WUSCHEL Transcription Research Articles

HAIRY MERISTEM proteins regulate the WUSCHEL protein levels in mediating CLAVATA3 expression.

The precise regulation of stem cells in the shoot apical meristems (SAMs) involves the function of the homeodomain transcription factor (TF)-WUSCHEL (WUS). WUS has been shown to move from the site of production-the rib-meristem (RM), into overlaying cells of the central zone (CZ), where it specifies stem cells and also regulates the transcription of CLAVATA3 (CLV3). The secreted signalling peptide CLV3 activates a receptor kinase signalling that restricts WUS transcription and also regulates the nuclear gradient of WUS by offsetting nuclear export. WUS has been shown to regulate both CLV3 levels and spatial activation, restricting its expression to a few cells in the CZ. The HAIRY MERISTEM (HAM), a GRASS-domain class of TFs expressed in the RM, has been shown to physically interact with WUS and regulate CLV3 expression. However, the mechanisms by which this interaction regulates CLV3 expression non-cell autonomously remain unclear. Here, we show that HAM function is required for regulating the WUS protein stability, and the CLV3 expression responds to altered WUS protein levels in ham mutants. Thus, HAM proteins non-cell autonomously regulates CLV3 expression.

Nitric oxide controls shoot meristem activity via regulation of DNA methylation

Despite the importance of Nitric Oxide (NO) as signaling molecule in both plant and animal development, the regulatory mechanisms downstream of NO remain largely unclear. Here, we show that NO is involved in Arabidopsis shoot stem cell control via modifying expression and activity of ARGONAUTE 4 (AGO4), a core component of the RNA-directed DNA Methylation (RdDM) pathway. Mutations in components of the RdDM pathway cause meristematic defects, and reduce responses of the stem cell system to NO signaling. Importantly, we find that the stem cell inducing WUSCHEL transcription factor directly interacts with AGO4 in a NO dependent manner, explaining how these two signaling systems may converge to modify DNA methylation patterns. Taken together, our results reveal that NO signaling plays an important role in controlling plant stem cell homeostasis via the regulation of de novo DNA methylation.

Paradigm and Framework of WUS-CLV Feedback Loop in Stem Cell Niche for SAM Maintenance and Cell Identity Transition

Shoot apical meristem (SAM) consists of stem cells that act as a reservoir for the aerial growth. It plays an important role in the differential architectural development in plants. SAM actively performs parallel functions by maintaining the pluripotent of stem cells and continuous organogenesis throughout the plant’s life cycle. Molecular mechanisms regulating the signaling networks of this dual function of the SAM have been progressively understood. In the SAM, the feedback loop of WUSCHEL (WUS)-CLAVATA (CLV) has been found to be the key regulator in stabilizing stem cell proliferation and differentiation. In general, WUS migrates into central zone (CZ) from organizing center (OC) and activates the expression of CLV3 by binding to the promoter elements. CLV3 acts as a ligand to interact with the CLV1, leucine rich repeats (LRR) receptor-like kinase (RLK) and LRR receptor-like protein CLV2, and protein kinase coryne (CRN) (CLV2/CRN) to restrict WUS transcription to the OC. Evolution of CLV3 is one of the main factors contributing to the transformation of two-dimensional (2D) to 3D plants. WUS-CLV loop is involved in several pathways and networks that integrate on meristem maintenance and cell identity transition. WUS-CLV maintains stem cells with simultaneous differentiation signals by the spatial-temporal signaling of the phytohormones. WUS-CLV loop has an interaction with reactive oxygen species (ROS), an important signaling molecules regulating cell proliferation and developmental transition. WUS also forms feedback loop with AGAMOUS (AG) for differentiation, proliferation, and termination of floral meristem. These loops might also involve in interaction with vernalization and its regulatory factors that oversees the precise timing of flowering after exposure to cold temperatures. In this review, we highlight the evolutionary and developmental importance of the WUS-CLV feedback loop on SAM maintenance and cell identity transition for inflorescence and floral meristem development.

Evolution of meristem zonation by CLE gene duplication in land plants.

In angiosperms, a negative feedback pathway involving CLAVATA3 (CLV3) peptide and WUSCHEL transcription factor maintains the stem-cell population in the shoot apical meristem and is central for continued shoot growth and organogenesis. An intriguing question is how this cell-signalling system was established during the evolution of land plants. On the basis of two recent studies on CLV3/ESR-related (CLE) genes, this paper proposes a model for the evolution of meristem zonation. The model suggests that a stem-cell-limiting CLV3 pathway is derived from stem-cell-promoting CLE pathways conserved in land pants by gene duplication in the angiosperm lineage. The model can be examined in the future by genomic and developmental studies on diverse plant species.

CLAVATA modulates auxin homeostasis and transport to regulate stem cell identity and plant shape in a moss.

Summary The CLAVATA pathway is a key regulator of stem cell function in the multicellular shoot tips of Arabidopsis, where it acts via the WUSCHEL transcription factor to modulate hormone homeostasis. Broad‐scale evolutionary comparisons have shown that CLAVATA is a conserved regulator of land plant stem cell function, but CLAVATA acts independently of WUSCHEL‐like (WOX) proteins in bryophytes. The relationship between CLAVATA, hormone homeostasis and the evolution of land plant stem cell functions is unknown.Here we show that in the moss, Physcomitrella (Physcomitrium patens), CLAVATA affects stem cell activity by modulating hormone homeostasis. CLAVATA pathway genes are expressed in the tip cells of filamentous tissues, regulating cell identity, filament branching, plant spread and auxin synthesis. The receptor‐like kinase PpRPK2 plays the major role, and Pprpk2 mutants have abnormal responses to cytokinin, auxin and auxin transport inhibition, and show reduced expression of PIN auxin transporters.We propose a model whereby PpRPK2 modulates auxin gradients in filaments to determine stem cell identity and overall plant form.Our data indicate that CLAVATA‐mediated auxin homeostasis is a fundamental property of plant stem cell function, probably exhibited by the last shared common ancestor of land plants.

CLAVATA3 mediated simultaneous control of transcriptional and post-translational processes provides robustness to the WUSCHEL gradient

Regulation of the homeodomain transcription factor WUSCHEL concentration is critical for stem cell homeostasis in Arabidopsis shoot apical meristems. WUSCHEL regulates the transcription of CLAVATA3 through a concentration-dependent activation-repression switch. CLAVATA3, a secreted peptide, activates receptor kinase signaling to repress WUSCHEL transcription. Considering the revised regulation, CLAVATA3 mediated repression of WUSCHEL transcription alone will lead to an unstable system. Here we show that CLAVATA3 signaling regulates nuclear-cytoplasmic partitioning of WUSCHEL to control nuclear levels and its diffusion into adjacent cells. Our work also reveals that WUSCHEL directly interacts with EXPORTINS via EAR-like domain which is also required for destabilizing WUSCHEL in the cytoplasm. We develop a combined experimental and computational modeling approach that integrates CLAVATA3-mediated transcriptional repression of WUSCHEL and post-translational control of nuclear levels with the WUSCHEL concentration-dependent regulation of CLAVATA3. We show that the dual control by the same signal forms a seamless connection between de novo WUSCHEL synthesis and sub-cellular partitioning in providing robustness to the WUSCHEL gradient.

Genetics of Shoot Meristem and Shoot Regeneration.

Plants exhibit remarkable lineage plasticity, allowing them to regenerate organs that differ from their respective origins. Such developmental plasticity is dependent on the activity of pluripotent founder cells or stem cells residing in meristems. At the shoot apical meristem (SAM), the constant flow of cells requires continuing cell specification governed by a complex genetic network, with the WUSCHEL transcription factor and phytohormone cytokinin at its core. In this review, I discuss some intriguing recent discoveries that expose new principles and mechanisms of patterning and cell specification acting both at the SAM and prior to meristem organogenesis during shoot regeneration. I also highlight unanswered questions and future challenges in the study of SAM and meristem regeneration. Finally, I put forward a model describing stochastic events mediated by epigenetic factors to explain how the gene regulatory network might be initiated at the onset of shoot regeneration.

WUSCHEL acts as an auxin response rheostat to maintain apical stem cells in Arabidopsis

To maintain the balance between long-term stem cell self-renewal and differentiation, dynamic signals need to be translated into spatially precise and temporally stable gene expression states. In the apical plant stem cell system, local accumulation of the small, highly mobile phytohormone auxin triggers differentiation while at the same time, pluripotent stem cells are maintained throughout the entire life-cycle. We find that stem cells are resistant to auxin mediated differentiation, but require low levels of signaling for their maintenance. We demonstrate that the WUSCHEL transcription factor confers this behavior by rheostatically controlling the auxin signaling and response pathway. Finally, we show that WUSCHEL acts via regulation of histone acetylation at target loci, including those with functions in the auxin pathway. Our results reveal an important mechanism that allows cells to differentially translate a potent and highly dynamic developmental signal into stable cell behavior with high spatial precision and temporal robustness.

Cytokinin stabilizes WUSCHEL by acting on the protein domains required for nuclear enrichment and transcription.

Concentration-dependent transcriptional regulation and the spatial regulation of transcription factor levels are poorly studied in plant development. WUSCHEL, a stem cell-promoting homeodomain transcription factor, accumulates at a higher level in the rib meristem than in the overlying central zone, which harbors stem cells in the shoot apical meristems of Arabidopsis thaliana. The differential accumulation of WUSCHEL in adjacent cells is critical for the spatial regulation and levels of CLAVATA3, a negative regulator of WUSCHEL transcription. Earlier studies have revealed that DNA-dependent dimerization, subcellular partitioning and protein destabilization control WUSCHEL protein levels and spatial accumulation. Moreover, the destabilization of WUSCHEL may also depend on the protein concentration. However, the roles of extrinsic spatial cues in maintaining differential accumulation of WUS are not understood. Through transient manipulation of hormone levels, hormone response patterns and analysis of the receptor mutants, we show that cytokinin signaling in the rib meristem acts through the transcriptional regulatory domains, the acidic domain and the WUSCHEL-box, to stabilize the WUS protein. Furthermore, we show that the same WUSCHEL-box functions as a degron sequence in cytokinin deficient regions in the central zone, leading to the destabilization of WUSCHEL. The coupled functions of the WUSCHEL-box in nuclear retention as described earlier, together with cytokinin sensing, reinforce higher nuclear accumulation of WUSCHEL in the rib meristem. In contrast a sub-threshold level may expose the WUSCHEL-box to destabilizing signals in the central zone. Thus, the cytokinin signaling acts as an asymmetric spatial cue in stabilizing the WUSCHEL protein to lead to its differential accumulation in neighboring cells, which is critical for concentration-dependent spatial regulation of CLAVATA3 transcription and meristem maintenance. Furthermore, our work shows that cytokinin response is regulated independently of the WUSCHEL function which may provide robustness to the regulation of WUSCHEL concentration.

Type-B ARABIDOPSIS RESPONSE REGULATORs Specify the Shoot Stem Cell Niche by Dual Regulation of WUSCHEL.

Plants are known for their capacity to regenerate the whole body through de novo formation of apical meristems from a mass of proliferating cells named callus. Exogenous cytokinin and auxin determine cell fate for the establishment of the stem cell niche, which is the vital step of shoot regeneration, but the underlying mechanisms remain unclear. Here, we show that type-B ARABIDOPSIS RESPONSE REGULATORs (ARRs), critical components of cytokinin signaling, activate the transcription of WUSCHEL (WUS), which encodes a key regulator for maintaining stem cells. In parallel, type-B ARRs inhibit auxin accumulation by repressing the expression of YUCCAs, which encode a key enzyme for auxin biosynthesis, indirectly promoting WUS induction. Both pathways are essential for de novo regeneration of the shoot stem cell niche. In addition, the dual regulation of type-B ARRs on WUS transcription is required for the maintenance of the shoot apical meristem in planta. Thus, our results reveal a long-standing missing link between cytokinin signaling and WUS regulator, and the findings provide critical information for understanding cell fate specification.

Shoot stem cell specification in roots by the WUSCHEL transcription factor.

The WUSCHEL homeobox transcription factor is required to specify stem-cell identity at the shoot apical meristem and its ectopic expression is sufficient to induce de novo shoot meristem formation. Yet, the manner by which WUS promotes stem-cell fate is not yet fully understood. In the present research we address this question by inducing WUS function outside of its domain. We show that activation of WUS function in the root inhibits the responses to exogenous auxin and suppresses the initiation and growth of lateral roots. Using time lapse movies to follow the cell-cycle marker CYCB1;1::GFP, we also show that activation of WUS function suppresses cell division and cell elongation. In addition, activation of WUS represses the auxin-induced expression of the PLETHORA1 root identity gene and promotes shoot fate. Shoot apical meristem formation requires a high cytokinin-to-auxin ratio. Our findings provide evidence for the manner by which WUS specifies stem-cell identity: by affecting auxin responses, by reducing the cell mitotic activity and by repressing other developmental pathways. At the meristem, the stem-cells which are characterized by low division rate are surrounded by the highly proliferative meristematic cells. Our results also provide a model for WUS establishing the differential mitotic rates between two cell populations at the minute structure of the meristem.

Stem Cell Fate versus Differentiation: the Missing Link.

The shoot apical meristem provides a microenvironment that ensures stem cell fate and proliferation via homeostasis between WUSCHEL (WUS) activity and CLAVATA signalling. New data from maize and arabidopsis reveal that an evolutionarily conserved signal deriving from primordium cells links WUS transcription to the morphogenetic programme.

Quantitative variation in maize kernel row number is controlled by the FASCIATED EAR2 locus

Domestication of cereal crops, such as maize, wheat and rice, had a profound influence on agriculture and the establishment of human civilizations. One major improvement was an increase in seed number per inflorescence, which enhanced yield and simplified harvesting and storage. The ancestor of maize, teosinte, makes 2 rows of kernels, and modern varieties make ∼8-20 rows. Kernel rows are initiated by the inflorescence shoot meristem, and shoot meristem size is controlled by a feedback loop involving the CLAVATA signaling proteins and the WUSCHEL transcription factor. We present a hypothesis that variation in inflorescence meristem size affects kernel row number (KRN), with the potential to increase yield. We also show that variation in the CLAVATA receptor-like protein FASCIATED EAR2 leads to increased inflorescence meristem size and KRN. These findings indicate that modulation of fundamental stem cell proliferation control pathways has the potential to enhance crop yields.

Plant stem cell maintenance involves direct transcriptional repression of differentiation program

In animal systems, master regulatory transcription factors (TFs) mediate stem cell maintenance through a direct transcriptional repression of differentiation promoting TFs. Whether similar mechanisms operate in plants is not known. In plants, shoot apical meristems serve as reservoirs of stem cells that provide cells for all above ground organs. WUSCHEL, a homeodomain TF produced in cells of the niche, migrates into adjacent cells where it specifies stem cells. Through high-resolution genomic analysis, we show that WUSCHEL represses a large number of genes that are expressed in differentiating cells including a group of differentiation promoting TFs involved in leaf development. We show that WUS directly binds to the regulatory regions of differentiation promoting TFs; KANADI1, KANADI2, ASYMMETRICLEAVES2 and YABBY3 to repress their expression. Predictions from a computational model, supported by live imaging, reveal that WUS-mediated repression prevents premature differentiation of stem cell progenitors, being part of a minimal regulatory network for meristem maintenance. Our work shows that direct transcriptional repression of differentiation promoting TFs is an evolutionarily conserved logic for stem cell regulation.

Identification of expressed sequences in the coffee genome potentially associated with somatic embryogenesis

Brazil possesses the most modern and productive coffee growing farms in the world, but technological development is desired to cope with the increasing world demand. One way to increase Brazilian coffee growing productivity is wide scale production of clones with superior genotypes, which can be obtained with in vitro propagation technique, or from tissue culture. These procedures can generate thousands of clones. However, the methodologies for in vitro cultivation are genotype-dependent, which leads to an almost empirical development of specific protocols for each species. Therefore, molecular markers linked to the biochemical events of somatic embryogenesis would greatly facilitate the development of such protocols. In this context, sequences potentially involved in embryogenesis processes in the coffee plant were identified in silico from libraries generated by the Brazilian Coffee Genome Project. Through these in silico analyses, we identified 15 EST-contigs related to the embryogenesis process. Among these, 5 EST-contigs (3605, 9850, 13686, 17240, and 17265) could readily be associated with plant embryogenesis. Sequence analysis of EST-contig 3605, 9850, and 17265 revealed similarity to a polygalacturonase, to a cysteine-proteinase, and to an allergenine, respectively. Results also show that EST-contig 17265 sequences presented similarity to an expansin. Finally, analysis of EST-contig 17240 revealed similarity to a protein of unknown function, but it grouped in the similarity dendrogram with the WUSCHEL transcription factor. The data suggest that these EST-contigs are related to the embryogenic process and have potential as molecular markers to increase methodological efficiency in obtaining coffee plant embryogenic materials.

WUSCHEL protein movement and stem cell homeostasis

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.

A timing mechanism for stem cell maintenance and differentiation in the Arabidopsis floral meristem

Developmental regulation of the floral meristem ensures that plants of the same species have similarly sized flowers with a fixed number of floral organs. The maintenance of stem cells in the floral meristem is terminated after the production of a fixed number of floral organ primordia. Precise repression of the Arabidopsis thaliana homeobox gene WUSCHEL (WUS) by the floral homeotic protein AGAMOUS (AG) plays a major part in this process. Here we show that KNUCKLES (KNU) mediates the repression of WUS in floral meristem determinacy control. AG directly induces the transcription of KNU, which encodes a C2H2-type zinc finger protein with a conserved transcriptional repression motif. In turn, KNU represses WUS transcription to abolish stem cell activity. We also show that the timing of KNU induction is key in balancing proliferation and differentiation in flower development. Delayed KNU expression results in an indeterminate meristem, whereas ectopic KNU expression prematurely terminates the floral meristem. Furthermore, KNU induction by AG is preceded by changes in repressive histone modification at the KNU locus, which occurs in an AG-dependent manner. This study provides a mechanistic link between transcriptional feedback and epigenetic regulation in plant stem cell proliferation.

Regulation ofWUSCHELTranscription in the Stem Cell Niche of the Arabidopsis Shoot Meristem

Pluripotent stem cells are localized in specialized microenvironments, called stem cell niches, where signals from surrounding cells maintain their undifferentiated status. In the Arabidopsis thaliana shoot meristem, the homeobox gene WUSCHEL (WUS) is expressed in the organizing center underneath the stem cells and integrates regulatory information from several pathways to define the boundaries of the stem cell niche. To investigate how these boundaries are precisely maintained within the proliferating cellular context of the shoot meristem, we analyzed the transcriptional control of the WUS gene. Our results show that the WUS promoter contains distinct regulatory regions that control tissue specificity and levels of transcription in a combinatorial manner. However, a 57-bp regulatory region is all that is required to control the boundaries of WUS transcription in the shoot meristem stem cell niche, and this activity can be further assigned to two adjacent short sequence motifs within this region. Our results indicate that the diverse regulatory pathways that control the stem cells in the shoot meristem converge at these two short sequence elements of the WUS promoter, suggesting that the integration of regulatory signals takes place at the level of a central transactivating complex.

Stem cell homeostasis in the Arabidopsis shoot meristem is regulated by intercellular movement of CLAVATA3 and its sequestration by CLAVATA1.

Stem cell maintenance in the Arabidopsis shoot meristem is regulated by communication between the apical stem cells and the underlying organizing centre. Expression of the homeobox gene WUSCHEL in the organizing centre induces stem cell identity in the overlying neighbours, which then express the CLAVATA3 gene whose activity in turn restricts the size of the WUSCHEL expression domain. We have analyzed how the stem cells and the organizing centre communicate, by studying the mode of action of CLAVATA3 protein. We provide direct evidence that CLAVATA3 protein functions as a mobile intercellular signal in the shoot apical meristem that spreads laterally from the stem cells and acts both on their lateral neighbours and on the stem cells themselves to repress WUSCHEL transcription. We also show that the spread and range of action of CLAVATA3 can be limited by binding to its receptor CLAVATA1, which offers an explanation for how CLAVATA3 is prevented from entering the organizing centre and repressing WUSCHEL transcription there. This regulated spread of a secreted signalling molecule enables the shoot meristem to permit the onset of cell differentiation in the periphery, but at the same time to maintain a stable niche for its stem cells in the center.