERIANTHIA (PAN) not only has a broader expression domain in SAM and flowers, but also carries out more diverse functions in meristem maintenance: pan mutants show alterations in environmental response, shoot meristem size, floral organ number, and exhibit severe defects in termination of floral stem cells in an environment dependent fashion. Genetic and genomic analyses indicate that PAN interacts with a plethora of developmental pathways including light, plant hormone, and meristem control systems, suggesting that PAN is as an important regulatory node in the network of plant stem cell control.
doi:10.3389/fpls.2011.00079
PMCID: PMC3355747  PMID: 22645551
Arabidopsis; meristem regulation; stem cells; auxin; cytokinin; PERIANTHIA; type-A ARR; SHOOTMERISTEMLESS
23.  Depletion of cellular brassinolide decreases embryo production and disrupts the architecture of the apical meristems in Brassica napus microspore-derived embryos 
Journal of Experimental Botany  2010;61(10):2779-2794.
Exogenous applications of brassinolide (BL) increased the number and quality of microspore-derived embryos (MDEs) whereas treatments with brassinazole (BrZ), a BL biosynthetic inhibitor, had the opposite effect. At the optimal concentration (4×10−6 M) BrZ decreased both embryo yield and conversion to less than half the value of control embryos. Metabolic studies revealed that BL levels had profound effects on glutathione and ascorbate metabolism by altering the amounts of their reduced forms (ASC and GSH) and oxidized forms [dehydroascorbate (DHA), ascorbate free radicals (AFRs), and GSSG]. Applications of BL switched the glutathione and ascorbate pools towards the oxidized forms, thereby lowering the ASC/ASC+DHA+AFR and GSH/GSH+GSSG ratios. These changes were ascribed to the ability of BL to increase the activity of ascorbate peroxidase (APX) and decrease that of glutathione reductase (GR). This trend was reversed in a BL-depleted environment, effected by BrZ applications. These metabolic alterations were associated with changes in embryo structure and performance. BL-treated MDEs developed zygotic-like shoot apical meristems (SAMs) whereas embryos treated with BrZ developed abnormal meristems. In the presence of BrZ, embryos either lacked a visible SAM, or formed SAMs in which the meristematic cells showed signs of differentiation, such as vacuolation and storage product accumulation. These abnormalities were accompanied by the lack or misexpression of three meristem marker genes isolated from Brassica napus (denoted as BnSTM, BnCLV1, and BnZLL-1) homologous to the Arabidopsis SHOOTMERISTEMLESS (STM), CLAVATA 1 (CLV1), and ZWILLE (ZLL). The expression of BnSTM and BnCLV1 increased after a few days in cultures in embryos treated with BL whereas an opposite tendency was observed with applications of BrZ. Compared with control embryos where these two genes exhibited abnormal localization patterns, BnSTM and BnCLV1 always localized throughout the subapical domains of BL-treated embryos in a zygotic-like fashion. Expression of both genes was often lost in the SAM of BrZ-treated embryos. The results suggest that maintenance of cellular BL levels is required to modulate the ascorbate and glutathione redox status during embryogenesis to ensure proper development of the embryos and formation of functional apical meristems.
doi:10.1093/jxb/erq110
PMCID: PMC2882269  PMID: 20435696
Brassica napus; brassinazole; brassinolide; meristem marker genes; meristems; microspore-derived embryos
24.  Fas-associated factor (Faf1) is a novel CD40 interactor that regulates CD40-induced NF-κB activation via a negative feedback loop 
Cell Death & Disease  2014;5(5):e1213-.
CD40-induced signalling through ligation with its natural ligand (CD40L/CD154) is dependent on recruitment of TRAF molecules to the cytoplasmic domain of the receptor. Here, we applied the yeast two-hybrid system to examine whether other proteins can interact with CD40. Fas-Associated Factor 1(FAF1) was isolated from a HeLa cDNA library using the CD40 cytoplasmic tail (216–278 aa) as a bait construct. FAF1 was able to interact with CD40 both in vitro and in vivo. The FAF1 N-terminal domain was sufficient to bind CD40 and required the TRAF6-binding domain within the cytoplasmic tail of CD40 for binding. CD40 ligation induced FAF1 expression in an NFκB-dependent manner. Knockdown of FAF1 prolonged CD40-induced NFκB, whereas overexpression of FAF1 suppressed CD40-induced NFκB activity and this required interaction of FAF1 with the CD40 receptor via its FID domain. Thus, we report a novel role for FAF1in regulating CD40-induced NFκB activation via a negative feedback loop. Loss of FAF1 function in certain human malignancies may contribute to oncogenesis through unchecked NFκB activation, and further understanding of this process may provide a biomarker of NFκB-targeted therapies for such malignancies.
doi:10.1038/cddis.2014.172
PMCID: PMC4047894  PMID: 24810049
CD40; FAF1; NFκB
25.  WUSCHEL-Responsive At5g65480 Interacts with CLAVATA Components In Vitro and in Transient Expression 
PLoS ONE  2013;8(6):e66345.
The CLAVATA (CLV) signaling pathway is essential for shoot meristem homeostasis in Arabidopsis. CLV acts to limit the expression domain of the stem cell-promoting gene WUSCHEL (WUS). The closely related receptor-kinases CLV1 and BAM1 are key components in this pathway; however, the downstream factors that link the receptors to WUS regulation are poorly understood. The Arabidopsis gene At5g65480 was recently identified as a direct transcriptional target up-regulated by WUS. We have independently identified this gene which we term CCI1 as a CLV1 and BAM1 interacting protein in vitro and in transient expression. CCI1 has phosphatidylinositide-binding activity in vitro and localizes to the plasma membrane in transient expression. Furthermore, CLV signaling components and CCI1 both partition to detergent-resistant membrane microdomains characterized as lipid rafts.
doi:10.1371/journal.pone.0066345
PMCID: PMC3679059  PMID: 23776660

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