Identification and improvement of isothiocyanate-based inhibitors on stomatal opening to act as drought tolerance-conferring agrochemicals.

Stomatal pores in the plant epidermis open and close to regulate gas exchange between leaves and the atmosphere. Upon light stimulation, the plasma membrane (PM) H+-ATPase is phosphorylated and activated via an intracellular signal transduction pathway in stomatal guard cells, providing a primary driving force for the opening movement. To uncover and manipulate this stomatal opening pathway, we screened a chemical library and identified benzyl isothiocyanate (BITC), a Brassicales-specific metabolite, as a potent stomatal-opening inhibitor that suppresses PM H+-ATPase phosphorylation. We further developed BITC derivatives with multiple isothiocyanate groups (multi-ITCs), which demonstrate inhibitory activity on stomatal opening up to 66 times stronger, as well as a longer duration of the effect and negligible toxicity. The multi-ITC treatment inhibits plant leaf wilting in both short (1.5 h) and long-term (24 h) periods. Our research elucidates the biological function of BITC and its use as an agrochemical that confers drought tolerance on plants by suppressing stomatal opening.

Morphological and antioxidant responses of Nopalea cochenillifera cv. Maya (edible Opuntia sp. "Kasugai Saboten") to chilling acclimatization.

To clarify the wintering ability of the cactus Nopalea cochenillifera cv. Maya (edible Opuntia sp., common name "Kasugai Saboten"), we investigated the effects of temperature and antioxidant capacity on chilling acclimatization. We analyzed the anatomy of cladode chlorenchyma tissue of plants exposed to light under chilling. We found that chilling acclimatization can be achieved by exposure to approximately 15 °C for 2 weeks and suggest that it is affected by whether or not antioxidant capacity can recover. The overwintering cacti had the thinnest cuticle but firm cuticular wax, which is important in the acquisition of low temperature tolerance under strong light. In cacti with severe chilling injury, round swollen nuclei with clumping chloroplasts were localized in the upper part (axial side) of the cell, as though pushed up by large vacuoles in the lower part. In overwintering cacti, chloroplasts were arranged on the lateral side of the cell as in control plants, but they formed pockets: invaginations with a thin layer of chloroplast stroma that surrounded mitochondria and peroxisomes. Specific cellular structural changes depended on the degree of chilling stress and provide useful insights linking chloroplast behavior and structural changes to the environmental stress response.

Overexpression of Plasma Membrane H+-ATPase in Guard Cells Enhances Light-Induced Stomatal Opening, Photosynthesis, and Plant Growth in Hybrid Aspen.

Stomata in the plant epidermis open in response to light and regulate CO2 uptake for photosynthesis and transpiration for uptake of water and nutrients from roots. Light-induced stomatal opening is mediated by activation of the plasma membrane (PM) H+-ATPase in guard cells. Overexpression of PM H+-ATPase in guard cells promotes light-induced stomatal opening, enhancing photosynthesis and growth in Arabidopsis thaliana. In this study, transgenic hybrid aspens overexpressing Arabidopsis PM H+-ATPase (AHA2) in guard cells under the strong guard cell promoter Arabidopsis GC1 (AtGC1) showed enhanced light-induced stomatal opening, photosynthesis, and growth. First, we confirmed that AtGC1 induces GUS expression specifically in guard cells in hybrid aspens. Thus, we produced AtGC1::AHA2 transgenic hybrid aspens and confirmed expression of AHA2 in AtGC1::AHA2 transgenic plants. In addition, AtGC1::AHA2 transgenic plants showed a higher PM H+-ATPase protein level in guard cells. Analysis using a gas exchange system revealed that transpiration and the photosynthetic rate were significantly increased in AtGC1::AHA2 transgenic aspen plants. AtGC1::AHA2 transgenic plants showed a>20% higher stem elongation rate than the wild type (WT). Therefore, overexpression of PM H+-ATPase in guard cells promotes the growth of perennial woody plants.

Three mutations repurpose a plant karrikin receptor to a strigolactone receptor.

Uncovering the basis of small-molecule hormone receptors' evolution is paramount to a complete understanding of how protein structure drives function. In plants, hormone receptors for strigolactones are well suited to evolutionary inquiries because closely related homologs have different ligand preferences. More importantly, because of facile plant transgenic systems, receptors can be swapped and quickly assessed functionally in vivo. Here, we show that only three mutations are required to turn the nonstrigolactone receptor, KAI2, into a receptor that recognizes the plant hormone strigolactone. This modified receptor still retains its native function to perceive KAI2 ligands. Our directed evolution studies indicate that only a few keystone mutations are required to increase receptor promiscuity of KAI2, which may have implications for strigolactone receptor evolution in parasitic plants.

Chemical control of stomatal function and development.

Stomata control trade-offs for plants: carbon dioxide uptake for photosynthetic growth and water loss via transpiration. While agrochemical control of transpiration is an old concept, recent discoveries of the core signaling components controlling stomatal function and numbers opened the door to develop chemical compounds with high potency and specificity. ABA agonists with potent anti-transpiration activity have been developed via in silico virtual screens and structure guided design and synthesis. Library-based chemical screens identified new compounds that influence stomatal movement in ABA-independent manners as well as those affecting stomatal numbers and division polarity. Subsequent hit compound derivatization can be employed to separate adverse side effects. Ultimately, such chemicals might help in optimizing plant productivity and water use in agriculture and florist industries.

SMAX1-dependent seed germination bypasses GA signalling in Arabidopsis and Striga.

Parasitic plant infestations dramatically reduce the yield of many major food crops of sub-Saharan Africa and pose a serious threat to food security on that continent1. The first committed step of a successful infestation is the germination of parasite seeds primarily in response to a group of related small-molecule hormones called strigolactones (SLs), which are emitted by host roots2. Despite the important role of SLs, it is not clear how host-derived SLs germinate parasitic plants. In contrast, gibberellins (GA) acts as the dominant hormone for stimulation of germination in non-parasitic plant species by inhibiting a set of DELLA repressors3. Here, we show that expression of SL receptors from the parasitic plant Striga hermonthica in the presence of SLs circumvents the GA requirement for germination of Arabidopsis thaliana seed. Striga receptors co-opt and enhance signalling through the HYPOSENSITIVE TO LIGHT/KARRIKIN INSENSITIVE 2 (AtHTL/KAI2) pathway, which normally plays a rudimentary role in Arabidopsis seed germination4,5. AtHTL/KAI2 negatively controls the SUPPRESSOR OF MAX2 1 (SMAX1) protein5, and loss of SMAX1 function allows germination in the presence of DELLA repressors. Our data suggest that ligand-dependent inactivation of SMAX1 in Striga and Arabidopsis can bypass GA-dependent germination in these species.

Regulation of stomatal opening and histone modification by photoperiod in Arabidopsis thaliana.

Stomatal movements are regulated by many environmental signals, such as light, CO2, temperature, humidity, and drought. Recently, we showed that photoperiodic flowering components have positive effects on light-induced stomatal opening in Arabidopsis thaliana. In this study, we determined that light-induced stomatal opening and increased stomatal conductance were larger in plants grown under long-day (LD) conditions than in those grown under short-day (SD) conditions. Gene expression analyses using purified guard cell protoplasts revealed that FT and SOC1 expression levels were significantly increased under LD conditions. Interestingly, the enhancement of light-induced stomatal opening and increased SOC1 expression in guard cells due to LD conditions persisted for at least 1 week after plants were transferred to SD conditions. We then investigated histone modification using chromatin immunoprecipitation-PCR, and observed increased trimethylation of lysine 4 on histone 3 (H3K4) around SOC1. We also found that LD-dependent enhancement of light-induced stomatal opening and H3K4 trimethylation in SOC1 were suppressed in the ft-2 mutant. These results indicate that photoperiod is an important environmental cue regulating stomatal opening, and that LD conditions enhance light-induced stomatal opening and epigenetic modification (H3K4 trimethylation) around SOC1, a positive regulator of stomatal opening, in an FT-dependent manner. Thus, this study provides novel insights into stomatal responses to photoperiod.

Arabidopsis ETHYLENE RESPONSE FACTOR 8 (ERF8) has dual functions in ABA signaling and immunity.

BACKGROUND: ETHYLENE RESPONSE FACTOR (ERF) 8 is a member of one of the largest transcription factor families in plants, the APETALA2/ETHYLENE RESPONSIVE FACTOR (AP2/ERF) superfamily. Members of this superfamily have been implicated in a wide variety of processes such as development and environmental stress responses. RESULTS: In this study we demonstrated that ERF8 is involved in both ABA and immune signaling. ERF8 overexpression induced programmed cell death (PCD) in Arabidopsis and Nicotiana benthamiana. This PCD was salicylic acid (SA)-independent, suggesting that ERF8 acts downstream or independent of SA. ERF8-induced PCD was abolished by mutations within the ERF-associated amphiphilic repression (EAR) motif, indicating ERF8 induces cell death through its transcriptional repression activity. Two immunity-related mitogen-activated protein kinases, MITOGEN-ACTIVATED PROTEIN KINASE 4 (MPK4) and MPK11, were identified as ERF8-interacting proteins and directly phosphorylated ERF8 in vitro. Four putative MPK phosphorylation sites were identified in ERF8, one of which (Ser103) was determined to be the predominantly phosphorylated residue in vitro, while mutation of all four putative phosphorylation sites partially suppressed ERF8-induced cell death in N. benthamiana. Genome-wide transcriptomic analysis and pathogen growth assays confirmed a positive role of ERF8 in mediating immunity, as ERF8 knockdown or overexpression lines conferred compromised or enhanced resistance against the hemibiotrophic bacterial pathogen Pseudomonas syringae, respectively. CONCLUSIONS: Together these data reveal that the ABA-inducible transcriptional repressor ERF8 has dual roles in ABA signaling and pathogen defense, and further highlight the complex influence of ABA on plant-microbe interactions.

Identification and Characterization of Compounds that Affect Stomatal Movements.

Regulation of stomatal aperture is essential for plant growth and survival in response to environmental stimuli. Opening of stomata induces uptake of CO2 for photosynthesis and transpiration, which enhances uptake of nutrients from roots. Light is the most important stimulus for stomatal opening. Under drought stress, the plant hormone ABA induces stomatal closure to prevent water loss. However, the molecular mechanisms of stomatal movements are not fully understood. In this study, we screened chemical libraries to identify compounds that affect stomatal movements in Commelina benghalensis and characterize the underlying molecular mechanisms. We identified nine stomatal closing compounds (SCL1-SCL9) that suppress light-induced stomatal opening by >50%, and two compounds (temsirolimus and CP-100356) that induce stomatal opening in the dark. Further investigations revealed that SCL1 and SCL2 had no effect on autophosphorylation of phototropin or the activity of the inward-rectifying plasma membrane (PM) K+ channel, KAT1, but suppressed blue light-induced phosphorylation of the penultimate residue, threonine, in PM H+-ATPase, which is a key enzyme for stomatal opening. SCL1 and SCL2 had no effect on ABA-dependent responses, including seed germination and expression of ABA-induced genes. These results suggest that SCL1 and SCL2 suppress light-induced stomatal opening at least in part by inhibiting blue light-induced activation of PM H+-ATPase, but not by the ABA signaling pathway. Interestingly, spraying leaves onto dicot and monocot plants with SCL1 suppressed wilting of leaves, indicating that inhibition of stomatal opening by these compounds confers tolerance to drought stress in plants.

α-Xylosidase plays essential roles in xyloglucan remodelling, maintenance of cell wall integrity, and seed germination in Arabidopsis thaliana.

Regulation and maintenance of cell wall physical properties are crucial for plant growth and environmental response. In the germination process, hypocotyl cell expansion and endosperm weakening are prerequisites for dicot seeds to complete germination. We have identified the Arabidopsis mutant thermoinhibition-resistant germination 1 (trg1), which has reduced seed dormancy and insensitivity to unfavourable conditions for germination owing to a loss-of-function mutation of TRG1/XYL1, which encodes an α-xylosidase. Compared to those of wild type, the elongating stem of trg1 showed significantly lower viscoelasticity, and the fruit epidermal cells were longitudinally shorter and horizontally enlarged. Actively growing tissues of trg1 over-accumulated free xyloglucan oligosaccharides (XGOs), and the seed cell wall had xyloglucan with a greatly reduced molecular weight. These observations suggest that XGOs reduce xyloglucan size by serving as an acceptor in transglycosylation and eventually enhancing cell wall loosening. TRG1/XYL1 gene expression was abundant in growing wild-type organs and tissues but relatively low in cells at most actively elongating part of the tissues, suggesting that α-xylosidase contributes to maintaining the mechanical integrity of the primary cell wall in the growing and pre-growing tissues. In germinating seeds of trg1, expression of genes encoding specific abscisic acid and gibberellin metabolism enzymes was altered in accordance with the aberrant germination phenotype. Thus, cell wall integrity could affect seed germination not only directly through the physical properties of the cell wall but also indirectly through the regulation of hormone gene expression.

Small-molecule antagonists of germination of the parasitic plant Striga hermonthica.

Striga spp. (witchweed) is an obligate parasitic plant that attaches to host roots to deplete them of nutrients. In Sub-Saharan Africa, the most destructive Striga species, Striga hermonthica, parasitizes major food crops affecting two-thirds of the arable land and over 100 million people. One potential weakness in the Striga infection process is the way it senses the presence of a host crop. Striga only germinates in the presence of the plant hormone strigolactone, which exudes from a host root. Hence small molecules that perturb strigolactone signaling may be useful tools for disrupting the Striga lifecycle. Here we developed a chemical screen to suppress strigolactone signaling in the model plant Arabidopsis. One compound, soporidine, specifically inhibited a S. hermonthica strigolactone receptor and inhibited the parasite's germination. This indicates that strigolactone-based screens using Arabidopsis are useful in identifying lead compounds to combat Striga infestations.

Structure-function analysis identifies highly sensitive strigolactone receptors in Striga.

Strigolactones are naturally occurring signaling molecules that affect plant development, fungi-plant interactions, and parasitic plant infestations. We characterized the function of 11 strigolactone receptors from the parasitic plant Striga hermonthica using chemical and structural biology. We found a clade of polyspecific receptors, including one that is sensitive to picomolar concentrations of strigolactone. A crystal structure of a highly sensitive strigolactone receptor from Striga revealed a larger binding pocket than that of the Arabidopsis receptor, which could explain the increased range of strigolactone sensitivity. Thus, the sensitivity of Striga to strigolactones from host plants is driven by receptor sensitivity. By expressing strigolactone receptors in Arabidopsis, we developed a bioassay that can be used to identify chemicals and crops with altered strigolactone levels.

PARASITIC PLANTS. Probing strigolactone receptors in Striga hermonthica with fluorescence.

Elucidating the signaling mechanism of strigolactones has been the key to controlling the devastating problem caused by the parasitic plant Striga hermonthica. To overcome the genetic intractability that has previously interfered with identification of the strigolactone receptor, we developed a fluorescence turn-on probe, Yoshimulactone Green (YLG), which activates strigolactone signaling and illuminates signal perception by the strigolactone receptors. Here we describe how strigolactones bind to and act via ShHTLs, the diverged family of α/ß hydrolase-fold proteins in Striga. Live imaging using YLGs revealed that a dynamic wavelike propagation of strigolactone perception wakes up Striga seeds. We conclude that ShHTLs function as the strigolactone receptors mediating seed germination in Striga. Our findings enable access to strigolactone receptors and observation of the regulatory dynamics for strigolactone signal transduction in Striga.

Detection of parasitic plant suicide germination compounds using a high-throughput Arabidopsis HTL/KAI2 strigolactone perception system.

Strigolactones are terpenoid-based plant hormones that act as communication signals within a plant, between plants and fungi, and between parasitic plants and their hosts. Here we show that an active enantiomer form of the strigolactone GR24, the germination stimulant karrikin, and a number of structurally related small molecules called cotylimides all bind the HTL/KAI2 α/ß hydrolase in Arabidopsis. Strigolactones and cotylimides also promoted an interaction between HTL/KAI2 and the F-box protein MAX2 in yeast. Identification of this chemically dependent protein-protein interaction prompted the development of a yeast-based, high-throughput chemical screen for potential strigolactone mimics. Of the 40 lead compounds identified, three were found to have in planta strigolactone activity using Arabidopsis-based assays. More importantly, these three compounds were all found to stimulate suicide germination of the obligate parasitic plant Striga hermonthica. These results suggest that screening strategies involving yeast/Arabidopsis models may be useful in combating parasitic plant infestations.

A mesoscale abscisic acid hormone interactome reveals a dynamic signaling landscape in Arabidopsis.

The sesquiterpenoid abscisic acid (ABA) mediates an assortment of responses across a variety of kingdoms including both higher plants and animals. In plants, where most is known, a linear core ABA signaling pathway has been identified. However, the complexity of ABA-dependent gene expression suggests that ABA functions through an intricate network. Here, using systems biology approaches that focused on genes transcriptionally regulated by ABA, we defined an ABA signaling network of over 500 interactions among 138 proteins. This map greatly expanded ABA core signaling but was still manageable for systematic analysis. For example, functional analysis was used to identify an ABA module centered on two sucrose nonfermenting (SNF)-like kinases. We also used coexpression analysis of interacting partners within the network to uncover dynamic subnetwork structures in response to different abiotic stresses. This comprehensive ABA resource allows for application of approaches to understanding ABA functions in higher plants.

ABA-insensitive3, ABA-insensitive5, and DELLAs Interact to activate the expression of SOMNUS and other high-temperature-inducible genes in imbibed seeds in Arabidopsis.

Seeds monitor the environment to germinate at the proper time, but different species respond differently to environmental conditions, particularly light and temperature. In Arabidopsis thaliana, light promotes germination but high temperature suppresses germination. We previously reported that light promotes germination by repressing SOMNUS (SOM). Here, we examined whether high temperature also regulates germination through SOM and found that high temperature activates SOM expression. Consistent with this, som mutants germinated more frequently than the wild type at high temperature. The induction of SOM mRNA at high temperature required abscisic acid (ABA) and gibberellic acid biosynthesis, and ABA-insensitive3 (ABI3), ABI5, and DELLAs positively regulated SOM expression. Chromatin immunoprecipitation assays indicated that ABI3, ABI5, and DELLAs all target the SOM promoter. At the protein level, ABI3, ABI5, and DELLAs all interact with each other, suggesting that they form a complex on the SOM promoter to activate SOM expression at high temperature. We found that high-temperature-inducible genes frequently have RY motifs and ABA-responsive elements in their promoters, some of which are targeted by ABI3, ABI5, and DELLAs in vivo. Taken together, our data indicate that ABI3, ABI5, and DELLAs mediate high-temperature signaling to activate the expression of SOM and other high-temperature-inducible genes, thereby inhibiting seed germination.

HY5 is involved in strigolactone-dependent seed germination in Arabidopsis.

Strigolactones (SLs) function as plant hormones that mediate a myriad of developmental responses in higher plants. SLs also act as an environmental signal to stimulate seed germination of parasitic plant species of genera Striga and Orobanche. In contrast to their hormonal roles, genetic mechanisms of how SLs stimulate parasitic seed germination are largely not known. Recently, we established a method to monitor the germination-stimulating activity of SLs in Arabidopsis using temperature as environmental constraint (thermoinhibition). Here, we show that SLs require HY5, a key transcription factor for light signal transduction, to stimulate Arabidopsis seed germination during thermoinhibiton. Genetic analysis suggests the HY5 dependent signaling pathway is independent of other known SL signaling pathways. Thermoinhibibed seeds expressed low level of HY5 while GR24 increase the level at both mRNA and protein level. A role of SLs on activating crucial light signaling components such as HY5 may hint the evolution of parasitism associated with SL usage.

Thermoinhibition uncovers a role for strigolactones in Arabidopsis seed germination.

Strigolactones are host factors that stimulate seed germination of parasitic plant species such as Striga and Orobanche. This hormone is also important in shoot branching architecture and photomorphogenic development. Strigolactone biosynthetic and signaling mutants in model systems, unlike parasitic plants, only show seed germination phenotypes under limited growth condition. To understand the roles of strigolactones in seed germination, it is necessary to develop a tractable experimental system using model plants such as Arabidopsis. Here, we report that thermoinhibition, which involves exposing seeds to high temperatures, uncovers a clear role for strigolactones in promoting Arabidopsis seed germination. Both strigolactone biosynthetic and signaling mutants showed increased sensitivity to seed thermoinhibition. The synthetic strigolactone GR24 rescued germination of thermoinbibited biosynthetic mutant seeds but not a signaling mutant. Hormone analysis revealed that strigolactones alleviate thermoinhibition by modulating levels of the two plant hormones, GA and ABA. We also showed that GR24 was able to counteract secondary dormancy in Arabidopsis ecotype Columbia (Col) and Cape Verde island (Cvi). Systematic hormone analysis of germinating Striga helmonthica seeds suggested a common mechanism between the parasitic and non-parasitic seeds with respect to how hormones regulate germination. Thus, our simple assay system using Arabidopsis thermoinhibition allows comparisons to determine similarities and differences between parasitic plants and model experimental systems for the use of strigolactones.

A small-molecule screen identifies new functions for the plant hormone strigolactone.

Parasitic weeds of the genera Striga and Orobanche are considered the most damaging agricultural agents in the developing world. An essential step in parasitic seed germination is sensing a group of structurally related compounds called strigolactones that are released by host plants. Although this makes strigolactone synthesis and action a major target of biotechnology, little fundamental information is known about this hormone. Chemical genetic screening using Arabidopsis thaliana as a platform identified a collection of related small molecules, cotylimides, which perturb strigolactone accumulation. Suppressor screens against select cotylimides identified light-signaling genes as positive regulators of strigolactone levels. Molecular analysis showed strigolactones regulate the nuclear localization of the COP1 ubiquitin ligase, which in part determines the levels of light regulators such as HY5. This information not only uncovers new functions for strigolactones but was also used to identify rice cultivars with reduced capacity to germinate parasitic seed.