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.

The homeostasis of meristems in flowering plants is maintained by cell-to-cell communication via CLE (CLAVATA3/EMBRYO SURROUNDING REGION-related) peptide hormones. In contrast, cell signals that regulate meristem activity remains elusive in bryophytes that maintain apical meristems in the gametophyte (haploid) body and undergo a gametophyte-dominant life cycle. We here show that MpCLE1 confines the proliferative activity of gametophytic meristem and affects the overall size of gametangiophores (reproductive organs) in Marchantia polymorpha, which is in sharp contrast with the meristem-promoting function of its ortholog TDIF/CLE41/CLE44 in Arabidopsis vascular meristems. Expression analysis suggests that MpCLE1 and its receptor gene MpTDR are expressed in distinct patterns across the apical meristem. These data suggest that local CLE peptide signaling may have had a role in regulating cell proliferation in the shoot meristem in the ancestral land plant and acts in both sporophytic and gametophytic meristems of extant plants.

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.

Allogenic hematopoietic stem cell transplantation (allo-HCT) is the standard treatment for acute myeloid leukemia (AML) in non-complete remission (non-CR); however, the prognosis is inconsistent. This study aimed to develop and validate nomograms and a web application to predict the overall survival (OS) of patients with non-CR AML undergoing allo-HCT (cord blood transplantation [CBT], bone marrow transplantation [BMT], and peripheral blood stem cell transplantation [PBSCT]). Data from 3052 patients were analyzed to construct and validate the prognostic models. The common significant prognostic factors among patients undergoing allo-HCT were age, performance status, percentage of peripheral blasts, cytogenetic risk, chemotherapy response, and number of transplantations. The conditioning regimen was a significant prognostic factor only in patients undergoing CBT. Compared with cyclophosphamide/total body irradiation, a conditioning regimen of ≥3 drugs, including fludarabine, with CBT exhibited the lowest hazard ratio for mortality (0.384; 95% CI, 0.266-0.554; p < 0.0001). A conditioning regimen of ≥3 drugs with CBT also showed the best leukemia-free survival among all conditioning regimens. Based on the results of the multivariable analysis, we developed prognostic models showing adequate calibration and discrimination (the c-indices for CBT, BMT, and PBSCT were 0.648, 0.600, and 0.658, respectively). Our prognostic models can help in assessing individual risks and designing future clinical studies. Furthermore, our study indicates the effectiveness of multi-drug conditioning regimens in patients undergoing CBT.

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.

Allogeneic hematopoietic stem cell transplantation is the sole curative therapy for myelodysplastic syndrome (MDS). However, there is concern regarding graft failure and relapse in patients who undergo cord blood transplantation (CBT). We conducted a retrospective study of the CBT outcomes in MDS patients using the Japanese Data Center for Hematopoietic Cell Transplantation database. Seven hundred fifty-two de novo MDS patients of ≥18 years of age (median, 58 years) undergoing their first CBT between 2001 and 2015 were examined. Two-thirds of the patients were male, and were RAEB. The cumulative incidences of neutrophil and platelet engraftment at day 100 were 77 and 59%, respectively. The 3-year overall survival (OS) was 41% and the median survival of the patients was 1.25 years. A multivariate analysis of pre-transplant variables showed that the age, gender, cytogenetic subgroups, number of RBC transfusions, HCT-CI and year of CBT significantly influenced the outcome. The cumulative incidence of acute graft-versus-host disease (aGVHD) and chronic GVHD (cGVHD) was 32 and 21%, respectively. A survival benefit was observed in patients who developed cGVHD, but not aGVHD. Our results suggest that CBT is an acceptable alternative graft and that a graft-versus-MDS effect can be expected, especially in patients who develop cGVHD.

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.

Mantle cell lymphoma is considered an aggressive B-cell lymphoma. The optimal induction regimen remains controversial as no randomized controlled trial has compared the efficacy of different induction therapies.

Acute erythroid leukemia (FAB-M6) and acute megakaryoblastic leukemia (FAB-M7) exhibit closely related properties in cells regarding morphology and the gene expression profile. Although allogeneic hematopoietic stem cell transplantation (allo-HSCT) is considered the mainstay of the treatment for both subtypes of leukemia due to their refractoriness to chemotherapy and high rates of relapse, it remains unclear whether allo-HSCT is curative in such cases due to their scarcity. We retrospectively examined the impact of allo-HSCT in 382 patients with M6 and 108 patients with M7 using nationwide HSCT data and found the overall survival (OS) and relapse rates of the M6 patients to be significantly better than those of the M7 patients after adjusting for confounding factors and statistically comparable with those of the patients with M0/M1/M2/M4/M5 disease. Consequently, the factors of age, gender, performance status, karyotype, disease status at HSCT and development of graft-vs.-host disease predicted the OS for the M6 patients, while the performance status and disease status at HSCT were predictive of the OS for the M7 patients. These findings substantiate the importance of distinguishing between M6 and M7 in the HSCT setting and suggest that unknown mechanisms influence the HSCT outcomes of these closely related subtypes of leukemia.