Assessing the High Temperature Effects on Stomatal Production.

The production of stomata, the epidermal pores of plants, is influenced by diverse environmental signals including high temperature. To assess its impact on stomatal formation, researchers need to grow plants in a carefully designed regime under controlled conditions and capture clear, microscopic views of the epidermis. Here, we describe a procedure to study the effect of high temperature on stomatal formation. This method can generate high-quality epidermal images of cotyledons, leaves, and hypocotyl of young Arabidopsis seedlings, which allow the determination of the pattern, density, and index of stomata on these tissues. Besides temperature, the protocol can serve as a general approach to examine stomatal phenotype and the effect of other external signals on stomatal formation.

Abscisic acid regulates stomatal production by imprinting a SnRK2 kinase-mediated phosphocode on the master regulator SPEECHLESS.

Stomata, the epidermal pores for gas exchange between plants and the atmosphere, are the major sites of water loss. During water shortage, plants limit the formation of new stoma via the phytohormone abscisic acid (ABA) to conserve water. However, how ABA suppresses stomatal production is largely unknown. Here, we demonstrate that three core SnRK2 kinases of ABA signaling inhibit the initiation and proliferation of the stomatal precursors in Arabidopsis. We show that the SnRK2s function within the precursors and directly phosphorylate SPEECHLESS (SPCH), the master transcription factor for stomatal initiation. We identify specific SPCH residues targeted by the SnRK2s, which mediate the ABA/drought-induced suppression of SPCH and stomatal production. This SnRK2-specific SPCH phosphocode connects stomatal development with ABA/drought signals and enables the independent control of this key water conservation response. Our work also highlights how distinct signaling activities can be specifically encoded on a master regulator to modulate developmental plasticity.

20 years of Developmental Cell: Looking forward.

In our 20th anniversary year, we reflect on how fields have changed since our first issue and here look to the future. In this collection of Voices, our writers speculate on the future: in terms of philosophy, cell states, cell processes, and then how to model cell systems.

Light regulates stomatal development by modulating paracrine signaling from inner tissues.

Developmental outcomes are shaped by the interplay between intrinsic and external factors. The production of stomata-essential pores for gas exchange in plants-is extremely plastic and offers an excellent system to study this interplay at the cell lineage level. For plants, light is a key external cue, and it promotes stomatal development and the accumulation of the master stomatal regulator SPEECHLESS (SPCH). However, how light signals are relayed to influence SPCH remains unknown. Here, we show that the light-regulated transcription factor ELONGATED HYPOCOTYL 5 (HY5), a critical regulator for photomorphogenic growth, is present in inner mesophyll cells and directly binds and activates STOMAGEN. STOMAGEN, the mesophyll-derived secreted peptide, in turn stabilizes SPCH in the epidermis, leading to enhanced stomatal production. Our work identifies a molecular link between light signaling and stomatal development that spans two tissue layers and highlights how an environmental signaling factor may coordinate growth across tissue types.

KIN10 promotes stomatal development through stabilization of the SPEECHLESS transcription factor.

Stomata are epidermal structures that modulate gas exchanges between plants and the atmosphere. The formation of stomata is regulated by multiple developmental and environmental signals, but how these signals are coordinated to control this process remains unclear. Here, we showed that the conserved energy sensor kinase SnRK1 promotes stomatal development under short-day photoperiod or in liquid culture conditions. Mutation of KIN10, the catalytic α-subunit of SnRK1, results in the decreased stomatal index; while overexpression of KIN10 significantly induces stomatal development. KIN10 displays the cell-type-specific subcellular location pattern. The nuclear-localized KIN10 proteins are highly enriched in the stomatal lineage cells to phosphorylate and stabilize SPEECHLESS, a master regulator of stomatal formation, thereby promoting stomatal development. Our work identifies a module links connecting the energy signaling and stomatal development and reveals that multiple regulatory mechanisms are in place for SnRK1 to modulate stomatal development in response to changing environments.

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 in the 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-INTERACTING FACTOR 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.

MOBE-ChIP: Probing Cell Type-Specific Binding Through Large-Scale Chromatin Immunoprecipitation.

In multicellular organisms, the initiation and maintenance of specific cell types often require the activity of cell type-specific transcriptional regulators. Understanding their roles in gene regulation is crucial but probing their DNA targets in vivo, especially in a genome-wide manner, remains a technical challenge with their limited expression. To improve the sensitivity of chromatin immunoprecipitation (ChIP) for detecting the cell type-specific signals, we have developed the Maximized Objects for Better Enrichment (MOBE)-ChIP, where ChIP is performed at a substantially larger experimental scale and under low background conditions. Here, we describe the procedure in the study of transcription factors in the model plant Arabidopsis. However, with some modifications, the technique should also be implemented in other systems. Besides cell type-specific studies, MOBE-ChIP can also be used as a general strategy to improve ChIP signals.

Characterization of Cell-Type-Specific DNA Binding Sites of Plant Transcription Factors Using Chromatin Immunoprecipitation.

The generation of diverse cell types in multicellular organisms often requires the activity of cell-type-specific transcription factors. Understanding where these transcription factors bind in controlling specific cellular programs is critical. However, probing these cell-type-specific factors in vivo with standard chromatin immunoprecipitation (ChIP) assays remains a challenge. We have developed an optimized ChIP assay termed Maximized Objects for Better Enrichment (MOBE)-ChIP, which improves ChIP sensitivity and allows the detection of cell-type-specific signals at a genome-wide scale. Here, I describe the procedure for implementing this method for the study of plant transcription factors. Besides being useful for cell-type-specific studies, MOBE-ChIP can also be employed as a general strategy for enhancing ChIP signals.

MOBE-ChIP: a large-scale chromatin immunoprecipitation assay for cell type-specific studies.

Cell type-specific transcriptional regulators play critical roles in the generation and maintenance of multicellularity. As they are often expressed at low levels, in vivo DNA-binding studies of these regulators by standard chromatin immunoprecipitation (ChIP) assays are technically challenging. We describe here an optimized ChIP protocol termed Maximized Objects for Better Enrichment (MOBE)-ChIP, which enhances the sensitivity of ChIP assays for detecting cell type-specific signals. The protocol, which is based on the disproportional increase of target signals over background at higher scales, uses substantially greater volume of starting materials than conventional ChIPs to achieve high signal enrichment. This technique can capture weak binding events that are ambiguous in standard ChIP assays, and is useful both in gene-specific and whole-genome analysis. This protocol has been optimized for Arabidopsis, but should be applicable to other model systems with minor modifications. The full procedure can be completed within 3 days.

Transcriptome dynamics of the stomatal lineage: birth, amplification, and termination of a self-renewing population.

Developmental transitions can be described in terms of morphology and the roles of individual genes, but also in terms of global transcriptional and epigenetic changes. Temporal dissections of transcriptome changes, however, are rare for intact, developing tissues. We used RNA sequencing and microarray platforms to quantify gene expression from labeled cells isolated by fluorescence-activated cell sorting to generate cell-type-specific transcriptomes during development of an adult stem-cell lineage in the Arabidopsis leaf. We show that regulatory modules in this early lineage link cell types that had previously been considered to be under separate control and provide evidence for recruitment of individual members of gene families for different developmental decisions. Because stomata are physiologically important and because stomatal lineage cells exhibit exemplary division, cell fate, and cell signaling behaviors, this dataset serves as a valuable resource for further investigations of fundamental developmental processes.

Irreversible fate commitment in the Arabidopsis stomatal lineage requires a FAMA and RETINOBLASTOMA-RELATED module.

The presumed totipotency of plant cells leads to questions about how specific stem cell lineages and terminal fates could be established. In the Arabidopsis stomatal lineage, a transient self-renewing phase creates precursors that differentiate into one of two epidermal cell types, guard cells or pavement cells. We found that irreversible differentiation of guard cells involves RETINOBLASTOMA-RELATED (RBR) recruitment to regulatory regions of master regulators of stomatal initiation, facilitated through interaction with a terminal stomatal lineage transcription factor, FAMA. Disrupting physical interactions between FAMA and RBR preferentially reveals the role of RBR in enforcing fate commitment over its role in cell-cycle control in this developmental context. Analysis of the phenotypes linked to the modulation of FAMA and RBR sheds new light on the way iterative divisions and terminal differentiation are coordinately regulated in a plant stem-cell lineage.

Direct roles of SPEECHLESS in the specification of stomatal self-renewing cells.

Lineage-specific stem cells are critical for the production and maintenance of specific cell types and tissues in multicellular organisms. In Arabidopsis, the initiation and proliferation of stomatal lineage cells is controlled by the basic helix-loop-helix transcription factor SPEECHLESS (SPCH). SPCH-driven asymmetric and self-renewing divisions allow flexibility in stomatal production and overall organ growth. How SPCH directs stomatal lineage cell behaviors, however, is unclear. Here, we improved the chromatin immunoprecipitation (ChIP) assay and profiled the genome-wide targets of Arabidopsis SPCH in vivo. We found that SPCH controls key regulators of cell fate and asymmetric cell divisions and modulates responsiveness to peptide and phytohormone-mediated intercellular communication. Our results delineate the molecular pathways that regulate an essential adult stem cell lineage in plants.

Conversion from CUL4-based COP1-SPA E3 apparatus to UVR8-COP1-SPA complexes underlies a distinct biochemical function of COP1 under UV-B.

The evolutionarily conserved constitutive photomorphogenesis 1 (COP1) is a RING and WD40 protein that functions as a substrate receptor of CULLIN4-damaged DNA binding protein 1 (CUL4-DDB1)-based E3 ubiquitin ligases in both plants and animals. In Arabidopsis, COP1 is a central repressor of photomorphogenesis in the form of COP1-suppressor of PHYA (SPA) complex(es). CUL4-DDB1-COP1-SPA suppresses the photomorphogenic program by targeting the transcription factor elongated hypocotyl 5 for degradation. Intriguingly, under photomorphogenic UV-B light, COP1 reverses its repressive role and promotes photomorphogenesis. However, the mechanism by which COP1 is functionally switched is still obscure. Here, we demonstrate that UV-B triggers the physical and functional disassociation of the COP1-SPA core complex(es) from CUL4-DDB1 and the formation of a unique complex(es) containing the UV-B receptor UV resistance locus 8 (UVR8). The establishment of this UV-B-dependent COP1 complex(es) is associated with its positive modulation of elongated hypocotyl 5 stability and activity, which sheds light on the mechanism of COP1's promotive action in UV-B-induced photomorphogenesis.

Arabidopsis FHY3 and HY5 positively mediate induction of COP1 transcription in response to photomorphogenic UV-B light.

As sessile organisms, higher plants have evolved the capacity to sense and interpret diverse light signals to modulate their development. In Arabidopsis thaliana, low-intensity and long-wavelength UV-B light is perceived as an informational signal to mediate UV-B-induced photomorphogenesis. Here, we report that the multifunctional E3 ubiquitin ligase, CONSTITUTIVE PHOTOMORPHOGENESIS1 (COP1), a known key player in UV-B photomorphogenic responses, is also a UV-B-inducible gene. Two transcription factors, FAR-RED ELONGATED HYPOCOTYL3 (FHY3) and ELONGATED HYPOCOTYL5 (HY5), directly bind to distinct regulatory elements within the COP1 promoter, which are essential for the induction of the COP1 gene mediated by photomorphogenic UV-B signaling. Absence of FHY3 results in impaired UV-B-induced hypocotyl growth and reduced tolerance against damaging UV-B. Thus, FHY3 positively regulates UV-B-induced photomorphogenesis by directly activating COP1 transcription, while HY5 promotes COP1 expression via a positive feedback loop. Furthermore, FHY3 and HY5 physically interact with each other, and this interaction is diminished by UV-B. Together, our findings reveal that COP1 gene expression in response to photomorphogenic UV-B is controlled by a combinatorial regulation of FHY3 and HY5, and this UV-B-specific working mode of FHY3 and HY5 is distinct from that in far-red light and circadian conditions.

Stomatal development: a plant's perspective on cell polarity, cell fate transitions and intercellular communication.

The plant stomatal lineage manifests features common to many developmental contexts: precursor cells are chosen from an initially equivalent field of cells, undergo asymmetric and self-renewing divisions, communicate among themselves and respond to information from a distance. As we review here, the experimental accessibility of these epidermal lineages, particularly in Arabidopsis, has made stomata a conceptual and technical framework for the study of cell fate, stem cells, and cell polarity in plants.

The photomorphogenic repressors COP1 and DET1: 20 years later.

COP1 and DET1 are among the first repressors of photomorphogenesis to be identified, more than 20 years ago. Discovery of these repressors as conserved regulators of the ubiquitin-proteasome system has established protein degradation as a central theme in light signal transduction. COP1 is a RING E3 ubiquitin ligase that targets key regulators for degradation, and DET1 complexes with COP10 and DDB1, which is proposed to aid in COP1-mediated degradation. Recent studies have strengthened the role of COP1 as a major signaling center. DET1 is also emerging as a chromatin regulator in repressing gene expression. Here, we review current understanding on COP1 and DET1, with a focus on their role as part of two distinct, multimeric CUL4-based E3 ligases.

Interaction of Arabidopsis DET1 with CCA1 and LHY in mediating transcriptional repression in the plant circadian clock.

The COP10-DET1-DDB1 (CDD) complex is an evolutionarily conserved protein complex discovered for its role in the repression of photomorphogenesis in Arabidopsis. It is important in many cellular and developmental processes in both plants and animals, but its molecular mode of action remains poorly understood. Here, we show that the CDD component DET1 possesses transcriptional repression activity and physically interacts with two closely related MYB transcription factors, CCA1 and LHY, which are core components of the plant circadian clock. DET1 associates with the promoter of CCA1/LHY target genes, such as TOC1, in a CCA1/LHY-dependent manner and is required for their repression, suggesting a recruitment of DET1 by the central oscillator components to regulate the clock. Our results reveal DET1 as a core transcriptional repression factor important for clock progression. Overall, the CDD complex may function as a transcriptional corepressor in diverse processes through direct interaction with distinct transcription factors.

Coordinated transcriptional regulation underlying the circadian clock in Arabidopsis.

The circadian clock controls many metabolic, developmental and physiological processes in a time-of-day-specific manner in both plants and animals. The photoreceptors involved in the perception of light and entrainment of the circadian clock have been well characterized in plants. However, how light signals are transduced from the photoreceptors to the central circadian oscillator, and how the rhythmic expression pattern of a clock gene is generated and maintained by diurnal light signals remain unclear. Here, we show that in Arabidopsis thaliana, FHY3, FAR1 and HY5, three positive regulators of the phytochrome A signalling pathway, directly bind to the promoter of ELF4, a proposed component of the central oscillator, and activate its expression during the day, whereas the circadian-controlled CCA1 and LHY proteins directly suppress ELF4 expression periodically at dawn through physical interactions with these transcription-promoting factors. Our findings provide evidence that a set of light- and circadian-regulated transcription factors act directly and coordinately at the ELF4 promoter to regulate its cyclic expression, and establish a potential molecular link connecting the environmental light-dark cycle to the central oscillator.