The trxG protein ULT1 regulates Arabidopsis organ size by interacting with TCP14/15 to antagonize the LIM peptidase DA1 for H3K4me3 on target genes.

Plant organ size is an important agronomic trait that makes a significant contribution to plant yield. Despite its central importance, the genetic and molecular mechanisms underlying organ size control remain to be fully clarified. Here, we report that the trithorax group protein ULTRAPETALA1 (ULT1) interacts with the TEOSINTE BRANCHED1/CYCLOIDEA/PCF14/15 (TCP14/15) transcription factors by antagonizing the LIN-11, ISL-1, and MEC-3 (LIM) peptidase DA1, thereby regulating organ size in Arabidopsis. Loss of ULT1 function significantly increases rosette leaf, petal, silique, and seed size, whereas overexpression of ULT1 results in reduced organ size. ULT1 associates with TCP14 and TCP15 to co-regulate cell size by affecting cellular endoreduplication. Transcriptome analysis revealed that ULT1 and TCP14/15 regulate common target genes involved in endoreduplication and leaf development. ULT1 can be recruited by TCP14/15 to promote lysine 4 of histone H3 trimethylation at target genes, activating their expression to determine final cell size. Furthermore, we found that ULT1 influences the interaction of DA1 and TCP14/15 and antagonizes the effect of DA1 on TCP14/15 degradation. Collectively, our findings reveal a novel epigenetic mechanism underlying the regulation of organ size in Arabidopsis.

Stem Cells: Engines of Plant Growth and Development.

The development of both animals and plants relies on populations of pluripotent stem cells that provide the cellular raw materials for organ and tissue formation. Plant stem cell reservoirs are housed at the shoot and root tips in structures called meristems, with the shoot apical meristem (SAM) continuously producing aerial leaf, stem, and flower organs throughout the life cycle. Thus, the SAM acts as the engine of plant development and has unique structural and molecular features that allow it to balance self-renewal with differentiation and act as a constant source of new cells for organogenesis while simultaneously maintaining a stem cell reservoir for future organ formation. Studies have identified key roles for intercellular regulatory networks that establish and maintain meristem activity, including the KNOX transcription factor pathway and the CLV-WUS stem cell feedback loop. In addition, the plant hormones cytokinin and auxin act through their downstream signaling pathways in the SAM to integrate stem cell activity and organ initiation. This review discusses how the various regulatory pathways collectively orchestrate SAM function and touches on how their manipulation can alter stem cell activity to improve crop yield.

The CLAVATA3/ESR-related peptide family in the biofuel crop pennycress.

CLAVATA3/ESR-related (CLE) peptides perform a variety of important functions in plant development and historically have been targeted during the domestication of existing crops. Pennycress (Thlaspi arvense) is an emerging biofuel crop currently undergoing domestication that offers novel monetary and environmental incentives as a winter cover crop during an otherwise fallow period of the corn/soybean farming rotation. Here we report the characterization of the CLE gene family in pennycress through homology comparison of the CLE motif with other dicot species by conducting a homology comparison and maximum likelihood phylogenetic analysis supplemented with manual annotation. Twenty-seven pennycress CLE genes were identified, and their expression analyzed through transcriptome profiling and RT-qPCR. Our study provides a genome-wide analysis of the CLE gene family in pennycress and carries significant value for accelerating the domestication of this crop through identification of potential key developmental regulatory genes.

Genetic and Phenotypic Analysis of Shoot Apical and Floral Meristem Development.

The shoot apical and floral meristems (SAM and FM, respectively) of Arabidopsis thaliana contain reservoirs of self-renewing stem cells that function as sources of progenitor cells for organ formation during development. The primary SAM produces all the aerial structures of the adult plant, while the FMs generate the four types of floral organs. Consequently, aberrant SAM and FM activity can profoundly affect vegetative and reproductive plant morphology. The embedded location and small size of Arabidopsis meristems make accessing these structures difficult, so specialized techniques have been developed to facilitate their analysis. Microscopic, histological, and molecular techniques provide both qualitative and quantitative data on meristem organization and function, which are crucial for the normal growth and development of the entire plant.

Unique and overlapping functions for the transcriptional regulators KANADI1 and ULTRAPETALA1 in Arabidopsis gynoecium and stamen gene regulation.

Plants generate their reproductive organs, the stamens and the carpels, de novo within the flowers that form when the plant reaches maturity. The carpels comprise the female reproductive organ, the gynoecium, a complex organ that develops along several axes of polarity and is crucial for plant reproduction, fruit formation, and seed dispersal. The epigenetic trithorax group (trxG) protein ULTRAPETALA1 (ULT1) and the GARP domain transcription factor KANADI1 (KAN1) act cooperatively to regulate Arabidopsis thaliana gynoecium patterning along the apical-basal polarity axis; however, the molecular pathways through which this patterning activity is achieved remain to be explored. In this study, we used transcriptomics to identify genome-wide ULT1 and KAN1 target genes during reproductive development. We discovered 278 genes in developing flowers that are regulated by ULT1, KAN1, or both factors together. Genes involved in developmental and reproductive processes are overrepresented among ULT1 and/or KAN1 target genes, along with genes involved in biotic or abiotic stress responses. Consistent with their function in regulating gynoecium patterning, a number of the downstream target genes are expressed in the developing gynoecium, including a unique subset restricted to the stigmatic tissue. Further, we also uncovered a number of KAN1- and ULT1-induced genes that are transcribed predominantly or exclusively in developing stamens. These findings reveal a potential cooperative role for ULT1 and KAN1 in male as well as female reproductive development that can be investigated with future genetic and molecular experiments.

Interactive CLV3, CLE16 and CLE17 signaling mediates stem cell homeostasis in the Arabidopsis shoot apical meristem.

The ability of plants to grow and form organs throughout their lifetime is dependent on their sustained stem cell activity. These stem cell populations are maintained by intricate networks of intercellular signaling pathways. In Arabidopsis thaliana, the small secreted peptide CLAVATA3 (CLV3) controls shoot apical meristem (SAM) maintenance by activating a signal transduction pathway that modulates the expression of the homeodomain transcription factor WUSCHEL (WUS). Here, we demonstrate that two CLV3-related peptides, CLE16 and CLE17, restrict stem cell accumulation in the absence of CLV3. CLE16 and CLE17 contribute independently to SAM maintenance and organ production in clv3 plants at all stages of development. We show that CLE16 and CLE17 signal through a subset of CLV3 receptors, the BARELY ANY MERISTEM (BAM) receptor kinases, and act upstream of WUS. Our study reveals that CLE16 and CLE17 function in a mechanism that partially compensates for CLV3 to maintain stem cell homeostasis and plant resiliency, and expands the potential targets for enhancing yield traits in crop species.

A group of CLE peptides regulates de novo shoot regeneration in Arabidopsis thaliana.

Known for their regulatory roles in stem cell homeostasis, CLAVATA3/ESR-RELATED (CLE) peptides also function as mediators of external stimuli such as hormones. De novo shoot regeneration, representing the remarkable plant cellular plasticity, involves reconstitution of stem cells under control of stem-cell regulators. Yet whether and how stem cell-regulating CLE peptides are implicated in plant regeneration remains unknown. By CRISPR/Cas9-induced loss-of-function studies, peptide application, precursor overexpression, and expression analyses, the role of CLE1-CLE7 peptides and their receptors in de novo shoot regeneration was studied in Arabidopsis thaliana. CLE1-CLE7 are induced by callus-induction medium and dynamically expressed in pluripotent callus. Exogenously-applied CLE1-CLE7 peptides or precursor overexpression effectively leads to shoot regeneration suppression, whereas their simultaneous mutation results in enhanced regenerative capacity, demonstrating that CLE1-CLE7 peptides redundantly function as negative regulators of de novo shoot regeneration. CLE1-CLE7-mediated shoot regeneration suppression is impaired in loss-of-function mutants of callus-expressed CLAVATA1 (CLV1) and BARELY ANY MERISTEM1 (BAM1) genes, indicating that CLV1/BAM1 are required for CLE1-CLE7-mediated shoot regeneration signaling. CLE1-CLE7 signaling resulted in transcriptional repression of WUSCHEL (WUS), a stem cell-promoting transcription factor known as a principal regulator of plant regeneration. Our results indicate that functionally-redundant CLE1-CLE7 peptides genetically act through CLV1/BAM1 receptors and repress WUS expression to modulate shoot-regeneration capacity, establishing the mechanistic basis for CLE1-CLE7-mediated shoot regeneration and a novel role for CLE peptides in hormone-dependent developmental plasticity.

Signaling peptides direct the art of rebirth.

Signaling peptide-mediated cell-cell communication is crucial for plant growth, development, and adaptive responses to environmental stimuli. Given the prominent roles signaling peptides play in stem cell homeostasis, we propose investigating their impact on plant regeneration, which requires cellular reprogramming of differentiated cells to stem cells and establishment of nascent meristems.

Recent Advances in Arabidopsis CLE Peptide Signaling.

Like communities of people, communities of cells must continuously communicate to thrive. Polypeptide signaling molecules that act as mobile ligands are widely used by eukaryotic organisms to transmit information between cells to coordinate developmental processes and responses to environmental cues. In plants, the CLAVATA3/EMBRYO SURROUNDING REGION-RELATED (CLE) genes encode a large family of extracellular signaling peptides that stimulate receptor-mediated signal transduction cascades to modulate diverse developmental and physiological processes. This review highlights the emerging roles of Arabidopsisthaliana CLE peptide signaling pathways in shoot stem cell homeostasis and root xylem development, as well as in root protophloem cell differentiation, vascular cambium activity, and stomatal formation and closure.

The Trithorax Group Factor ULTRAPETALA1 Regulates Developmental as Well as Biotic and Abiotic Stress Response Genes in Arabidopsis.

In eukaryotes, Polycomb group (PcG) and trithorax group (trxG) factors oppositely regulate gene transcription during development through histone modifications, with PcG factors repressing and trxG factors activating the expression of their target genes. Although plant trxG factors regulate many developmental and physiological processes, their downstream targets are poorly characterized. Here we use transcriptomics to identify genome-wide targets of the Arabidopsis thaliana trxG factor ULTRAPETALA1 (ULT1) during vegetative and reproductive development and compare them with those of the PcG factor CURLY LEAF (CLF). We find that genes involved in development and transcription regulation are over-represented among ULT1 target genes. In addition, stress response genes and defense response genes such as those in glucosinolate metabolic pathways are enriched, revealing a previously unknown role for ULT1 in controlling biotic and abiotic response pathways. Finally, we show that many ULT1 target genes can be oppositely regulated by CLF, suggesting that ULT1 and CLF may have antagonistic effects on plant growth and development in response to various endogenous and environmental cues.

The CLV-WUS Stem Cell Signaling Pathway: A Roadmap to Crop Yield Optimization.

The shoot apical meristem at the growing shoot tip acts a stem cell reservoir that provides cells to generate the entire above-ground architecture of higher plants. Many agronomic plant yield traits such as tiller number, flower number, fruit number, and kernel row number are therefore defined by the activity of the shoot apical meristem and its derivatives, the floral meristems. Studies in the model plant Arabidopsis thaliana demonstrated that a molecular negative feedback loop called the CLAVATA (CLV)-WUSCHEL (WUS) pathway regulates stem cell maintenance in shoot and floral meristems. CLV-WUS pathway components are associated with quantitative trait loci (QTL) for yield traits in crop plants such as oilseed, tomato, rice, and maize, and may have played a role in crop domestication. The conservation of these pathway components across the plant kingdom provides an opportunity to use cutting edge techniques such as genome editing to enhance yield traits in a wide variety of agricultural plant species.

The signaling peptide-encoding genes CLE16, CLE17 and CLE27 are dispensable for Arabidopsis shoot apical meristem activity.

The shoot apical meristem produces all of the leaves, stems and flowers of a flowering plant from a reservoir of stem cells at its growing tip. In Arabidopsis, the small polypeptide signaling molecule CLAVATA3 (CLV3), a member of the CLV3/EMBRYO SURROUNDING REGION-RELATED (CLE) gene family, is a key component of a negative feedback loop that maintains stem cell activity in shoot and floral meristems throughout development. Because in some plant species multiple CLE genes are involved in regulating shoot apical meristem activity, we tested the hypothesis that CLE genes other than CLV3 might function in stem cell homeostasis in Arabidopsis. We identified three Arabidopsis CLE genes expressed in the post-embryonic shoot apical meristem, generated loss-of-function alleles using genome editing, and analyzed the meristem phenotypes of the resulting mutant plants. We found that null mutations in CLE16, CLE17 or CLE27 affected neither vegetative nor reproductive shoot meristem activity under normal growth conditions, although CLE27 appears to slightly prolong vegetative growth. Our results indicate that the CLE16, CLE17 and CLE27 genes have largely redundant roles in the Arabidopsis shoot apical meristem and/or regulate meristem activity only under specific environmental conditions.

Trithorax Group Proteins Act Together with a Polycomb Group Protein to Maintain Chromatin Integrity for Epigenetic Silencing during Seed Germination in Arabidopsis.

Polycomb group (PcG) and trithorax group (trxG) proteins have been shown to act antagonistically to epigenetically regulate gene expression in eukaryotes. The trxG proteins counteract PcG-mediated floral repression in Arabidopsis, but their roles in other developmental processes are poorly understood. We investigated the interactions between the trxG genes, ARABIDOPSIS HOMOLOG OF TRITHORAX1 (ATX1) and ULTRAPETALA1 (ULT1), and the PcG gene EMBRYONIC FLOWER 1 (EMF1) during early development. Unexpectedly, we found that mutations in the trxG genes failed to rescue the early-flowering phenotype of emf1 mutants. Instead, emf1 atx1 ult1 seedlings showed a novel swollen root phenotype and massive deregulation of gene expression. Greater ectopic expression of seed master regulatory genes in emf1 atx1 ult1 triple than in emf1 single mutants indicates that PcG and trxG factors together repress seed gene expression after germination. Furthermore, we found that the widespread gene derepression is associated with reduced levels of H3K27me3, an epigenetic repressive mark of gene expression, and with globally altered chromatin organization. EMF1, ATX1, and ULT1 are able to bind the chromatin of seed genes and ULT1 can physically interact with ATX1 and EMF1, suggesting that the trxG and EMF1 proteins directly associate at target gene loci for EMF1-mediated gene silencing. Thus, while ATX1, ULT1, and EMF1 interact antagonistically to regulate flowering, they work together to maintain chromatin integrity and prevent precocious seed gene expression after germination.

Peptide signaling molecules CLE5 and CLE6 affect Arabidopsis leaf shape downstream of leaf patterning transcription factors and auxin.

Intercellular signaling mediated by small peptides is critical to coordinate organ formation in animals, but whether extracellular polypeptides play similar roles in plants is unknown. Here we describe a role in Arabidopsis leaf development for two members of the CLAVATA3/ESR-RELATED peptide family, CLE5 and CLE6, which lie adjacent to each other on chromosome 2. Uniquely among the CLE genes, CLE5 and CLE6 are expressed specifically at the base of developing leaves and floral organs, adjacent to the boundary with the shoot apical meristem. During vegetative development CLE5 and CLE6 transcription is regulated by the leaf patterning transcription factors BLADE-ON-PETIOLE1 (BOP1) and ASYMMETRIC LEAVES2 (AS2), as well as by the WUSCHEL-RELATED HOMEOBOX (WOX) transcription factors WOX1 and PRESSED FLOWER (PRS). Moreover, CLE5 and CLE6 transcript levels are differentially regulated in various genetic backgrounds by the phytohormone auxin. Analysis of loss-of-function mutations generated by genome engineering reveals that CLE5 and CLE6 independently and together have subtle effects on rosette leaf shape. Our study indicates that the CLE5 and CLE6 peptides function downstream of leaf patterning factors and phytohormones to modulate the final leaf morphology.

State of the Art: trxG Factor Regulation of Post-embryonic Plant Development.

Multicellular organisms rely on the precise and consistent regulation of gene expression to direct their development in tissue- and cell-type specific patterns. This regulatory activity involves arrays of DNA-binding transcription factors and epigenetic factors that modify chromatin structure. Among the chromatin modifiers, trithorax (trxG) and Polycomb (PcG) group proteins play important roles in orchestrating the stable activation and repression of gene expression, respectively. These proteins have generally antagonistic functions in maintaining cell and tissue homeostasis as well as in mediating widespread transcriptional reprogramming during developmental transitions. Plants utilize multiple trxG factors to regulate gene transcription as they modulate their development in response to both endogenous and environmental cues. Here, I will discuss the roles of trxG factors and their associated proteins in post-embryonic plant development.

Calpain-Mediated Positional Information Directs Cell Wall Orientation to Sustain Plant Stem Cell Activity, Growth and Development.

Eukaryotic development and stem cell control depend on the integration of cell positional sensing with cell cycle control and cell wall positioning, yet few factors that directly link these events are known. The DEFECTIVE KERNEL1 (DEK1) gene encoding the unique plant calpain protein is fundamental for development and growth, being essential to confer and maintain epidermal cell identity that allows development beyond the globular embryo stage. We show that DEK1 expression is highest in the actively dividing cells of seeds, meristems and vasculature. We further show that eliminating Arabidopsis DEK1 function leads to changes in developmental cues from the first zygotic division onward, altered microtubule patterns and misshapen cells, resulting in early embryo abortion. Expression of the embryonic marker genes WOX2, ATML1, PIN4, WUS and STM, related to axis organization, cell identity and meristem functions, is also altered in dek1 embryos. By monitoring cell layer-specific DEK1 down-regulation, we show that L1- and 35S-induced down-regulation mainly affects stem cell functions, causing severe shoot apical meristem phenotypes. These results are consistent with a requirement for DEK1 to direct layer-specific cellular activities and set downstream developmental cues. Our data suggest that DEK1 may anchor cell wall positions and control cell division and differentiation, thereby balancing the plant's requirement to maintain totipotent stem cell reservoirs while simultaneously directing growth and organ formation. A role for DEK1 in regulating microtubule-orchestrated cell wall orientation during cell division can explain its effects on embryonic development, and suggests a more general function for calpains in microtubule organization in eukaryotic cells.

The ULTRAPETALA1 trxG factor contributes to patterning the Arabidopsis adaxial-abaxial leaf polarity axis.

The SAND domain protein ULTRAPETALA1 (ULT1) functions as a trithorax group factor that regulates a variety of developmental processes in Arabidopsis. We have recently shown that ULT1 regulates developmental patterning in the gynoecia and leaves. ULT1 acts together with the KANADI1 (KAN1) transcription factor to pattern the apical-basal axis during gynoecium formation, whereas the 2 genes act antagonistically to pattern the adaxial-abaxial axis during both gynoecium and leaf formation. In particular, our data showed that ULT1 is necessary for the kan1 adaxialized organ phenotype. Here, we observe the internal structure of ult1, kan1 and ult1 kan1 rosette leaves to better understand the suppression of the kan1 adaxialized leaf polarity defect by ult1 mutations. Our results indicate that ULT1 and KAN1 act antagonistically to pattern the adaxial-abaxial axis in leaves by establishing the asymmetry of the internal cell layers.

Polypeptide signaling molecules in plant development.

Intercellular communication mediated by small signaling molecules is a key mechanism for coordinating plant growth and development. In the past few years, polypeptide signals have been shown to play prominent roles in processes as diverse as shoot and root meristem maintenance, vascular differentiation, lateral root emergence, and seed formation. Signaling components such as CLV1 and the IDA-HAE/HSL2 signaling module have been discovered to regulate distinct developmental processes in different tissues. Recent studies have also uncovered novel polypeptide-receptor interactions, intracellular components and downstream target genes, adding complexity to our picture of polypeptide signaling networks. Finally, new families of plant polypeptides, such as the GLV/RGF/CLEL and ESF factors, are being identified, the functions of which we are only beginning to understand.

The ULT trxG factors play a role in Arabidopsis fertilization.

Trithorax group (trxG) and Polycomb group (PcG) proteins are epigenetic modifiers that play key roles in eukaryotic development by promoting active or repressive gene expression states, respectively. Although PcG proteins have well-defined roles in controlling developmental transitions, cell fate decisions and cellular differentiation in plants, relatively little is known about the functions of plant trxG factors. We recently determined the biological roles for the ULT1 and ULT2 trxG genes during Arabidopsis vegetative and reproductive development. Our study revealed that ULT1 and ULT2 genes have overlapping activities in regulating Arabidopsis shoot and floral stem cell activity, and that they have a redundant function in establishing the apical-basal polarity axis of the gynoecium. Here we present data that ult1 and ult1 ult2 siliques contain a significant proportion of aborted ovules, supporting an additional role for ULT1 in Arabidopsis fertility. Our results add to the number of plant developmental processes that are regulated by trxG activity.

ULTRAPETALA trxG genes interact with KANADI transcription factor genes to regulate Arabidopsis gynoecium patterning.

Organ formation relies upon precise patterns of gene expression that are under tight spatial and temporal regulation. Transcription patterns are specified by several cellular processes during development, including chromatin remodeling, but little is known about how chromatin-remodeling factors contribute to plant organogenesis. We demonstrate that the trithorax group (trxG) gene ULTRAPETALA1 (ULT1) and the GARP transcription factor gene KANADI1 (KAN1) organize the Arabidopsis thaliana gynoecium along two distinct polarity axes. We show that ULT1 activity is required for the kan1 adaxialized polarity defect, indicating that ULT1 and KAN1 act oppositely to regulate the adaxial-abaxial axis. Conversely, ULT1 and KAN1 together establish apical-basal polarity by promoting basal cell fate in the gynoecium, restricting the expression domain of the basic helix-loop-helix transcription factor gene SPATULA. Finally, we show that ult alleles display dose-dependent genetic interactions with kan alleles and that ULT and KAN proteins can associate physically. Our findings identify a dual role for plant trxG factors in organ patterning, with ULT1 and KAN1 acting antagonistically to pattern the adaxial-abaxial polarity axis but jointly to pattern the apical-basal axis. Our data indicate that the ULT proteins function to link chromatin-remodeling factors with DNA binding transcription factors to regulate target gene expression.