The stomatal fates: Understanding initiation and enforcement of stomatal cell fate transitions.

In the stomatal lineage, repeated arcs of initiation, stem-cell proliferation, and terminal cell fate commitment are displayed on the surface of aerial organs. Over the past two decades, the core transcription and signaling elements that guide cell divisions, patterning, and fate transitions were defined. Here we highlight recent work that extends the core using a variety of cutting-edge techniques in different plant species. New work has discovered transcriptional circuits that initiate and reinforce stomatal fate transitions, while also enabling the lineage to interpret and respond to environmental inputs. Recent developments show that some key stomatal factors are more flexible or potentially even interchangeable, opening up avenues to explore stomatal fates and regulatory networks.

A cell size threshold triggers commitment to stomatal fate in Arabidopsis.

How flexible developmental programs integrate information from internal and external factors to modulate stem cell behavior is a fundamental question in developmental biology. Cells of the Arabidopsis stomatal lineage modify the balance of stem cell proliferation and differentiation to adjust the size and cell type composition of mature leaves. Here, we report that meristemoids, one type of stomatal lineage stem cell, trigger the transition from asymmetric self-renewing divisions to commitment and terminal differentiation by crossing a critical cell size threshold. Through computational simulation, we demonstrate that this cell size-mediated transition allows robust, yet flexible termination of stem cell proliferation, and we observe adjustments in the number of divisions before the differentiation threshold under several genetic manipulations. We experimentally evaluate several mechanisms for cell size sensing, and our data suggest that this stomatal lineage transition is dependent on a nuclear factor that is sensitive to DNA content.

Extensive embryonic patterning without cellular differentiation primes the plant epidermis for efficient post-embryonic stomatal activities.

Plant leaves feature epidermal stomata that are organized in stereotyped patterns. How does the pattern originate? We provide transcriptomic, imaging, and genetic evidence that Arabidopsis embryos engage known stomatal fate and patterning factors to create regularly spaced stomatal precursor cells. Analysis of embryos from 36 plant species indicates that this trait is widespread among angiosperms. Embryonic stomatal patterning in Arabidopsis is established in three stages: first, broad SPEECHLESS (SPCH) expression; second, coalescence of SPCH and its targets into discrete domains; and third, one round of asymmetric division to create stomatal precursors. Lineage progression is then halted until after germination. We show that the embryonic stomatal pattern enables fast stomatal differentiation and photosynthetic activity upon germination, but it also guides the formation of additional stomata as the leaf expands. In addition, key stomatal regulators are prevented from driving the fate transitions they can induce after germination, identifying stage-specific layers of regulation that control lineage progression during embryogenesis.

De novo stem cell establishment in meristems requires repression of organ boundary cell fate.

Stem cells play important roles in animal and plant biology, as they sustain morphogenesis and tissue replenishment following aging or injury. In plants, stem cells are embedded in multicellular structures called meristems. The formation of new meristems is essential for the plastic expansion of the highly branched shoot and root systems. In particular, axillary meristems (AMs) that produce lateral shoots arise from the division of boundary domain cells at the leaf base. The CUP-SHAPED COTYLEDON (CUC) genes are major determinants of the boundary domain and are required for AM initiation. However, how AMs get structured and how stem cells become established de novo remain elusive. Here, we show that two NGATHA-LIKE (NGAL) transcription factors, DEVELOPMENT-RELATED PcG TARGET IN THE APEX4 (DPA4)/NGAL3 and SUPPRESSOR OF DA1-1 7 (SOD7)/NGAL2, redundantly repress CUC expression in initiating AMs of Arabidopsis thaliana. Ectopic boundary fate leads to abnormal growth and organization of the AM and prevents de novo stem cell establishment. Floral meristems of the dpa4 sod7 double mutant show a similar delay in de novo stem cell establishment. Altogether, while boundary fate is required for the initiation of AMs, our work reveals how it is later repressed to allow proper meristem establishment and de novo stem cell niche formation.

A network of transcriptional repressors modulates auxin responses.

The regulation of signalling capacity, combined with the spatiotemporal distribution of developmental signals themselves, is pivotal in setting developmental responses in both plants and animals1. The hormone auxin is a key signal for plant growth and development that acts through the AUXIN RESPONSE FACTOR (ARF) transcription factors2-4. A subset of these, the conserved class A ARFs5, are transcriptional activators of auxin-responsive target genes that are essential for regulating auxin signalling throughout the plant lifecycle2,3. Although class A ARFs have tissue-specific expression patterns, how their expression is regulated is unknown. Here we show, by investigating chromatin modifications and accessibility, that loci encoding these proteins are constitutively open for transcription. Through yeast one-hybrid screening, we identify the transcriptional regulators of the genes encoding class A ARFs from Arabidopsis thaliana and demonstrate that each gene is controlled by specific sets of transcriptional regulators. Transient transformation assays and expression analyses in mutants reveal that, in planta, the majority of these regulators repress the transcription of genes encoding class A ARFs. These observations support a scenario in which the default configuration of open chromatin enables a network of transcriptional repressors to regulate expression levels of class A ARF proteins and modulate auxin signalling output throughout development.

Specification and regulation of vascular tissue identity in the Arabidopsis embryo.

Development of plant vascular tissues involves tissue identity specification, growth, pattern formation and cell-type differentiation. Although later developmental steps are understood in some detail, it is still largely unknown how the tissue is initially specified. We used the early Arabidopsis embryo as a simple model to study this process. Using a large collection of marker genes, we found that vascular identity was specified in the 16-cell embryo. After a transient precursor state, however, there was no persistent uniform tissue identity. Auxin is intimately connected to vascular tissue development. We found that, although an AUXIN RESPONSE FACTOR5/MONOPTEROS (ARF5/MP)-dependent auxin response was required, it was not sufficient for tissue specification. We therefore used a large-scale enhanced yeast one-hybrid assay to identify potential regulators of vascular identity. Network and functional analysis of candidate regulators suggest that vascular identity is under robust, complex control. We found that one candidate regulator, the G-class bZIP transcription factor GBF2, can modulate vascular gene expression by tuning MP output through direct interaction. Our work uncovers components of a gene regulatory network that controls the initial specification of vascular tissue identity.

A PXY-Mediated Transcriptional Network Integrates Signaling Mechanisms to Control Vascular Development in Arabidopsis.

The cambium and procambium generate the majority of biomass in vascular plants. These meristems constitute a bifacial stem cell population from which xylem and phloem are specified on opposing sides by positional signals. The PHLOEM INTERCALATED WITH XYLEM (PXY) receptor kinase promotes vascular cell division and organization. However, how these functions are specified and integrated is unknown. Here, we mapped a putative PXY-mediated transcriptional regulatory network comprising 690 transcription factor-promoter interactions in Arabidopsis (Arabidopsis thaliana). Among these interactions was a feedforward loop containing transcription factors WUSCHEL HOMEOBOX RELATED14 (WOX14) and TARGET OF MONOPTEROS6 (TMO6), each of which regulates the expression of the gene encoding a third transcription factor, LATERAL ORGAN BOUNDARIES DOMAIN4 (LBD4). PXY signaling in turn regulates the WOX14, TMO6, and LBD4 feedforward loop to control vascular proliferation. Genetic interaction between LBD4 and PXY suggests that LBD4 marks the phloem-procambium boundary, thus defining the shape of the vascular bundle. These data collectively support a mechanism that influences the recruitment of cells into the phloem lineage, and they define the role of PXY signaling in this context in determining the arrangement of vascular tissue.

A brief history of the TDIF-PXY signalling module: balancing meristem identity and differentiation during vascular development.

474 I. 474 II. 475 III. 475 IV. 477 V. 477 VI. 477 VII. 479 VIII. 481 482 References 482 SUMMARY: A significant proportion of terrestrial biomass is constituted of xylem cells that make up woody plant tissue. Xylem is required for water transport, and is present in the vascular tissue with a second conductive tissue, phloem, required primarily for nutrient transport. Both xylem and phloem are derived from cell divisions in vascular meristems known as the cambium and procambium. One major component that influences several aspects of plant vascular development, including cell division in the vascular meristem, vascular organization and differentiation of vascular cell types, is a signalling module characterized by a peptide ligand called TRACHEARY ELEMENT DIFFERENTIATION INHIBITORY FACTOR (TDIF) and its cognate receptor, PHLOEM INTERCALATED WITH XYLEM (PXY). In this review, we explore the literature that describes signalling components, phytohormones and transcription factors that interact with these two central factors, to control the varying outputs required in vascular tissues for normal organization and elaboration of plant vascular tissue.

The role of auxin signaling in early embryo pattern formation.

Pattern formation of the early Arabidopsis embryo generates precursors to all major cell types, and is profoundly controlled by the signaling molecule auxin. Here we discuss recent milestones in our understanding of auxin-dependent embryo patterning. Auxin biosynthesis, transport and response mechanisms interact to generate local auxin accumulation in the early embryo. New auxin-dependent reporters help identifying these sites, while atomic structures of transcriptional response mediators help explain the diverse outputs of auxin signaling. Key auxin outputs are control of cell identity and cell division orientation, and progress has been made towards understanding the cellular basis of each. Importantly, a number of studies have combined computational modeling and experiments to analyze the developmental role, genetic circuitry and molecular mechanisms of auxin-dependent cell division control.

Plant development. Integration of growth and patterning during vascular tissue formation in Arabidopsis.

Coordination of cell division and pattern formation is central to tissue and organ development, particularly in plants where walls prevent cell migration. Auxin and cytokinin are both critical for division and patterning, but it is unknown how these hormones converge upon tissue development. We identify a genetic network that reinforces an early embryonic bias in auxin distribution to create a local, nonresponding cytokinin source within the root vascular tissue. Experimental and theoretical evidence shows that these cells act as a tissue organizer by positioning the domain of oriented cell divisions. We further demonstrate that the auxin-cytokinin interaction acts as a spatial incoherent feed-forward loop, which is essential to generate distinct hormonal response zones, thus establishing a stable pattern within a growing vascular tissue.