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Stomata-small pores generally found on the leaves of plants-control gas exchange between plant and the atmosphere. Elucidating the mechanism that underlies such control through the regulation of stomatal opening/closing is important to understand how plants regulate photosynthesis and tolerate against drought. However, up-to-date, molecular components and their function involved in stomatal regulation are not fully understood. We challenged such problem through a chemical genetic approach by isolating and characterizing synthetic molecules that influence stomatal movement. Here, we describe that a small chemical collection, prepared during the development of C-H amination reactions, lead to the discovery of a Stomata Influencing Molecule (SIM); namely, a sulfonimidated oxazole that inhibits stomatal opening. The starting molecule SIM1 was initially isolated from screening of compounds that inhibit light induced opening of dayflower stomata. A range of SIM molecules were rapidly accessed using our state-of-the-art C-H amination technologies. This enabled an efficient structure-activity relationship (SAR) study, culminating in the discovery of a sulfonamidated oxazole derivative (SIM*) having higher activity and enhanced specificity against stomatal regulation. Biological assay results have shed some light on the mode of action of SIM molecules within the cell, which may ultimately lead to drought tolerance-conferring agrochemicals through the control of stomatal movement.

Growth regulation tailors development in plants to their environment. A prominent example of this is the response to gravity, in which shoots bend up and roots bend down.sup.1. This paradox is based on opposite effects of the phytohormone auxin, which promotes cell expansion in shoots while inhibiting it in roots via a yet unknown cellular mechanism.sup.2. Here, by combining microfluidics, live imaging, genetic engineering and phosphoproteomics in Arabidopsis thaliana, we advance understanding of how auxin inhibits root growth. We show that auxin activates two distinct, antagonistically acting signalling pathways that converge on rapid regulation of apoplastic pH, a causative determinant of growth. Cell surface-based TRANSMEMBRANE KINASE1 (TMK1) interacts with and mediates phosphorylation and activation of plasma membrane H.sup.+-ATPases for apoplast acidification, while intracellular canonical auxin signalling promotes net cellular H.sup.+ influx, causing apoplast alkalinization. Simultaneous activation of these two counteracting mechanisms poises roots for rapid, fine-tuned growth modulation in navigating complex soil environments. Auxin rapidly modulates root growth through simultaneous activation of two opposing mechanisms--TMK1-mediated apoplast acidification and TIR1/AFB-mediated apoplast alkalinization., Author(s): Lanxin Li [sup.1] , Inge Verstraeten [sup.1] , Mark Roosjen [sup.2] , Koji Takahashi [sup.3] [sup.4] , Lesia Rodriguez [sup.1] , Jack Merrin [sup.1] , Jian Chen [sup.5] [sup.6] [...]

The phytohormone auxin controls many processes in plants, at least in part through its regulation of cell expansion.sup.1. The acid growth hypothesis has been proposed to explain auxin-stimulated cell expansion for five decades, but the mechanism that underlies auxin-induced cell-wall acidification is poorly characterized. Auxin induces the phosphorylation and activation of the plasma membrane H.sup.+-ATPase that pumps protons into the apoplast.sup.2, yet how auxin activates its phosphorylation remains unclear. Here we show that the transmembrane kinase (TMK) auxin-signalling proteins interact with plasma membrane H.sup.+-ATPases, inducing their phosphorylation, and thereby promoting cell-wall acidification and hypocotyl cell elongation in Arabidopsis. Auxin induced interactions between TMKs and H.sup.+-ATPases in the plasma membrane within seconds, as well as TMK-dependent phosphorylation of the penultimate threonine residue on the H+-ATPases. Our genetic, biochemical and molecular evidence demonstrates that TMKs directly phosphorylate plasma membrane H.sup.+-ATPase and are required for auxin-induced H.sup.+-ATPase activation, apoplastic acidification and cell expansion. Thus, our findings reveal a crucial connection between auxin and plasma membrane H.sup.+-ATPase activation in regulating apoplastic pH changes and cell expansion through TMK-based cell surface auxin signalling. Auxin induces transmembrane-kinase-dependent activation of H.sup.+-ATPase in the plasma membrane through phosphorylation of its penultimate threonine residue, promoting apoplastic acidification and hypocotyl cell elongation in Arabidopsis., Author(s): Wenwei Lin [sup.1] [sup.2] , Xiang Zhou [sup.1] [sup.2] , Wenxin Tang [sup.1] , Koji Takahashi [sup.3] [sup.4] , Xue Pan [sup.2] , Jiawei Dai [sup.1] , Hong Ren [...]

The evolution of land flora transformed the terrestrial environment. Land plants evolved from an ancestral charophycean alga from which they inherited developmental, biochemical, and cell biological attributes. Additional biochemical and physiological adaptations to land, and a life cycle with an alternation between multicellular haploid and diploid generations that facilitated efficient dispersal of desiccation tolerant spores, evolved in the ancestral land plant. We analyzed the genome of the liverwort Marchantia polymorpha, a member of a basal land plant lineage. Relative to charophycean algae, land plant genomes are characterized by genes encoding novel biochemical pathways, new phytohormone signaling pathways (notably auxin), expanded repertoires of signaling pathways, and increased diversity in some transcription factor families. Compared with other sequenced land plants, M. polymorpha exhibits low genetic redundancy in most regulatory pathways, with this portion of its genome resembling that predicted for the ancestral land plant. PAPERCLIP.

Plasma membrane (PM) H+-ATPase is crucial for light-induced stomatal opening and phosphorylation of a penultimate residue, Thr948 (pen-Thr, numbering according to Arabidopsis AHA1) is required for enzyme activation. In this study, a comprehensive phosphoproteomic analysis using guard cell protoplasts from Vicia faba shows that both red and blue light increase the phosphorylation of Thr881, of PM H+-ATPase. Light-induced stomatal opening and the blue light-induced increase in stomatal conductance are reduced in transgenic Arabidopsis plants expressing mutant AHA1-T881A in aha1–9, whereas the blue light-induced phosphorylation of pen-Thr is unaffected. Auxin and photosynthetically active radiation induce the phosphorylation of both Thr881 and pen-Thr in etiolated seedlings and leaves, respectively. The dephosphorylation of phosphorylated Thr881 and pen-Thr are mediated by type 2 C protein phosphatase clade D isoforms. Taken together, Thr881 phosphorylation, in addition of the pen-Thr phosphorylation, are important for PM H+-ATPase function during physiological responses, such as light-induced stomatal opening in Arabidopsis thaliana. Comprehensive phosphoproteomic analysis using guard cell protoplasts revealed that Thr881 phosphorylation in addition of a penultimate residue, Thr948 of plasma membrane H+-ATPase are important for its function and light-induced stomatal opening. [ABSTRACT FROM AUTHOR]

Plants adopt optimal tolerance strategies depending on the intensity and duration of stress. Retaining water is a priority under short-term drought conditions, whereas maintaining growth and reproduction processes takes precedence over survival under conditions of prolonged drought. However, the mechanism underlying changes in the stress response depending on the degree of drought is unclear. Here, we report that SNF1-related protein kinase 2 (SnRK2) substrate 1 (SNS1) is involved in this growth regulation under conditions of drought stress. SNS1 is phosphorylated and stabilized by SnRK2 protein kinases reflecting drought conditions. It contributes to the maintenance of growth and promotion of flowering as drought escape by repressing stress-responsive genes and inducing FLOWERING LOCUS T (FT) expression, respectively. SNS1 interacts with the histone methylation reader proteins MORF-related gene 1 (MRG1) and MRG2, and the SNS1–MRG1/2 module cooperatively regulates abscisic acid response. Taken together, these observations suggest that the phosphorylation and accumulation of SNS1 in plants reflect the intensity and duration of stress and can serve as a molecular scale for maintaining growth and adopting optimal drought tolerance strategies under stress conditions. [ABSTRACT FROM AUTHOR]

Circadian clocks are biological timekeeping systems that coordinate genetic, metabolic and physiological behaviors with the external day–night cycle. The clock in plants relies on the transcriptional-translational feedback loops transcription-translation feedback loop (TTFL), consisting of transcription factors including PSUEDO-RESPONSE REGULATOR (PRR) proteins, plant lineage–specific transcriptional repressors. Here, we report that a novel synthetic small-molecule modulator, 5-(3,4-dichlorophenyl)-1-phenyl-1,7-dihydro-4H-pyrazolo[3,4-d] pyrimidine-4,6(5H)-dione (TU-892), affects the PRR7 protein amount. A clock reporter line of Arabidopsis was screened against the 10,000 small molecules in the Maybridge Hitfinder 10K chemical library. This screening identified TU-892 as a period-lengthening molecule. Gene expression analyses showed that TU-892 treatment upregulates CIRCADIAN CLOCK–ASSOCIATED 1 (CCA1) mRNA expression. TU-892 treatment reduced the amount of PRR7 protein, a transcriptional repressor of CCA1. Other PRR proteins including TIMING OF CAB EXPRESSION 1 were altered less by TU-892 treatment. TU-892-dependent CCA1 upregulation was attenuated in mutants impaired in PRR7. Collectively, TU-892 is a novel type of clock modulator that reduces the levels of PRR7 protein. [ABSTRACT FROM AUTHOR]

The quantification of stomatal pore size has long been a fundamental approach to understand the physiological response of plants in the context of environmental adaptation. Automation of such methodologies not only alleviates human labor and bias but also realizes new experimental research methods through massive analysis. Here, we present an image analysis pipeline that automatically quantifies stomatal aperture of Arabidopsis thaliana leaves from bright-field microscopy images containing mesophyll tissue as noisy backgrounds. By combining a You Only Look Once X–based stomatal detection submodule and a U-Net-based pore segmentation submodule, we achieved a mean average precision with an intersection of union (IoU) threshold of 50% value of 0.875 (stomata detection performance) and an IoU of 0.745 (pore segmentation performance) against images of leaf discs taken with a bright-field microscope. Moreover, we designed a portable imaging device that allows easy acquisition of stomatal images from detached/undetached intact leaves on-site. We demonstrated that this device in combination with fine-tuned models of the pipeline we generated here provides robust measurements that can substitute for manual measurement of stomatal responses against pathogen inoculation. Utilization of our hardware and pipeline for automated stomatal aperture measurements is expected to accelerate research on stomatal biology of model dicots. [ABSTRACT FROM AUTHOR]