Reactive oxygen species (ROS) are harmful to all living organisms and therefore they must be removed to ensure normal growth and development. ROS are also signaling molecules, but so far little is known about the mechanisms of ROS perception and developmental response in plants. We here report that hydrogen peroxide induces cortex proliferation in the Arabidopsis root and that SPINDLY (SPY), an O-linked glucosamine acetyltransferase, regulates cortex proliferation by maintaining cellular redox homeostasis. We also found that mutation in the leucine-rich receptor kinase ERECTA and its putative peptide ligand STOMAGEN block the effect of hydrogen peroxide on root cortex proliferation. However, ERECTA and STOMAGEN are expressed in the vascular tissue, whereas extra cortex cells are produced from the endodermis, suggesting the involvement of intercellular signaling. SPY appears to act downstream of ERECTA, because the spy mutation still caused cortex proliferation in the erecta mutant background. We therefore have not only gained insight into the mechanism by which SPY regulates root development but also uncovered a novel pathway for ROS signaling in plants. The importance of redox-mediated cortex proliferation as a protective mechanism against oxidative stress is also discussed.

Cell fate in eukaryotes is controlled by mitogen-activated protein kinases (MAPKs) that translate external cues into cellular responses. In plants, two MAPKs-MPK3 and MPK6-regulate diverse processes of development, environmental response and immunity. However, the mechanism that bridges these shared signalling components with a specific target remains unresolved. Focusing on the development of stomata-epidermal valves that are essential for gas exchange and transpiration-here, we report that the basic helix-loop-helix protein SCREAM functions as a scaffold that recruits MPK3/6 to downregulate SPEECHLESS, a transcription factor that initiates stomatal cell lineages. SCREAM directly binds to MPK3/6 through an evolutionarily conserved, yet unconventional, bipartite motif. Mutations in this motif abrogate association, phosphorylation and degradation of SCREAM, unmask hidden non-redundancies between MPK3 and MPK6, and result in uncontrolled stomatal differentiation. Structural analyses of MPK6 with a resolution of 2.75 Å showed bipartite binding of SCREAM to MPK6 that is distinct from an upstream MAPKK. Our findings elucidate, at the atomic resolution, the mechanism that directly links extrinsic signals to transcriptional reprogramming during the establishment of stomatal cell fate, and highlight a unique substrate-binding mode adopted by plant MAPKs.

Land plants have evolved the ability to cope with submergence. Amphibious plants are adapted to both aerial and aquatic environments through phenotypic plasticity in leaf form and function, known as heterophylly. In general, underwater leaves of amphibious plants are devoid of stomata, yet their molecular regulatory mechanisms remain elusive. Using the emerging model of the Brassicaceae amphibious species Rorippa aquatica, we lay the foundation for the molecular physiological basis of the submergence-triggered inhibition of stomatal development. A series of temperature shift experiments showed that submergence-induced inhibition of stomatal development is largely uncoupled from morphological heterophylly and likely regulated by independent pathways. Submergence-responsive transcriptome analysis revealed rapid reprogramming of gene expression, exemplified by the suppression of RaSPEECHLESS and RaMUTE within 1 h and the involvement of light and hormones in the developmental switch from terrestrial to submerged leaves. Further physiological studies place ethylene as a central regulator of the submergence-triggered inhibition of stomatal development. Surprisingly, red and blue light have opposing functions in this process: blue light promotes, whereas red light inhibits stomatal development, through influencing the ethylene pathway. Finally, jasmonic acid counteracts the inhibition of stomatal development, which can be attenuated by the red light. The actions and interactions of light and hormone pathways in regulating stomatal development in R. aquatica are different from those in the terrestrial species, Arabidopsis thaliana. Thus, our work suggests that extensive rewiring events of red light to ethylene signaling might underlie the evolutionary adaption to water environment in Brassicaceae.

Paraoxonase 2 (PON2) is an intracellular antioxidant enzyme located at the inner mitochondrial membrane. Previous studies have found PON2 to be an important antioxidant in a variety of cellular systems, such as the cardiovascular and renal system. Recent work has also suggested that PON2 plays an important role in the central nervous system (CNS), as decreased PON2 expression in the CNS leads to higher oxidative stress and subsequent cell toxicity. However, the precise role of PON2 in the CNS is still largely unknown, and what role it may play in specific regions of the brain remains unexamined. Dopamine metabolism generates considerable oxidative stress and antioxidant function is critical to the survival of dopaminergic neurons, providing a potential mechanism for PON2 in the dopaminergic system.

Chromatin architecture and transcription factor (TF) binding underpin cell-fate specification during development, but their mutual regulatory relationships remain unclear. Here we report an atlas of dynamic chromatin landscapes during stomatal cell-lineage progression, in which sequential cell-state transitions are governed by lineage-specific bHLH TFs. Major reprogramming of chromatin accessibility occurs at the proliferation-to-differentiation transition. We discover novel co-cis regulatory elements (CREs) signifying the early precursor stage, BBR/BPC (GAGA) and bHLH (E-box) motifs, where master-regulatory bHLH TFs, SPEECHLESS and MUTE, consecutively bind to initiate and terminate the proliferative state, respectively. BPC TFs complex with MUTE to repress SPEECHLESS expression through a local deposition of repressive histone marks. We elucidate the mechanism by which cell-state-specific heterotypic TF complexes facilitate cell-fate commitment by recruiting chromatin modifiers via key co-CREs.

The phytohormone auxin indole-3-acetic acid (IAA) regulates nearly all aspects of plant growth and development. Despite substantial progress in our understanding of auxin biology, delineating specific auxin response remains a major challenge. Auxin regulates transcriptional response via its receptors, TIR1 and AFB F-box proteins. Here we report an engineered, orthogonal auxin-TIR1 receptor pair, developed through a bump-and-hole strategy, that triggers auxin signaling without interfering with endogenous auxin or TIR1/AFBs. A synthetic, convex IAA (cvxIAA) hijacked the downstream auxin signaling in vivo both at the transcriptomic level and in specific developmental contexts, only in the presence of a complementary, concave TIR1 (ccvTIR1) receptor. Harnessing the cvxIAA-ccvTIR1 system, we provide conclusive evidence for the role of the TIR1-mediated pathway in auxin-induced seedling acid growth. The cvxIAA-ccvTIR1 system serves as a powerful tool for solving outstanding questions in auxin biology and for precise manipulation of auxin-mediated processes as a controllable switch.

Precise cell division control is critical for developmental patterning. For the differentiation of a functional stoma, a cellular valve for efficient gas exchange, the single symmetric division of an immediate precursor is absolutely essential. Yet, the mechanism governing this event remains unclear. Here we report comprehensive inventories of gene expression by the Arabidopsis bHLH protein MUTE, a potent inducer of stomatal differentiation. MUTE switches the gene expression program initiated by SPEECHLESS. MUTE directly induces a suite of cell-cycle genes, including CYCD5;1, in which introduced expression triggers the symmetric divisions of arrested precursor cells in mute, and their transcriptional repressors, FAMA and FOUR LIPS. The regulatory network initiated by MUTE represents an incoherent type 1 feed-forward loop. Our mathematical modeling and experimental perturbations support a notion that MUTE orchestrates a transcriptional cascade leading to a tightly restricted pulse of cell-cycle gene expression, thereby ensuring the single cell division to create functional stomata.

ERECTA (ER) receptor-like kinase (RLK) regulates Arabidopsis thaliana organ growth, and inflorescence and stomatal development by interacting with the ERECTA-family genes (ERf) paralogs, ER-like 1 (ERL1) and ERL2, and the receptor-like protein (RLP) TOO MANY MOUTHS (TMM). ER also controls immune responses and resistance to pathogens such as the bacterium Pseudomonas syringae pv. tomato DC3000 (Pto) and the necrotrophic fungus Plectosphaerella cucumerina BMM (PcBMM). We found that er null-mutant plants overexpressing an ER dominant-negative version lacking the cytoplasmic kinase domain (ERΔK) showed an enhanced susceptibility to PcBMM, suggesting that ERΔK associates and forms inactive complexes with additional RLKs/RLPs required for PcBMM resistance. Genetic analyses demonstrated that ER acts in a combinatorial specific manner with ERL1, ERL2, and TMM to control PcBMM resistance. Moreover, BAK1 (BRASSINOSTEROID INSENSITIVE 1-associated kinase 1) RLK, which together with ERf/TMM regulates stomatal patterning and resistance to Pto, was also found to have an unequal contribution with ER in regulating immune responses and resistance to PcBMM. Co-immunoprecipitation experiments in Nicotiana benthamiana further demonstrated BAK1-ER protein interaction. The secreted epidermal pattern factor peptides (EPF1 and EPF2), which are perceived by ERf members to specify stomatal patterning, do not seem to regulate ER-mediated immunity to PcBMM, since their inducible overexpression in A. thaliana did not impact on PcBMM resistance. Our results indicate that the multiproteic receptorsome formed by ERf, TMM and BAK1 modulates A. thaliana resistance to PcBMM, and suggest that the cues underlying ERf/TMM/BAK1-mediated immune responses are distinct from those regulating stomatal pattering.

Plants develop in the absence of cell migration. As such, cell division and differentiation need to be coordinated for functional tissue formation. Cellular valves on the plant epidermis, stomata, are generated through a stereotypical sequence of cell division and differentiation events. In Arabidopsis, three master regulatory transcription factors, SPEECHLESS (SPCH), MUTE and FAMA, sequentially drive initiation, proliferation and differentiation of stomata. Among them, MUTE switches the cell cycle mode from proliferative asymmetric division to terminal symmetric division and orchestrates the execution of the single symmetric division event. However, it remains unclear to what extent MUTE regulates the expression of cell cycle genes through the symmetric division and whether MUTE accumulation itself is gated by the cell cycle. Here, we show that MUTE directly upregulates the expression of cell cycle components throughout the terminal cell cycle phases of a stomatal precursor, not only core cell cycle engines but also check-point regulators. Time-lapse live imaging using the multicolor Plant Cell Cycle Indicator revealed that MUTE accumulates up to the early G2 phase, whereas its successor and direct target, FAMA, accumulate at late G2 through terminal mitosis. In the absence of MUTE, meristemoids fail to differentiate and their G1 phase elongates as they reiterate asymmetric divisions. Together, our work provides the framework of cell cycle and master regulatory transcription factors to coordinate a single symmetric cell division and suggests a mechanism for the eventual cell cycle arrest of an uncommitted stem-cell-like precursor at the G1 phase.

Multicellular organisms develop specialized cell types to achieve complex functions of tissues and organs. The basic helix-loop-helix (bHLH) proteins act as master regulatory transcription factors of such specialized cell types. Plant stomata are cellular valves in the aerial epidermis for efficient gas exchange and water control. Stomatal differentiation is governed by sequential actions of three lineage-specific bHLH proteins, SPEECHLESS (SPCH), MUTE, and FAMA, specifying initiation and proliferation, commitment, and terminal differentiation, respectively. A broadly expressed bHLH, SCREAM (SCRM), heterodimerizes with SPCH/MUTE/FAMA and drives stomatal differentiation via switching its partners. Yet nothing is known about its heterodimerization properties or partner preference. Here, we report the role of the SCRM C-terminal ACT-like (ACTL) domain for heterodimerization selectivity. Our intragenic suppressor screen of a dominant scrm-D mutant identified the ACTL domain as a mutation hotspot. Removal of this domain or loss of its structural integrity abolishes heterodimerization with MUTE, but not with SPCH or FAMA, and selectively abrogates the MUTE direct target gene expression. Consequently, the scrm-D ACTL mutants confer massive clusters of arrested stomatal precursor cells that cannot commit to differentiation when redundancy is removed. Structural and biophysical studies further show that SPCH, MUTE, and FAMA also possess the C-terminal ACTL domain, and that ACTL•ACTL heterodimerization is sufficient for partner selectivity. Our work elucidates a role for the SCRM ACTL domain in the MUTE-governed proliferation-differentiation switch and suggests mechanistic insight into the biological function of the ACTL domain, a module uniquely associated with plant bHLH proteins, as a heterodimeric partner selectivity interface.

Paraoxonase 2 (PON2) is an intracellular antioxidant enzyme shown to play an important role in mitigating oxidative stress in the brain. Oxidative stress is a common mechanism of toxicity for neurotoxicants and is increasingly implicated in the etiology of multiple neurological diseases. While PON2 deficiency increases oxidative stress in the brain in-vitro, little is known about its effects on behavior in-vivo and what global transcript changes occur from PON2 deficiency. We sought to characterize the effects of PON2 deficiency on behavior in mice, with an emphasis on locomotion, and evaluate transcriptional changes with RNA-Seq. Behavioral endpoints included home-cage behavior (Noldus PhenoTyper), motor coordination (Rotarod) and various gait metrics (Noldus CatWalk). Home-cage behavior analysis showed PON2 deficient mice had increased activity at night compared to wildtype controls and spent more time in the center of the cage, displaying a possible anxiolytic phenotype. PON2 deficient mice had significantly shorter latency to fall when tested on the rotarod, suggesting impaired motor coordination. Minimal gait alterations were observed, with decreased girdle support posture noted as the only significant change in gait with PON2 deficiency. Beyond one home-cage metric, no significant sex-based behavioral differences were found in this study. Finally, A subset of samples were utilized for RNA-Seq analysis, looking at three discrete brain regions: cerebral cortex, striatum, and cerebellum. Highly regional- and sex-specific changes in RNA expression were found when comparing PON2 deficient and wildtype mice, suggesting PON2 may play distinct regional roles in the brain in a sex-specific manner. Taken together, these findings demonstrates that PON2 deficiency significantly alters the brain on both a biochemical and phenotypic level, with a specific impact on motor function. These data have implications for future gene-environment toxicological studies and warrants further investigation of the role of PON2 in the brain.

Evolution often diversifies a peptide hormone family into multiple subfamilies, which exert distinct activities by exclusive interaction with specific receptors. Here we show that systematic swapping of pre-existing variation in a subfamily of plant CLE peptide hormones leads to a synthetic bifunctional peptide that exerts activities beyond the original subfamily by interacting with multiple receptors. This approach provides new insights into the complexity and specificity of peptide signalling.

The auxin-inducible degron (AID) system enables rapid depletion of target proteins within the cell by applying the natural auxin IAA. The AID system is useful for investigating the physiological functions of essential proteins; however, this system generally requires high dose of auxin to achieve effective depletion in vertebrate cells. Here, we describe a super-sensitive AID system that incorporates the synthetic auxin derivative 5-Ad-IAA and its high-affinity-binding partner OsTIR1F74A. The super-sensitive AID system enabled more than a 1000-fold reduction of the AID inducer concentrations in chicken DT40 cells. To apply this system to various mammalian cell lines including cancer cells containing multiple sets of chromosomes, we utilized a single-step method where CRISPR/Cas9-based gene knockout is combined with insertion of a pAID plasmid. The single-step method coupled with the super-sensitive AID system enables us to easily and rapidly generate AID-based conditional knockout cells in a wide range of vertebrate cell lines. Our improved method that incorporates the super-sensitive AID system and the single-step method provides a powerful tool for elucidating the roles of essential genes.

Multicellular organisms use cell-surface receptor kinases to sense and process extracellular signals. Many plant receptor kinases are activated by the formation of ligand-induced complexes with shape-complementary co-receptors1. The best-characterized co-receptor is BRASSINOSTEROID INSENSITIVE 1-ASSOCIATED KINASE 1 (BAK1), which associates with numerous leucine-rich repeat receptor kinases (LRR-RKs) to control immunity, growth and development2. Here we report key regulatory events that control the function of BAK1 and, more generally, LRR-RKs. Through a combination of phosphoproteomics and targeted mutagenesis, we identified conserved phosphosites that are required for the immune function of BAK1 in Arabidopsis thaliana. Notably, these phosphosites are not required for BAK1-dependent brassinosteroid-regulated growth. In addition to revealing a critical role for the phosphorylation of the BAK1 C-terminal tail, we identified a conserved tyrosine phosphosite that may be required for the function of the majority of Arabidopsis LRR-RKs, and which separates them into two distinct functional classes based on the presence or absence of this tyrosine. Our results suggest a phosphocode-based dichotomy of BAK1 function in plant signalling, and provide insights into receptor kinase activation that have broad implications for our understanding of how plants respond to their changing environment.

Development of stomata, valves on the plant epidermis for optimal gas exchange and water control, is fine-tuned by multiple signaling peptides with unique, overlapping, or antagonistic activities. EPIDERMAL PATTERNING FACTOR1 (EPF1) is a founding member of the secreted peptide ligands enforcing stomatal patterning. Yet, its exact role remains unclear. Here, we report that EPF1 and its primary receptor ERECTA-LIKE1 (ERL1) target MUTE, a transcription factor specifying the proliferation-to-differentiation switch within the stomatal cell lineages. In turn, MUTE directly induces ERL1. The absolute co-expression of ERL1 and MUTE, with the co-presence of EPF1, triggers autocrine inhibition of stomatal fate. During normal stomatal development, this autocrine inhibition prevents extra symmetric divisions of stomatal precursors likely owing to excessive MUTE activity. Our study reveals the unexpected role of self-inhibition as a mechanism for ensuring proper stomatal development and suggests an intricate signal buffering mechanism underlying plant tissue patterning.

The phytohormone auxin is the information carrier in a plethora of developmental and physiological processes in plants1. It has been firmly established that canonical, nuclear auxin signalling acts through regulation of gene transcription2. Here, we combined microfluidics, live imaging, genetic engineering and computational modelling to reanalyse the classical case of root growth inhibition3 by auxin. We show that Arabidopsis roots react to addition and removal of auxin by extremely rapid adaptation of growth rate. This process requires intracellular auxin perception but not transcriptional reprogramming. The formation of the canonical TIR1/AFB-Aux/IAA co-receptor complex is required for the growth regulation, hinting to a novel, non-transcriptional branch of this signalling pathway. Our results challenge the current understanding of root growth regulation by auxin and suggest another, presumably non-transcriptional, signalling output of the canonical auxin pathway.

Receptor endocytosis is important for signal activation, transduction, and deactivation. However, how a receptor interprets conflicting signals to adjust cellular output is not clearly understood. Using genetic, cell biological, and pharmacological approaches, we report here that ERECTA-LIKE1 (ERL1), the major receptor restricting plant stomatal differentiation, undergoes dynamic subcellular behaviors in response to different EPIDERMAL PATTERNING FACTOR (EPF) peptides. Activation of ERL1 by EPF1 induces rapid ERL1 internalization via multivesicular bodies/late endosomes to vacuolar degradation, whereas ERL1 constitutively internalizes in the absence of EPF1. The co-receptor, TOO MANY MOUTHS is essential for ERL1 internalization induced by EPF1 but not by EPFL6. The peptide antagonist, Stomagen, triggers retention of ERL1 in the endoplasmic reticulum, likely coupled with reduced endocytosis. In contrast, the dominant-negative ERL1 remained dysfunctional in ligand-induced subcellular trafficking. Our study elucidates that multiple related yet unique peptides specify cell fate by deploying the differential subcellular dynamics of a single receptor.

Stomata are epidermal valves that facilitate gas exchange between plants and their environment. Stomatal patterning is regulated by the EPIDERMAL PATTERING FACTOR (EPF) family of secreted peptides: EPF1 enforces stomatal spacing, whereas EPIDERMAL PATTERNING FACTOR-LIKE9 (EPFL9), also known as Stomagen, promotes stomatal development. It remains unknown, however, how far these signaling peptides act. Utilizing Cre-lox recombination-based mosaic sectors that overexpress either EPF1 or Stomagen in Arabidopsis cotyledons, we reveal a range within the epidermis and across the cell layers in which these peptides influence patterns. To determine their effective ranges quantitatively, we developed a computational pipeline, SPACE (stomata patterning autocorrelation on epidermis), that describes probabilistic two-dimensional stomatal distributions based upon spatial autocorrelation statistics used in astrophysics. The SPACE analysis shows that, whereas both peptides act locally, the inhibitor EPF1 exerts longer range effects than the activator Stomagen. Furthermore, local perturbation of stomatal development has little influence on global two-dimensional stomatal patterning. Our findings conclusively demonstrate the nature and extent of EPF peptides as non-cell autonomous local signals and provide a means for quantitative characterization of complex spatial patterns in development.This article has an associated 'The people behind the papers' interview.

Differentiation of specialized cell types requires precise cell-cycle control. Plant stomata are generated through asymmetric divisions of a stem-cell-like precursor followed by a single symmetric division that creates paired guard cells surrounding a pore. The stomatal-lineage-specific transcription factor MUTE terminates the asymmetric divisions and commits to differentiation. However, the role of cell-cycle machineries in this transition remains unknown. We discover that the symmetric division is slower than the asymmetric division in Arabidopsis. We identify a plant-specific cyclin-dependent kinase inhibitor, SIAMESE-RELATED4 (SMR4), as a MUTE-induced molecular brake that decelerates the cell cycle. SMR4 physically and functionally associates with CYCD3;1 and extends the G1 phase of asymmetric divisions. By contrast, SMR4 fails to interact with CYCD5;1, a MUTE-induced G1 cyclin, and permits the symmetric division. Our work unravels a molecular framework of the proliferation-to-differentiation switch within the stomatal lineage and suggests that a timely proliferative cell cycle is critical for stomatal-lineage identity.

Mitogen-activated protein kinase (MAPK) and leucine-rich repeat receptor-like kinase (LRR-RLK) signaling pathways have been shown to regulate diverse aspects of plant growth and development. In Arabidopsis, proper anther development relies on intercellular communication to coordinate cell proliferation and differentiation. Two closely related genes encoding MAPKs, MPK3 and MPK6, function redundantly in regulating stomatal patterning. Although the mpk6 mutant has reduced fertility, the function of MPK3 and MPK6 in anther development has not been characterized. Similarly, the ERECTA (ER), ERECTA-LIKE1 (ERL1) and ERL2 genes encoding LRR-RLKs function together to direct stomatal cell fate specification and the er-105 erl1-2 erl2-1 triple mutant is sterile. Because the mpk3 mpk6 double null mutant is embryo lethal, anther development was characterized in the viable mpk3/+ mpk6/- and er-105 erl1-2 erl2-1 mutants. We found that both mutant anthers usually fail to form one or more of the four anther lobes, with the er-105 erl1-2 erl2-1 triple mutant exhibiting more severe phenotypes than those of the mpk3/+ mpk6/- mutant. The somatic cell layers of the differentiated mutant lobes appeared larger and more disorganized than that of wild-type. In addition, the er-105 erl1-2 erl2-1 triple mutant has a reduced number of stamens, the majority of which possess completely undifferentiated or under-differentiated anthers. Furthermore, sometimes, the mpk3/+ mpk6/- mutant anthers do not dehisce, and the er-105 erl1-2 erl2-1 anthers were not observed to dehisce. Therefore, our results indicate that both ER/ERL1/ERL2 and MPK3/MPK6 play important roles in normal anther lobe formation and anther cell differentiation. The close functional relationship between these genes in other developmental processes and the similarities in anther developmental phenotypes of the two types of mutants reported here further suggest the possibility that these genes might also function in the same pathway to regulate anther cell division and differentiation.