Stomatal Lineage Research Articles

SOL1 and SOL2 regulate fate transition and cell divisions in the Arabidopsis stomatal lineage.

In the Arabidopsis stomatal lineage, cells transit through several distinct precursor identities, each characterized by unique cell division behaviors. Flexibility in the duration of these precursor phases enables plants to alter leaf size and stomatal density in response to environmental conditions; however, transitions between phases must be complete and unidirectional to produce functional and correctly patterned stomata. Among direct transcriptional targets of the stomatal initiating factor SPEECHLESS, a pair of genes, SOL1 and SOL2, are required for effective transitions in the lineage. We show that these two genes, which are homologs of the LIN54 DNA-binding components of the mammalian DREAM complex, are expressed in a cell cycle-dependent manner and regulate cell fate and division properties in the self-renewing early lineage. In the terminal division of the stomatal lineage, however, these two proteins appear to act in opposition to their closest paralog, TSO1, revealing complexity in the gene family that may enable customization of cell divisions in coordination with development.

The CLE9/10 secretory peptide regulates stomatal and vascular development through distinct receptors.

The frequency and orientation of cell division are regulated by intercellular signalling molecules; however, tissue-specific regulatory systems for cell divisions are only partially understood. Here, we report that the peptide hormone CLAVATA3/ESR-RELATED 9/10 (CLE9/10) regulates two different developmental processes, stomatal lineage development and xylem development, through two distinct receptor systems in Arabidopsis thaliana. We show that the receptor kinase HAESA-LIKE 1 (HSL1) is a CLE9/10 receptor that regulates stomatal lineage cell division, and BARELY NO MERISTEM (BAM) class receptor kinases are CLE9/10 receptors that regulate periclinal cell division of xylem precursor cells. Both HSL1 and BAM1 bind to CLE9/10, but only HSL1 recruits SOMATIC EMBRYOGENESIS RECEPTOR KINASES as co-receptors in the presence of CLE9/10, suggesting different signalling modes for these receptor systems.

Long-Distance and Trans-Generational Stomatal Patterning by CO2 Across Arabidopsis Organs.

Stomata control water loss and carbon dioxide uptake by both altering pore aperture and developmental patterning. Stomatal patterning is regulated by environmental factors including atmospheric carbon dioxide (p[CO2]), which is increasing globally at an unprecedented rate. Mature leaves are known to convey developmental cues to immature leaves in response to p[CO2], but the developmental mechanisms are unknown. To characterize changes in stomatal patterning resulting from signals moving from mature to developing leaves, we constructed a dual-chamber growth system in which rosette and cauline leaves of Arabidopsis thaliana were subjected to differing p[CO2]. Young rosette tissue was found to adjust stomatal index (SI, the proportion of stomata to total cell number) in response to both the current environment and the environment experienced by mature rosette tissue, whereas cauline leaves appear to be insensitive to p[CO2] treatment. It is likely that cauline leaves and cotyledons deploy mechanisms for controlling stomatal development that share common but also deploy distinctive mechanisms to that operating in rosette leaves. The effect of p[CO2] on stomatal development is retained in cotyledons of the next generation, however, this effect does not occur in pre-germination stomatal lineage cells but only after germination. Finally, these data suggest that p[CO2] affects regulation of stomatal development specifically through the development of satellite stomata (stomata induced by signals from a neighboring stomate) during spacing divisions and not the basal pathway. To our knowledge, this is the first report identifying developmental steps responsible for altered stomatal patterning to p[CO2] and its trans-generational inheritance.

POLAR-guided signalling complex assembly and localization drive asymmetric cell division.

Stomatal cell lineage is an archetypal example of asymmetric cell division (ACD), which is necessary for plant survival1-4. In Arabidopsis thaliana, the GLYCOGEN SYNTHASE KINASE3 (GSK3)/SHAGGY-like kinase BRASSINOSTEROID INSENSITIVE 2 (BIN2) phosphorylates both the mitogen-activated protein kinase (MAPK) signalling module5,6 and its downstream target, the transcription factor SPEECHLESS (SPCH)7, to promote and restrict ACDs, respectively, in the same stomatal lineage cell. However, the mechanisms that balance these mutually exclusive activities remain unclear. Here we identify the plant-specific protein POLAR as a stomatal lineage scaffold for a subset of GSK3-like kinases that confines them to the cytosol and subsequently transiently polarizes them within the cell, together with BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL), before ACD. As a result, MAPK signalling is attenuated, enabling SPCH to drive ACD in the nucleus. Moreover, POLAR turnover requires phosphorylation on specific residues, mediated by GSK3. Our study reveals a mechanism by which the scaffolding protein POLAR ensures GSK3 substrate specificity, and could serve as a paradigm for understanding regulation of GSK3 in plants.

Spatiotemporal coordination of cell division and growth during organ morphogenesis.

A developing plant organ exhibits complex spatiotemporal patterns of growth, cell division, cell size, cell shape, and organ shape. Explaining these patterns presents a challenge because of their dynamics and cross-correlations, which can make it difficult to disentangle causes from effects. To address these problems, we used live imaging to determine the spatiotemporal patterns of leaf growth and division in different genetic and tissue contexts. In the simplifying background of the speechless (spch) mutant, which lacks stomatal lineages, the epidermal cell layer exhibits defined patterns of division, cell size, cell shape, and growth along the proximodistal and mediolateral axes. The patterns and correlations are distinctive from those observed in the connected subepidermal layer and also different from the epidermal layer of wild type. Through computational modelling we show that the results can be accounted for by a dual control model in which spatiotemporal control operates on both growth and cell division, with cross-connections between them. The interactions between resulting growth and division patterns lead to a dynamic distributions of cell sizes and shapes within a deforming leaf. By modulating parameters of the model, we illustrate how phenotypes with correlated changes in cell size, cell number, and organ size may be generated. The model thus provides an integrated view of growth and division that can act as a framework for further experimental study.

Modulation of Asymmetric Division Diversity through Cytokinin and SPEECHLESS Regulatory Interactions in the Arabidopsis Stomatal Lineage

Coordinated growth of organs requires communication among cells within and between tissues. In plants, leaf growth is largely dictated by the epidermis; here, asymmetric and self-renewing divisions of the stomatal lineage create two essential cell types-pavement cells and guard cells-in proportions reflecting inputs from local, systemic, and environmental cues. The transcription factor SPEECHLESS (SPCH) is the primeregulator of divisions, but whether and how it is influenced by external cues to provide flexible development is enigmatic. Here, we show that the phytohormone cytokinin (CK) can act as an endogenous signal to affect the extent and types of stomatal lineage divisions and forms a regulatory circuit withSPCH. Local domains of low CK signaling are created by SPCH-dependent cell-type-specific activity of two repressive type-A ARABIDOPSIS RESPONSE REGULATORs (ARRs), ARR16 and ARR17, and two secreted peptides, CLE9 and CLE10, which, together with SPCH, can customize epidermal cell-type composition.

Ectopic BASL Reveals Tissue Cell Polarity throughout Leaf Development in Arabidopsis thaliana

SummaryTissue-wide polarity fields, in which cell polarity is coordinated across the tissue, have been described for planar organs such as the Drosophila wing and are considered important for coordinating growth and differentiation [1]. In planar plant organs, such as leaves, polarity fields have been identified for subgroups of cells, such as stomatal lineages [2], trichomes [3, 4], serrations [5], or early developmental stages [6]. Here, we show that ectopic induction of the stomatal protein BASL (BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE) reveals a tissue-wide epidermal polarity field in leaves throughout development. Ectopic GFP-BASL is typically localized toward the proximal end of cells and to one lobe of mature pavement cells, revealing a polarity field that aligns with the proximodistal axis of the leaf (base to tip). The polarity field is largely parallel to the midline of the leaf but diverges in more lateral positions, particularly at later stages in development, suggesting it may be deformed during growth. The polarity field is observed in the speechless mutant, showing that it is independent of stomatal lineages, and is observed in isotropic cells, showing that cell shape anisotropy is not required for orienting polarity. Ectopic BASL forms convergence and divergence points at serrations, mirroring epidermal PIN polarity patterns, suggesting a common underlying polarity mechanism. Thus, we show that similar to the situation in animals, planar plant organs have a tissue-wide cell polarity field, and this may provide a general cellular mechanism for guiding growth and differentiation.

MAP KINASE PHOSPHATASE1 Controls Cell Fate Transition during Stomatal Development.

Stomata on the plant epidermis control gas and water exchange and are formed by MAPK-dependent processes. Although the contribution of MAP KINASE3 (MPK3) and MPK6 (MPK3/MPK6) to the control of stomatal patterning and differentiation in Arabidopsis (Arabidopsis thaliana) has been examined extensively, how they are inactivated and regulate distinct stages of stomatal development is unknown. Here, we identify a dual-specificity phosphatase, MAP KINASE PHOSPHATASE1 (MKP1), which promotes stomatal cell fate transition by controlling MAPK activation at the early stage of stomatal development. Loss of function of MKP1 creates clusters of small cells that fail to differentiate into stomata, resulting in the formation of patches of pavement cells. We show that MKP1 acts downstream of YODA (a MAPK kinase kinase) but upstream of MPK3/MPK6 in the stomatal signaling pathway and that MKP1 deficiency causes stomatal signal-induced MAPK hyperactivation in vivo. By expressing MKP1 in the three discrete cell types of stomatal lineage, we further identified that MKP1-mediated deactivation of MAPKs in early stomatal precursor cells directs cell fate transition leading to stomatal differentiation. Together, our data reveal the important role of MKP1 in controlling MAPK signaling specificity and cell fate decision during stomatal development.

A2-type cyclin is required for the asymmetric entry division in rice stomatal development.

In rice, and other major cereal grass crops, stomata are arranged in linear files parallel to the long growth axis of leaves. Each stomatal unit comprises two dumbbell-shaped guard cells flanked by two subsidiary cells. These morphological and developmental characteristics enable grass stomata to respond to environmental changes more efficiently. Cyclin-dependent kinases (CDKs) and their cyclin partners co-ordinate cell proliferation and differentiation during the development of multicellular organisms. In contrast to animals, plants have many more types and members of cyclins. In Arabidopsis, four A2-type cyclins (CYCA2s) function redundantly in regulating CDKB1 activity to promote the asymmetric division for stomatal initiation and the symmetric division of guard mother cells (GMCs). In this study, we examine the function of the single A2-type cyclin in rice, OsCYCA2;1, as well the single B1-type CDK, OsCDKB1;1. Cross-species complementation tests demonstrated that OsCYCA2;1 and OsCDKB1;1 could complement the defective stomatal phenotypes of Arabidopsis cyca2 and cdkb1 mutants, but also could suppress DNA endoduplication and cell enlargement. The early asymmetric divisions that establish the stomatal lineages are often missing within the stomatal cell files of OsCYCA2;1-RNAi rice transgenic lines, leading to a significantly reduced stomatal production. However, GMC divisions are not disrupted either in OsCYCA2;1-RNAi or in OsCDKB1;1-RNAi rice transgenic lines as expected. Our results demonstrate a conserved but diverged function and behavior of rice A2-type cyclins, which might be associated with the distinct stomatal development pathways between rice and Arabidopsis.

Direct Control of SPEECHLESS by PIF4 in the High-Temperature Response of Stomatal Development

Environmental factors shape the phenotypes of multicellular organisms. The production of stomata-the epidermal pores required for gas exchange in plants-is highly plastic and provides a powerful platform to address environmental influence on cell differentiation [1-3]. Rising temperatures are already impacting plant growth, a trend expected to worsen inthe near future [4]. High temperature inhibits stomatal production, but the underlying mechanism is not known [5]. Here, we show that elevated temperature suppresses the expression of SPEECHLESS (SPCH), the basic-helix-loop-helix (bHLH) transcription factor that serves as the master regulator of stomatal lineage initiation [6, 7]. Our genetic and expression analyses indicate that the suppression of SPCH and stomatal production is mediated by the bHLH transcription factor PHYTOCHROME-INTERACTINGFACTOR 4 (PIF4), a core component of high-temperature signaling [8]. Importantly, we demonstrate that, upon exposure to high temperature, PIF4 accumulates in the stomatal precursors and binds to the promoter of SPCH. In addition, we find SPCH feeds back negatively to the PIF4 gene. We propose a model where warm-temperature-activated PIF4 binds and represses SPCH expression to restrict stomatal production at elevated temperatures. Our work identifies a molecular link connecting high-temperature signaling and stomatal development and reveals a direct mechanism by which production of a specific cell lineage can be controlled by a broadly expressed environmental signaling factor.

Lineage- and stage-specific expressed CYCD7;1 coordinates the single symmetric division that creates stomatal guard cells.

Plants, with cells fixed in place by rigid walls, often utilize spatial and temporally distinct cell division programs to organize and maintain organs. This leads to the question of how developmental regulators interact with the cell cycle machinery to link cell division events with particular developmental trajectories. In Arabidopsis leaves, the development of stomata, two-celled epidermal valves that mediate plant-atmosphere gas exchange, relies on a series of oriented stem cell-like asymmetric divisions followed by a single symmetric division. The stomatal lineage is embedded in a tissue in which other cells transition from proliferation to postmitotic differentiation earlier, necessitating stomatal lineage-specific factors to prolong competence to divide. We show that the D-type cyclin, CYCD7;1, is specifically expressed just prior to the symmetric guard cell-forming division, and that it is limiting for this division. Further, we find that CYCD7;1 is capable of promoting divisions in multiple contexts, likely through RBR1-dependent promotion of the G1/S transition, but that CYCD7;1 is regulated at the transcriptional level by cell type-specific transcription factors that confine its expression to the appropriate developmental window.

Ultrastructure and development of non-contiguous stomatal clusters and helicocytic patterning in Begonia.

Helicocytic stomata are characterized by an inward spiral of mesogenous cells surrounding a central stomatal pore. They represent a relatively rare feature that occurs in some drought-tolerant angiosperm species. In some Begonia species with thick leaves, the stomata are not only helicocytic but also clustered into groups that are spaced apart by at least one cell. This paper presents a detailed ontogenetic study of this characteristic non-contiguous stomatal patterning in a developmental and phylogenetic context. Light microscopy and both scanning and transmission electron microscopy were used to examine stomatal development in several species of Begonia. Published reports of stomatal development in Begonia and other angiosperms were reviewed to provide a comprehensive discussion of the evolution of stomatal patterning. Helicocytic stomata develop from meristemoids that undergo a series of oriented asymmetric divisions to produce a spiral of mesogene stomatal lineage ground cells (SLGCs) surrounding a stoma. A clear developmental similarity between anisocytic and helicocytic stomata is positively correlated with the number of iterations of amplifying divisions that result in SLGCs. Stomatal clusters develop from asymmetric divisions in neighbouring SLGCs. Within each cluster, non-contiguous spacing of meristemoids is maintained by asymmetric divisions oriented away from each developing meristemoid. Formation of non-contiguous stomatal clusters in Begonia relies on two primary developmental factors in the epidermis: an inwardly spiralling series of amplifying divisions that result in helicocytic stomata, and the development of a variable number of meristemoids from neighbouring SLGCs within each cluster. Optimization of these features on an angiosperm phylogeny indicates that the occurrence of amplifying divisions could be pre-adaptive for these factors. Both factors have been thoroughly studied in terms of developmental genetics in Arabidopsis, suggesting gene orthologues that could be implicated in Begonia stomatal patterning.

Disruption of stomatal lineage signaling or transcriptional regulators has differential effects on mesophyll development, but maintains coordination of gas exchange.

Summary Stomata are simultaneously tasked with permitting the uptake of carbon dioxide for photosynthesis while limiting water loss from the plant. This process is mainly regulated by guard cell control of the stomatal aperture, but recent advancements have highlighted the importance of several genes that control stomatal development.Using targeted genetic manipulations of the stomatal lineage and a combination of gas exchange and microscopy techniques, we show that changes in stomatal development of the epidermal layer lead to coupled changes in the underlying mesophyll tissues. This coordinated response tends to match leaf photosynthetic potential (V cmax) with gas‐exchange capacity (g smax), and hence the uptake of carbon dioxide for water lost.We found that different genetic regulators systematically altered tissue coordination in separate ways: the transcription factor SPEECHLESS (SPCH) primarily affected leaf size and thickness, whereas peptides in the EPIDERMAL PATTERNING FACTOR (EPF) family altered cell density in the mesophyll. It was also determined that interlayer coordination required the cell‐surface receptor TOO MANY MOUTHS (TMM).These results demonstrate that stomata‐specific regulators can alter mesophyll properties, which provides insight into how molecular pathways can organize leaf tissues to coordinate gas exchange and suggests new strategies for improving plant water‐use efficiency.

A Mutation in the bHLH Domain of the SPCH Transcription Factor Uncovers a BR-Dependent Mechanism for Stomatal Development.

The asymmetric cell divisions necessary for stomatal lineage initiation and progression in Arabidopsis (Arabidopsis thaliana) require the function of the basic helix-loop-helix (bHLH) transcription factor SPEECHLESS (SPCH). Mutants lacking SPCH do not produce stomata or lineages. Here, we isolated a new spch-5 allele carrying a point mutation in the bHLH domain that displayed normal growth, but had an extremely low number of sometimes clustered stomata in the leaves, whereas the hypocotyls did not have any stomata. In vivo tracking of leaf epidermal cell divisions, combined with marker lines and genetic analysis, showed that the spch-5 leaf phenotype is dosage dependent and results from the decreased ability to initiate and amplify lineages, defects in asymmetric cell fate allocation, and misorientation of asymmetric division planes. Notably, application of brassinosteroids (BRs) partly rescued the stomatal leaf phenotype of spch-5 Transcriptomic analysis combining spch-5 with BR treatments revealed that the expression of a set of SPCH target genes was restored by BRs. Our results also show that BR-dependent stomata formation and expression of some, but not all, SPCH target genes require the integrity of the bHLH domain of SPCH.

The chemical compound bubblin induces stomatal mispatterning in Arabidopsis by disrupting the intrinsic polarity of stomatal lineage cells.

Stem cell polarization is a crucial step in asymmetric cell division, which is a universal system for generating cellular diversity in multicellular organisms. Several conventional genetics studies have attempted to elucidate the mechanisms underlying cell polarization in plants, but it remains largely unknown. In plants, stomata, which are valves for gas exchange, are generated through several rounds of asymmetric divisions. In this study, we identified and characterized a chemical compound that affects stomatal stem cell polarity. High-throughput screening for bioactive molecules identified a pyridine-thiazole derivative, named bubblin, which induced stomatal clustering in Arabidopsis epidermis. Bubblin perturbed stomatal asymmetric division, resulting in the generation of two identical daughter cells. Both cells continued to express the stomatal fate determinant SPEECHLESS, and then differentiated into mispatterned stomata. Bubblin-treated cells had a defect in the polarized localization of BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL), which is required for asymmetric cell fate determination. Our results suggest that bubblin induces stomatal lineage cells to divide without BASL-dependent pre-mitotic establishment of polarity. Bubblin is a potentially valuable tool for investigating cell polarity establishment in stomatal asymmetric division.

Plant Development: Suppression the Key to Asymmetric Cell Fate

A new study shows that SPEECHLESS determines cell fate in the stomatal lineage but is inherited equally by daughter cells following an asymmetric cell division. The polarity determinant BASL acts as a MAPK scaffold, targeting SPEECHLESS for degradation in the larger daughter cell.

Phosphorylation of the Polarity Protein BASL Differentiates Asymmetric Cell Fate through MAPKs and SPCH

Cell polarization is commonly used for the regulation of stem cell asymmetric division in both animals and plants. Stomatal development in Arabidopsis, a process that produces breathing pores in the epidermis, requires asymmetric cell division to differentiate highly specialized guard cells while maintaining a stem cell population [1, 2]. The BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL) protein exhibits a polarized localization pattern in the cell and is required for differential cell fates resulting from asymmetric cell division [3]. The polarization of BASL is made possible by a positive feedback loop with a canonical mitogen-activated protein kinase (MAPK) pathway that recruits the MAPKK kinase YODA (YDA) and MAPK 6 (MPK6) to the cortical polarity site [4]. Here, we study BASL intracellular dynamics and show that the membrane-associated BASL is slowly replenished at the cortical polarity site and that the mobility is tightly linked to its phosphorylation status. Because BASL polarity is only exhibited by one daughter cell after an asymmetric cell division, we study how BASL differentiallyfunctions in the two daughter cells. The YDA MAPKcascade transduces upstream ligand-receptor signaling [5-13] to the transcription factor SPEECHLESS (SPCH), which controls stomatal initiation and is directly suppressed by MPK3/6-mediated phosphorylation [14, 15]. We show that BASL polarization leads to elevated nuclear MPK6 signaling and lowered SPCH abundance in one of the two daughter cells. Therefore, two daughter cells are differentiated by BASL polarity-mediated differential suppression of SPCH, which may provide developmental plasticity in plant stem cell asymmetric cell division (ACD).

Modulators of Stomatal Lineage Signal Transduction Alter Membrane Contact Sites and Reveal Specialization among ERECTA Kinases

Signal transduction from a cell's surface to its interior requires dedicated signaling elements and a cellular environment conducive to signal propagation. Plant development, defense, and homeostasis rely on plasma membrane receptor-like kinases to perceive endogenous and environmental signals, but little is known about their immediate downstream targets and signaling modifiers. Using genetics, biochemistry, and live-cell imaging, we show that the VAP-RELATED SUPPRESSOR OF TMM (VST) family is required for ERECTA-mediated signaling in growth and cell-fate determination and reveal a role for ERECTA-LIKE2 in modulating signaling by its sister kinases. We show that VSTs are peripheral plasma membrane proteins that can form complexes with integral ER-membrane proteins, thereby potentially influencing the organization of the membrane milieu to promote efficient and differential signaling from the ERECTA-family members to their downstream intracellular targets.

Fine-scale dissection of the subdomains of polarity protein BASL in stomatal asymmetric cell division.

Cell polarity is a prerequisite for asymmetric cell divisions (ACDs) that generate cell type diversity during development of multicellular organisms. In Arabidopsis, stomatal lineage ACDs are regulated by the plant-specific protein BREAKING OF ASYMMETRY IN THE STOMATAL LINEAGE (BASL). BASL exhibits dynamic subcellular localization, accumulating initially in the nucleus, but then additionally in a highly polarized crescent at the cell cortex before division. BASL polarization requires a phosphorylation-mediated activation process, but how this is achieved remains unknown. In this study, we performed a fine-scale dissection of BASL protein subdomains and elucidated a nuclear localization sequence for nuclear import and a critical FxFP motif for cortical polarity formation, respectively. Artificially tethering BASL subdomains to the plasma membrane suggests that novel protein partner/s might exist and bind to an internal region of BASL. In addition, we suspect the existence of a protein degradation mechanism associated with the amino terminal domain of BASL that accounts for restricting its predominant expression to the stomatal lineage cells of the epidermis. Taken together, our results revealed that BASL, through its distinct subdomains, integrates multiple regulatory inputs to provide a mechanism that promotes difference during stomatal lineage ACDs.

Arabidopsis CSLD5 Functions in Cell Plate Formation in a Cell Cycle-Dependent Manner.

In plants, the presence of a load-bearing cell wall presents unique challenges during cell division. Unlike other eukaryotes, which undergo contractile cytokinesis upon completion of mitosis, plants instead synthesize and assemble a new dividing cell wall to separate newly formed daughter cells. Here, we mine transcriptome data from individual cell types in the Arabidopsis thaliana stomatal lineage and identify CSLD5, a member of the Cellulose Synthase Like-D family, as a cell wall biosynthesis enzyme uniquely enriched in rapidly dividing cell populations. We further show that CSLD5 is a direct target of SPEECHLESS, the master transcriptional regulator of these divisions during stomatal development. Using a combination of genetic analysis and in vivo localization of fluorescently tagged fusion proteins, we show that CSLD5 preferentially accumulates in dividing plant cells where it participates in the construction of newly forming cell plates. We show that CSLD5 is an unstable protein that is rapidly degraded upon completion of cell division and that the protein turnover characteristics of CSLD5 are altered in ccs52a2 mutants, indicating that CSLD5 turnover may be regulated by a cell cycle-associated E3-ubiquitin ligase, the anaphase-promoting complex.