Ectopic expression of BOTRYTIS SUSCEPTIBLE1 reveals its function as a positive regulator of wound-induced cell death and plant susceptibility to Botrytis.

Programmed cell death (PCD) is integral to plant life and required for stress responses, immunity, and development. Our understanding of the regulation of PCD is incomplete, especially concerning regulators involved in multiple divergent processes. The botrytis-susceptible (bos1) mutant of Arabidopsis is highly susceptible to fungal infection by Botrytis cinerea (Botrytis). BOS1 (also known as MYB108) regulates cell death propagation during plant responses to wounding. The bos1-1 allele contains a T-DNA insertion in the 5'-untranslated region upstream of the start codon. This insertion results in elevated expression of BOS1/MYB108. We used clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated nuclease 9 (Cas9) system (CRISPR/Cas9) to create new bos1 alleles with disrupted exons, and found that these lines lacked the typical bos1-1 wounding and Botrytis phenotypes. They did exhibit reduced fertility, as was previously observed in other bos1 alleles. Resequencing of the bos1-1 genome confirmed the presence of a mannopine synthase (MAS) promoter at the T-DNA left border. Expression of the BOS1 gene under control of the MAS promoter in wild-type plants conferred the characteristic phenotypes of bos1-1: Botrytis sensitivity and response to wounding. Multiple overexpression lines demonstrated that BOS1 was involved in regulation of cell death propagation in a dosage-dependent manner. Our data indicate that bos1-1 is a gain-of-function mutant and that BOS1 function in regulation of fertility and Botrytis response can both be understood as misregulated cell death.

ALMT-independent guard cell R-type anion currents.

Plant transpiration is controlled by stomata, with S- and R-type anion channels playing key roles in guard cell action. Arabidopsis mutants lacking the ALMT12/QUAC1 R-type anion channel function in guard cells show only a partial reduction in R-type channel currents. The molecular nature of these remaining R-type anion currents is still unclear. To further elucidate this, patch clamp, transcript and gas-exchange measurements were performed with wild-type (WT) and different almt mutant plants. The R-type current fraction in the almt12 mutant exhibited the same voltage dependence, susceptibility to ATP block and lacked a chloride permeability as the WT. Therefore, we asked whether the R-type anion currents in the ALMT12/QUAC1-free mutant are caused by additional ALMT isoforms. In WT guard cells, ALMT12, ALMT13 and ALMT14 transcripts were detected, whereas only ALMT13 was found expressed in the almt12 mutant. Substantial R-type anion currents still remained active in the almt12/13 and almt12/14 double mutants as well as the almt12/13/14 triple mutant. In good agreement, CO2 -triggered stomatal closure required the activity of ALMT12 but not ALMT13 or ALMT14. The results suggest that, with the exception of ALMT12, channel species other than ALMTs carry the guard cell R-type anion currents.

Jasmonic acid and salicylic acid play minor roles in stomatal regulation by CO2 , abscisic acid, darkness, vapor pressure deficit and ozone.

Jasmonic acid (JA) and salicylic acid (SA) regulate stomatal closure, preventing pathogen invasion into plants. However, to what extent abscisic acid (ABA), SA and JA interact, and what the roles of SA and JA are in stomatal responses to environmental cues, remains unclear. Here, by using intact plant gas-exchange measurements in JA and SA single and double mutants, we show that stomatal responsiveness to CO2 , light intensity, ABA, high vapor pressure deficit and ozone either did not or, for some stimuli only, very slightly depended upon JA and SA biosynthesis and signaling mutants, including dde2, sid2, coi1, jai1, myc2 and npr1 alleles. Although the stomata in the mutants studied clearly responded to ABA, CO2 , light and ozone, ABA-triggered stomatal closure in npr1-1 was slightly accelerated compared with the wild type. Stomatal reopening after ozone pulses was quicker in the coi1-16 mutant than in the wild type. In intact Arabidopsis plants, spraying with methyl-JA led to only a modest reduction in stomatal conductance 80 min after treatment, whereas ABA and CO2 induced pronounced stomatal closure within minutes. We could not document a reduction of stomatal conductance after spraying with SA. Coronatine-induced stomatal opening was initiated slowly after 1.5-2.0 h, and reached a maximum by 3 h after spraying intact plants. Our results suggest that ABA, CO2 and light are major regulators of rapid guard cell signaling, whereas JA and SA could play only minor roles in the whole-plant stomatal response to environmental cues in Arabidopsis and Solanum lycopersicum (tomato).

Combined action of guard cell plasma membrane rapid- and slow-type anion channels in stomatal regulation.

Initiation of stomatal closure by various stimuli requires activation of guard cell plasma membrane anion channels, which are defined as rapid (R)- and slow (S)-type. The single-gene loss-of-function mutants of these proteins are well characterized. However, the impact of suppressing both the S- and R-type channels has not been studied. Here, by generating and studying double and triple Arabidopsis thaliana mutants of SLOW ANION CHANNEL1 (SLAC1), SLAC1 HOMOLOG3 (SLAH3), and ALUMINUM-ACTIVATED MALATE TRANSPORTER 12/QUICK-ACTIVATING ANION CHANNEL 1 (QUAC1), we show that impairment of R- and S-type channels gradually increased whole-plant steady-state stomatal conductance. Ozone-induced cell death also increased gradually in higher-order mutants with the highest levels observed in the quac1 slac1 slah3 triple mutant. Strikingly, while single mutants retained stomatal responsiveness to abscisic acid, darkness, reduced air humidity, and elevated CO2, the double mutant lacking SLAC1 and QUAC1 was nearly insensitive to these stimuli, indicating the need for coordinated activation of both R- and S-type anion channels in stomatal closure.

Ozone responses in Arabidopsis: beyond stomatal conductance.

Tropospheric ozone (O3) is a major air pollutant that decreases yield of important crops worldwide. Despite long-lasting research of its negative effects on plants, there are many gaps in our knowledge on how plants respond to O3. In this study, we used natural variation in the model plant Arabidopsis (Arabidopsis thaliana) to characterize molecular and physiological mechanisms underlying O3 sensitivity. A key parameter in models for O3 damage is stomatal uptake. Here we show that the extent of O3 damage in the sensitive Arabidopsis accession Shahdara (Sha) does not correspond with O3 uptake, pointing toward stomata-independent mechanisms for the development of O3 damage. We compared tolerant (Col-0) versus sensitive accessions (Sha, Cvi-0) in assays related to photosynthesis, cell death, antioxidants, and transcriptional regulation. Acute O3 exposure increased cell death, development of lesions in the leaves, and decreased photosynthesis in sensitive accessions. In both Sha and Cvi-0, O3-induced lesions were associated with decreased maximal chlorophyll fluorescence and low quantum yield of electron transfer from Photosystem II to plastoquinone. However, O3-induced repression of photosynthesis in these two O3-sensitive accessions developed in different ways. We demonstrate that O3 sensitivity in Arabidopsis is influenced by genetic diversity given that Sha and Cvi-0 developed accession-specific transcriptional responses to O3. Our findings advance the understanding of plant responses to O3 and set a framework for future studies to characterize molecular and physiological mechanisms allowing plants to respond to high O3 levels in the atmosphere as a result of high air pollution and climate change.

Dissecting Contrasts in Cell Death, Hormone, and Defense Signaling in Response to Botrytis cinerea and Reactive Oxygen Species.

Plants require interaction between signaling pathways to differentiate and integrate stress responses and deploy appropriate defenses. The hormones ethylene, salicylic acid (SA), and jasmonic acid (JA) are important regulators of plant defenses. Numerous interactions between these signaling pathways are the cornerstone of robust plant immunity. Additionally, during the early response to pathogens, reactive oxygen species (ROS) act as signaling molecules. Here, we examined the extent of signal interaction in the early stages of Botrytis cinerea infection. To enable a comparison between B. cinerea infection with ROS signaling, we subjected plants to ozone treatment, which stimulates an apoplastic ROS burst. We used a collection of single, double, and triple signaling mutants defective in hormone signaling and biosynthesis and subjected them to B. cinerea infection and ozone treatment at different timepoints. We examined lesion size, cell death, and gene expression (both quantitatively and spatially). The two treatments shared many similarities, especially in JA-insensitive mutants, which were sensitive to both treatments. Unexpectedly, a B. cinerea-susceptible JA-insensitive mutant (coi1), became tolerant when both SA biosynthesis and signaling was impaired (coi1 npr1 sid2), demonstrating that JA responses may be under the control of SA. Extensive marker gene analysis indicated JA as the main regulator of both B. cinerea and ozone defenses. In addition, we identified the transcription factor SR1 as a crucial regulator of PLANT DEFENSIN expression and cell-death regulation, which contributes to resistance to B. cinerea. Overall, our work further defines the context of ROS in plant defense signaling.[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.

Rapid depolarization and cytosolic calcium increase go hand-in-hand in mesophyll cells' ozone response.

Plant stress signalling involves bursts of reactive oxygen species (ROS), which can be mimicked by the application of acute pulses of ozone. Such ozone-pulses inhibit photosynthesis and trigger stomatal closure in a few minutes, but the signalling that underlies these responses remains largely unknown. We measured changes in Arabidopsis thaliana gas exchange after treatment with acute pulses of ozone and set up a system for simultaneous measurement of membrane potential and cytosolic calcium with the fluorescent reporter R-GECO1. We show that within 1 min, prior to stomatal closure, O3 triggered a drop in whole-plant CO2 uptake. Within this early phase, O3 pulses (200-1000 ppb) elicited simultaneous membrane depolarization and cytosolic calcium increase, whereas these pulses had no long-term effect on either stomatal conductance or photosynthesis. In contrast, pulses of 5000 ppb O3 induced cell death, systemic Ca2+ signals and an irreversible drop in stomatal conductance and photosynthetic capacity. We conclude that mesophyll cells respond to ozone in a few seconds by distinct pattern of plasma membrane depolarizations accompanied by an increase in the cytosolic calcium ion (Ca2+ ) level. These responses became systemic only at very high ozone concentrations. Thus, plants have rapid mechanism to sense and discriminate the strength of ozone signals.

PROTEIN PHOSPHATASE 2A-B'γ Controls Botrytis cinerea Resistance and Developmental Leaf Senescence.

Plants optimize their growth and survival through highly integrated regulatory networks that coordinate defensive measures and developmental transitions in response to environmental cues. Protein phosphatase 2A (PP2A) is a key signaling component that controls stress reactions and growth at different stages of plant development, and the PP2A regulatory subunit PP2A-B'γ is required for negative regulation of pathogenesis responses and for maintenance of cell homeostasis in short-day conditions. Here, we report molecular mechanisms by which PP2A-B'γ regulates Botrytis cinerea resistance and leaf senescence in Arabidopsis (Arabidopsis thaliana). We extend the molecular functionality of PP2A-B'γ to a protein kinase-phosphatase interaction with the defense-associated calcium-dependent protein kinase CPK1 and present indications this interaction may function to control CPK1 activity. In presenescent leaf tissues, PP2A-B'γ is also required to negatively control the expression of salicylic acid-related defense genes, which have recently proven vital in plant resistance to necrotrophic fungal pathogens. In addition, we find the premature leaf yellowing of pp2a-b'γ depends on salicylic acid biosynthesis via SALICYLIC ACID INDUCTION DEFICIENT2 and bears the hallmarks of developmental leaf senescence. We propose PP2A-B'γ age-dependently controls salicylic acid-related signaling in plant immunity and developmental leaf senescence.

The photoreceptor UVR8 mediates the perception of both UV-B and UV-A wavelengths up to 350 nm of sunlight with responsivity moderated by cryptochromes.

The photoreceptors UV RESISTANCE LOCUS 8 (UVR8) and CRYPTOCHROMES 1 and 2 (CRYs) play major roles in the perception of UV-B (280-315 nm) and UV-A/blue radiation (315-500 nm), respectively. However, it is poorly understood how they function in sunlight. The roles of UVR8 and CRYs were assessed in a factorial experiment with Arabidopsis thaliana wild-type and photoreceptor mutants exposed to sunlight for 6 or 12 hr under five types of filters with cut-offs in UV and blue-light regions. Transcriptome-wide responses triggered by UV-B and UV-A wavelengths shorter than 350 nm (UV-Asw ) required UVR8 whereas those induced by blue and UV-A wavelengths longer than 350 nm (UV-Alw ) required CRYs. UVR8 modulated gene expression in response to blue light while lack of CRYs drastically enhanced gene expression in response to UV-B and UV-Asw . These results agree with our estimates of photons absorbed by these photoreceptors in sunlight and with in vitro monomerization of UVR8 by wavelengths up to 335 nm. Motif enrichment analysis predicted complex signaling downstream of UVR8 and CRYs. Our results highlight that it is important to use UV waveband definitions specific to plants' photomorphogenesis as is routinely done in the visible region.

Genetic controls of short- and long-term stomatal CO2 responses in Arabidopsis thaliana.

BACKGROUND AND AIMS: The stomatal conductance (gs) of most plant species decreases in response to elevated atmospheric CO2 concentration. This response could have a significant impact on plant water use in a future climate. However, the regulation of the CO2-induced stomatal closure response is not fully understood. Moreover, the potential genetic links between short-term (within minutes to hours) and long-term (within weeks to months) responses of gs to increased atmospheric CO2 have not been explored. METHODS: We used Arabidopsis thaliana recombinant inbred lines originating from accessions Col-0 (strong CO2 response) and C24 (weak CO2 response) to study short- and long-term controls of gs. Quantitative trait locus (QTL) mapping was used to identify loci controlling short- and long-term gs responses to elevated CO2, as well as other stomata-related traits. KEY RESULTS: Short- and long-term stomatal responses to elevated CO2 were significantly correlated. Both short- and long-term responses were associated with a QTL at the end of chromosome 2. The location of this QTL was confirmed using near-isogenic lines and it was fine-mapped to a 410-kb region. The QTL did not correspond to any known gene involved in stomatal closure and had no effect on the responsiveness to abscisic acid. Additionally, we identified numerous other loci associated with stomatal regulation. CONCLUSIONS: We identified and confirmed the effect of a strong QTL corresponding to a yet unknown regulator of stomatal closure in response to elevated CO2 concentration. The correlation between short- and long-term stomatal CO2 responses and the genetic link between these traits highlight the importance of understanding guard cell CO2 signalling to predict and manipulate plant water use in a world with increasing atmospheric CO2 concentration. This study demonstrates the power of using natural variation to unravel the genetic regulation of complex traits.

Mitogen-activated protein kinases MPK4 and MPK12 are key components mediating CO2 -induced stomatal movements.

Respiration in leaves and the continued elevation in the atmospheric CO2 concentration cause CO2 -mediated reduction in stomatal pore apertures. Several mutants have been isolated for which stomatal responses to both abscisic acid (ABA) and CO2 are simultaneously defective. However, there are only few mutations that impair the stomatal response to elevated CO2 , but not to ABA. Such mutants are invaluable in unraveling the molecular mechanisms of early CO2 signal transduction in guard cells. Recently, mutations in the mitogen-activated protein (MAP) kinase, MPK12, have been shown to partially impair CO2 -induced stomatal closure. Here, we show that mpk12 plants, in which MPK4 is stably silenced specifically in guard cells (mpk12 mpk4GC homozygous double-mutants), completely lack CO2 -induced stomatal responses and have impaired activation of guard cell S-type anion channels in response to elevated CO2 /bicarbonate. However, ABA-induced stomatal closure, S-type anion channel activation and ABA-induced marker gene expression remain intact in the mpk12 mpk4GC double-mutants. These findings suggest that MPK12 and MPK4 act very early in CO2 signaling, upstream of, or parallel to the convergence of CO2 and ABA signal transduction. The activities of MPK4 and MPK12 protein kinases were not directly modulated by CO2 /bicarbonate in vitro, suggesting that they are not direct CO2 /bicarbonate sensors. Further data indicate that MPK4 and MPK12 have distinguishable roles in Arabidopsis and that the previously suggested role of RHC1 in stomatal CO2 signaling is minor, whereas MPK4 and MPK12 act as key components of early stomatal CO2 signal transduction.

Cell death regulation but not abscisic acid signaling is required for enhanced immunity to Botrytis in Arabidopsis cuticle-permeable mutants.

Prevailing evidence indicates that abscisic acid (ABA) negatively influences immunity to the fungal pathogen Botrytis cinerea in most but not all cases. ABA is required for cuticle biosynthesis, and cuticle permeability enhances immunity to Botrytis via unknown mechanisms. This complex web of responses obscures the role of ABA in Botrytis immunity. Here, we addressed the relationships between ABA sensitivity, cuticle permeability, and Botrytis immunity in the Arabidopsis thaliana ABA-hypersensitive mutants protein phosphatase2c quadruple mutant (pp2c-q) and enhanced response to aba1 (era1-2). Neither pp2c-q nor era1-2 exhibited phenotypes predicted by the known roles of ABA; conversely, era1-2 had a permeable cuticle and was Botrytis resistant. We employed RNA-seq analysis in cuticle-permeable mutants of differing ABA sensitivities and identified a core set of constitutively activated genes involved in Botrytis immunity and susceptibility to biotrophs, independent of ABA signaling. Furthermore, botrytis susceptible1 (bos1), a mutant with deregulated cell death and enhanced ABA sensitivity, suppressed the Botrytis immunity of cuticle permeable mutants, and this effect was linearly correlated with the extent of spread of wound-induced cell death in bos1. Overall, our data demonstrate that Botrytis immunity conferred by cuticle permeability can be genetically uncoupled from PP2C-regulated ABA sensitivity, but requires negative regulation of a parallel ABA-dependent cell-death pathway.

How do cryptochromes and UVR8 interact in natural and simulated sunlight?

Cryptochromes (CRYs) and UV RESISTANCE LOCUS 8 (UVR8) photoreceptors perceive UV-A/blue (315-500 nm) and UV-B (280-315 nm) radiation in plants, respectively. While the roles of CRYs and UVR8 have been studied in separate controlled-environment experiments, little is known about the interaction between these photoreceptors. Here, Arabidopsis wild-type Ler, CRYs and UVR8 photoreceptor mutants (uvr8-2, cry1cry2 and cry1cry2uvr8-2), and a flavonoid biosynthesis-defective mutant (tt4) were grown in a sun simulator. Plants were exposed to filtered radiation for 17 d or for 6 h, to study the effects of blue, UV-A, and UV-B radiation. Both CRYs and UVR8 independently enabled growth and survival of plants under solar levels of UV, while their joint absence was lethal under UV-B. CRYs mediated gene expression under blue light. UVR8 mediated gene expression under UV-B radiation, and in the absence of CRYs, also under UV-A. This negative regulation of UVR8-mediated gene expression by CRYs was also observed for UV-B. The accumulation of flavonoids was also consistent with this interaction between CRYs and UVR8. In conclusion, we provide evidence for an antagonistic interaction between CRYs and UVR8 and a role of UVR8 in UV-A perception.

A Dominant Mutation in the HT1 Kinase Uncovers Roles of MAP Kinases and GHR1 in CO2-Induced Stomatal Closure.

Activation of the guard cell S-type anion channel SLAC1 is important for stomatal closure in response to diverse stimuli, including elevated CO2 The majority of known SLAC1 activation mechanisms depend on abscisic acid (ABA) signaling. Several lines of evidence point to a parallel ABA-independent mechanism of CO2-induced stomatal regulation; however, molecular details of this pathway remain scarce. Here, we isolated a dominant mutation in the protein kinase HIGH LEAF TEMPERATURE1 (HT1), an essential regulator of stomatal CO2 responses, in an ozone sensitivity screen of Arabidopsis thaliana The mutation caused constitutively open stomata and impaired stomatal CO2 responses. We show that the mitogen-activated protein kinases (MPKs) MPK4 and MPK12 can inhibit HT1 activity in vitro and this inhibition is decreased for the dominant allele of HT1. We also show that HT1 inhibits the activation of the SLAC1 anion channel by the protein kinases OPEN STOMATA1 and GUARD CELL HYDROGEN PEROXIDE-RESISTANT1 (GHR1) in Xenopus laevis oocytes. Notably, MPK12 can restore SLAC1 activation in the presence of HT1, but not in the presence of the dominant allele of HT1. Based on these data, we propose a model for sequential roles of MPK12, HT1, and GHR1 in the ABA-independent regulation of SLAC1 during CO2-induced stomatal closure.

Natural Variation in Arabidopsis Cvi-0 Accession Reveals an Important Role of MPK12 in Guard Cell CO2 Signaling.

Plant gas exchange is regulated by guard cells that form stomatal pores. Stomatal adjustments are crucial for plant survival; they regulate uptake of CO2 for photosynthesis, loss of water, and entrance of air pollutants such as ozone. We mapped ozone hypersensitivity, more open stomata, and stomatal CO2-insensitivity phenotypes of the Arabidopsis thaliana accession Cvi-0 to a single amino acid substitution in MITOGEN-ACTIVATED PROTEIN (MAP) KINASE 12 (MPK12). In parallel, we showed that stomatal CO2-insensitivity phenotypes of a mutant cis (CO2-insensitive) were caused by a deletion of MPK12. Lack of MPK12 impaired bicarbonate-induced activation of S-type anion channels. We demonstrated that MPK12 interacted with the protein kinase HIGH LEAF TEMPERATURE 1 (HT1)-a central node in guard cell CO2 signaling-and that MPK12 functions as an inhibitor of HT1. These data provide a new function for plant MPKs as protein kinase inhibitors and suggest a mechanism through which guard cell CO2 signaling controls plant water management.

Tip-localized Ca2+ -permeable channels control pollen tube growth via kinase-dependent R- and S-type anion channel regulation.

Pollen tubes (PTs) are characterized by having tip-focused cytosolic calcium ion (Ca2+ ) concentration ([Ca2+ ]cyt ) gradients, which are believed to control PT growth. However, the mechanisms by which the apical [Ca2+ ]cyt orchestrates PT growth are not well understood. Here, we aimed to identify these mechanisms by combining reverse genetics, cell biology, electrophysiology, and live-cell Ca2+ and anion imaging. We triggered Ca2+ -channel activation by applying hyperpolarizing voltage pulses and observed that the evoked [Ca2+ ]cyt increases were paralleled by high anion channel activity and a decrease in the cytosolic anion concentration at the PT tip. We confirmed a functional correlation between these patterns by showing that inhibition of Ca2+ -permeable channels eliminated the [Ca2+ ]cyt increase, resulting in the abrogation of anion channel activity via Ca2+ -dependent protein kinases (CPKs). Functional characterization of CPK and anion-channel mutants revealed a CPK2/20/6-dependent activation of SLAH3 and ALMT12/13/14 anion channels. The impaired growth phenotypes of anion channel and CPK mutants support the physiological significance of a kinase- and Ca2+ -dependent pathway to control PT growth via anion channel activation. Other than unveiling this functional link, our membrane hyperpolarization method allows for unprecedented manipulation of the [Ca2+ ]cyt gradient or oscillations in the PT tips and opens an array of opportunities for channel screenings.

The Role of ENHANCED RESPONSES TO ABA1 (ERA1) in Arabidopsis Stomatal Responses Is Beyond ABA Signaling.

Proper stomatal responses are essential for plant function in an altered environment. The core signaling pathway for abscisic acid (ABA)-induced stomatal closure involves perception of the hormone that leads to the activation of guard cell anion channels by the protein kinase OPEN STOMATA1. Several other regulators are suggested to modulate the ABA signaling pathway, including the protein ENHANCED RESPONSE TO ABA1 (ERA1), that encodes the farnesyl transferase ß-subunit. The era1 mutant is hypersensitive to ABA during seed germination and shows a more closed stomata phenotype. Using a genetics approach with the double mutants era1 abi1-1 and era1 ost1, we show that while era1 suppressed the high stomatal conductance of abi1-1 and ost1, the ERA1 function was not required for stomatal closure in response to ABA and environmental factors. Further experiments indicated a role for ERA1 in blue light-induced stomatal opening. In addition, we show that ERA1 function in disease resistance was independent of its role in stomatal regulation. Our results indicate a function for ERA1 in stomatal opening and pathogen immunity.

Interaction points in plant stress signaling pathways.

Plants live in a world where they are challenged by abiotic and biotic stresses. In response to unfavorable conditions or an acute challenge like a pathogen attack, plants use various signaling pathways that regulate expression of defense genes and other mechanisms to provide resistance or stress adaptation. Identification of the regulatory steps in defense signaling has seen much progress in recent years. Many of the identified signaling pathways show interactions with each other, exemplified by the modulation of the jasmonic acid response by salicylic acid. Accordingly, defense regulation is more appropriately thought of as a web of interactions, rather than linear pathways. Here we describe various regulatory components and how they interact to provide an appropriate defense response. One of the common assays to monitor the output of defense signaling, as well as interaction between signaling pathways, is the measurement of altered gene expression. We illustrate that, while this is a suitable assay to monitor defense regulation, it can also inadvertently provide overstated conclusions about interaction among signaling pathways.

Large-Scale Phenomics Identifies Primary and Fine-Tuning Roles for CRKs in Responses Related to Oxidative Stress.

Cysteine-rich receptor-like kinases (CRKs) are transmembrane proteins characterized by the presence of two domains of unknown function 26 (DUF26) in their ectodomain. The CRKs form one of the largest groups of receptor-like protein kinases in plants, but their biological functions have so far remained largely uncharacterized. We conducted a large-scale phenotyping approach of a nearly complete crk T-DNA insertion line collection showing that CRKs control important aspects of plant development and stress adaptation in response to biotic and abiotic stimuli in a non-redundant fashion. In particular, the analysis of reactive oxygen species (ROS)-related stress responses, such as regulation of the stomatal aperture, suggests that CRKs participate in ROS/redox signalling and sensing. CRKs play general and fine-tuning roles in the regulation of stomatal closure induced by microbial and abiotic cues. Despite their great number and high similarity, large-scale phenotyping identified specific functions in diverse processes for many CRKs and indicated that CRK2 and CRK5 play predominant roles in growth regulation and stress adaptation, respectively. As a whole, the CRKs contribute to specificity in ROS signalling. Individual CRKs control distinct responses in an antagonistic fashion suggesting future potential for using CRKs in genetic approaches to improve plant performance and stress tolerance.