Condensation of STM is critical for shoot meristem maintenance and salt tolerance in Arabidopsis.

The shoot meristem generates the entire shoot system and is precisely maintained throughout the life cycle under various environmental challenges. In this study, we identified a prion-like domain (PrD) in the key shoot meristem regulator SHOOT MERISTEMLESS (STM), which distinguishes STM from other related KNOX1 proteins. We demonstrated that PrD stimulates STM to form nuclear condensates, which are required for maintaining the shoot meristem. STM nuclear condensate formation is stabilized by selected PrD-containing STM-interacting BELL proteins in vitro and in vivo. Moreover, condensation of STM promotes its interaction with the Mediator complex subunit MED8 and thereby enhances its transcriptional activity. Thus, condensate formation emerges as a novel regulatory mechanism of shoot meristem functions. Furthermore, we found that the formation of STM condensates is enhanced upon salt stress, which allows enhanced salt tolerance and increased shoot branching. Our findings highlight that the transcription factor partitioning plays an important role in cell fate determination and might also act as a tunable environmental acclimation mechanism.

Differential growth dynamics control aerial organ geometry.

How gene activities and biomechanics together direct organ shapes is poorly understood. Plant leaf and floral organs develop from highly similar initial structures and share similar gene expression patterns, yet they gain drastically different shapes later-flat and bilateral leaf primordia and radially symmetric floral primordia, respectively. We analyzed cellular growth patterns and gene expression in young leaves and flowers of Arabidopsis thaliana and found significant differences in cell growth rates, which correlate with convergence sites of phytohormone auxin that require polar auxin transport. In leaf primordia, the PRESSED-FLOWER-expressing middle domain grows faster than adjacent adaxial domain and coincides with auxin convergence. In contrast, in floral primordia, the LEAFY-expressing domain shows accelerated growth rates and pronounced auxin convergence. This distinct cell growth dynamics between leaf and flower requires changes in levels of cell-wall pectin de-methyl-esterification and mechanical properties of the cell wall. Data-driven computer model simulations at organ and cellular levels demonstrate that growth differences are central to obtaining distinct organ shape, corroborating in planta observations. Together, our study provides a mechanistic basis for the establishment of early aerial organ symmetries through local modulation of differential growth patterns with auxin and biomechanics.

A Self-Activation Loop Maintains Meristematic Cell Fate for Branching.

In plants and animals, self-renewing stem cell populations play fundamental roles in many developmental contexts. Plants differ from most animals in their retained ability to initiate new cycles of growth and development, which relies on the establishment and activity of branch meristems. In seed plants, branching is achieved by stem-cell-containing axillary meristems, which are initiated from a leaf axil meristematic cell population originally detached from the shoot apical meristem. It remains unclear how the meristematic cell fate is maintained. Here, we show that ARABIDOPSISTHALIANAHOMEOBOXGENE1 (ATH1) maintains the meristem marker gene SHOOT MERISTEMLESS (STM) expression in the leaf axil to enable meristematic cell fate maintenance. Furthermore, ATH1 protein interacts with STM protein to form a STM self-activation loop. Genetic and biochemical data suggest that ATH1 anchors STM to activate STM as well as other axillary meristem regulatory genes. This auto-regulation allows the STM locus to remain epigenetically active. Taken together, our findings provide a striking example of a self-activation loop that maintains the flexibility required for stem cell niche re-establishment during organogenesis.

Control of cell fate during axillary meristem initiation.

Axillary meristems (AMs) are located in the leaf axil and can establish new growth axes. Whereas their neighboring cells are differentiated, the undifferentiated cells in the AM endow the AM with the same developmental potential as the shoot apical meristem. The AM is, therefore, an excellent system to study stem cell fate maintenance in plants. In this review, we summarize the current knowledge of AM initiation. Recent findings have shown that AMs derive from a stem cell lineage that is maintained in the leaf axil. This review covers AM progenitor cell fate maintenance, reactivation, and meristem establishment. We also highlight recent work that links transcription factors, phytohormones, and epigenetic regulation to AM initiation.

A chromatin loop represses WUSCHEL expression in Arabidopsis.

WUSCHEL (WUS) is critical for plant meristem maintenance and determinacy in Arabidopsis, and the regulation of its spatiotemporal expression patterns is complex. We previously found that AGAMOUS (AG), a key MADS-domain transcription factor in floral organ identity and floral meristem determinacy, can directly suppress WUS expression through the recruitment of the Polycomb group (PcG) protein TERMINAL FLOWER 2 (TFL2, also known as LIKE HETEROCHROMATIN PROTEIN 1, LHP1) at the WUS locus; however, the mechanism by which WUS is repressed remains unclear. Here, using chromosome conformation capture (3C) and chromatin immunoprecipitation 3C, we found that two specific regions flanking the WUS gene body bound by AG and TFL2 form a chromatin loop that is directly promoted by AG during flower development in a manner independent of the physical distance and sequence content of the intervening region. Moreover, AG physically interacts with TFL2, and TFL2 binding to the chromatin loop is dependent on AG. Transgenic and CRISPR/Cas9-edited lines showed that the WUS chromatin loop represses gene expression by blocking the recruitment of RNA polymerase II at the locus. The findings uncover the WUS chromatin loop as another regulatory mechanism controlling WUS expression, and also shed light on the factors required for chromatin conformation change and their recruitment.

AUXIN RESPONSE FACTOR3 Regulates Floral Meristem Determinacy by Repressing Cytokinin Biosynthesis and Signaling.

Successful floral meristem (FM) determinacy is critical for subsequent reproductive development and the plant life cycle. Although the phytohormones cytokinin and auxin interact to coregulate many aspects of plant development, whether and how cytokinin and auxin function in FM determinacy remain unclear. Here, we show that in Arabidopsis thaliana, cytokinin homeostasis is critical for FM determinacy. In this developmental context, auxin promotes the expression of AUXIN RESPONSE FACTOR3 (ARF3) to repress cytokinin activity. ARF3 directly represses the expression of ISOPENTENYLTRANSFERASE (IPT) family genes and indirectly represses LONELY GUY (LOG) family genes, both of which encode enzymes required for cytokinin biosynthesis. ARF3 also directly inhibits the expression of ARABIDOPSIS HISTIDINE KINASE4, a cytokinin receptor gene, resulting in reduced cytokinin activity. Consequently, ARF3 controls cell division by regulating cell cycle gene expression through cytokinin. In flowers, we show that AGAMOUS (AG) dynamically regulates the expression of ARF3 and IPTs, resulting in coordinated regulation of FM maintenance and termination through cell division. Moreover, genome-wide transcriptional profiling revealed both repressive and active roles for ARF3 in early flower development. Our findings establish a molecular link between AG and auxin/cytokinin and shed light on the mechanisms of stem cell maintenance and termination in the FM.

Cytokinin Signaling Activates WUSCHEL Expression during Axillary Meristem Initiation.

The homeodomain transcription factor WUSCHEL (WUS) defines the shoot stem cell niche, but the mechanisms underlying the establishment of WUS expression remain unclear. Here, we show that cytokinin signaling precedes WUS expression in leaf axils and activates WUS expression de novo in the leaf axil to promote axillary meristem initiation. Furthermore, type-B Arabidopsis response regulator proteins, which are transcriptional activators in the cytokinin signaling pathway, directly bind to the WUS promoter and activate its expression. Finally, we show that cytokinin activation of WUS in the leaf axil correlates with increased histone acetylation and methylation markers associated with transcriptional activation, supporting the fact that WUS expression requires a permissive epigenetic environment to restrict it to highly defined meristematic tissues. Taken together, these findings explain how cytokinin regulates axillary meristem initiation and establish a mechanistic framework for the postembryonic establishment of the shoot stem cell niche.

FAR-RED ELONGATED HYPOCOTYL3 activates SEPALLATA2 but inhibits CLAVATA3 to regulate meristem determinacy and maintenance in Arabidopsis.

Plant meristems are responsible for the generation of all plant tissues and organs. Here we show that the transcription factor (TF) FAR-RED ELONGATED HYPOCOTYL3 (FHY3) plays an important role in both floral meristem (FM) determinacy and shoot apical meristem maintenance in Arabidopsis, in addition to its well-known multifaceted roles in plant growth and development during the vegetative stage. Through genetic analyses, we show that WUSCHEL (WUS) and CLAVATA3 (CLV3), two central players in the establishment and maintenance of meristems, are epistatic to FHY3 Using genome-wide ChIP-seq and RNA-seq data, we identify hundreds of FHY3 target genes in flowers and find that FHY3 mainly acts as a transcriptional repressor in flower development, in contrast to its transcriptional activator role in seedlings. Binding motif-enrichment analyses indicate that FHY3 may coregulate flower development with three flower-specific MADS-domain TFs and four basic helix-loop-helix TFs that are involved in photomorphogenesis. We further demonstrate that CLV3, SEPALLATA1 (SEP1), and SEP2 are FHY3 target genes. In shoot apical meristem, FHY3 directly represses CLV3, which consequently regulates WUS to maintain the stem cell pool. Intriguingly, CLV3 expression did not change significantly in fhy3 and phytochrome B mutants before and after light treatment, indicating that FHY3 and phytochrome B are involved in light-regulated meristem activity. In FM, FHY3 directly represses CLV3, but activates SEP2, to ultimately promote FM determinacy. Taken together, our results reveal insights into the mechanisms of meristem maintenance and determinacy, and illustrate how the roles of a single TF may vary in different organs and developmental stages.

Epigenetic Mechanisms Are Critical for the Regulation of WUSCHEL Expression in Floral Meristems.

The floral meristem (FM), which develops from the inflorescence meristem upon completion of the floral transition, terminates after producing a defined number of floral organs. This is in contrast to the shoot apical meristem, which is active throughout the entire life span of plants. WUSCHEL (WUS) encodes a homeodomain-containing protein and plays a critical role in shoot apical meristem, inflorescence meristem, and FM establishment and maintenance as well as FM determinacy. Although many genes have been implicated in FM determinacy through the regulation of WUS expression, precisely how these genes are coordinated to regulate WUS and consequently dictate FM fate remains unclear. Emerging lines of evidence indicate that epigenetic mechanisms, such as histone modification, chromatin remodeling, noncoding RNAs, and DNA methylation, play vital roles in meristem maintenance and termination. Here, recent findings demonstrating the involvement of the epigenetic network in the regulation of WUS expression in the context of FM determinacy are summarized and discussed.

AUXIN RESPONSE FACTOR 3 integrates the functions of AGAMOUS and APETALA2 in floral meristem determinacy.

In Arabidopsis, AUXIN RESPONSE FACTOR 3 (ARF3) belongs to the auxin response factor (ARF) family that regulates the expression of auxin-responsive genes. ARF3 is known to function in leaf polarity specification and gynoecium patterning. In this study, we discovered a previously unknown role for ARF3 in floral meristem (FM) determinacy through the isolation and characterization of a mutant of ARF3 that enhanced the FM determinacy defects of agamous (ag)-10, a weak ag allele. Central players in FM determinacy include WUSCHEL (WUS), a gene critical for FM maintenance, and AG and APETALA2 (AP2), which regulate FM determinacy by repression and promotion of WUS expression, respectively. We showed that ARF3 confers FM determinacy through repression of WUS expression, and associates with the WUS locus in part in an AG-dependent manner. We demonstrated that ARF3 is a direct target of AP2 and partially mediates AP2's function in FM determinacy. ARF3 exhibits dynamic and complex expression patterns in floral organ primordia; altering the patterns spatially compromised FM determinacy. This study uncovered a role for ARF3 in FM determinacy and revealed relationships among genes in the genetic network governing FM determinacy.