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.

Ozone and nitrogen dioxide regulate similar gene expression responses in Arabidopsis but natural variation in the extent of cell death is likely controlled by different genetic loci.

High doses of ozone (O3) and nitrogen dioxide (NO2) cause damage and cell death in plants. These two gases are among the most harmful air pollutants for ecosystems and therefore it is important to understand how plant resistance or sensitivity to these gases work at the molecular level and its genetic control. We compared transcriptome data from O3 and NO2 fumigations to other cell death related treatments, as well as individual marker gene transcript level in different Arabidopsis thaliana accessions. Our analysis revealed that O3 and NO2 trigger very similar gene expression responses that include genes involved in pathogen resistance, cell death and ethylene signaling. However, we also identified exceptions, for example RBOHF encoding a reactive oxygen species producing RESPIRATORY BURST OXIDASE PROTEIN F. This gene had increased transcript levels by O3 but decreased transcript levels by NO2, showing that plants can identify each of the gases separately and activate distinct signaling pathways. To understand the genetics, we conducted a genome wide association study (GWAS) on O3 and NO2 tolerance of natural Arabidopsis accessions. Sensitivity to both gases seem to be controlled by several independent small effect loci and we did not find an overlap in the significantly associated regions. Further characterization of the GWAS candidate loci identified new regulators of O3 and NO2 induced cell death including ABH1, a protein that functions in abscisic acid signaling, mRNA splicing and miRNA processing. The GWAS results will facilitate further characterization of the control of programmed cell death and differences between oxidative and nitrosative stress in plants.

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.

Simultaneous Ozone and High Light Treatments Reveal an Important Role for the Chloroplast in Co-ordination of Defense Signaling.

Plants live in a world of changing environments, where they are continuously challenged by alternating biotic and abiotic stresses. To transfer information from the environment to appropriate protective responses, plants use many different signaling molecules and pathways. Reactive oxygen species (ROS) are critical signaling molecules in the regulation of plant stress responses, both inside and between cells. In natural environments, plants can experience multiple stresses simultaneously. Laboratory studies on stress interaction and crosstalk at regulation of gene expression, imply that plant responses to multiple stresses are distinctly different from single treatments. We analyzed the expression of selected marker genes and reassessed publicly available datasets to find signaling pathways regulated by ozone, which produces apoplastic ROS, and high light treatment, which produces chloroplastic ROS. Genes related to cell death regulation were differentially regulated by ozone versus high light. In a combined ozone + high light treatment, the light treatment enhanced ozone-induced cell death in leaves. The distinct responses from ozone versus high light treatments show that plants can activate stress signaling pathways in a highly precise manner.

Chromosome-level genome assembly of the diploid blueberry Vaccinium darrowii provides insights into its subtropical adaptation and cuticle synthesis.

Vaccinium darrowii is a subtropical wild blueberry species that has been used to breed economically important southern highbush cultivars. The adaptive traits of V. darrowii to subtropical climates can provide valuable information for breeding blueberry and perhaps other plants, especially against the background of global warming. Here, we assembled the V. darrowii genome into 12 pseudochromosomes using Oxford Nanopore long reads complemented with Hi-C scaffolding technologies, and we predicted 41 815 genes using RNA-sequencing evidence. Syntenic analysis across three Vaccinium species revealed a highly conserved genome structure, with the highest collinearity between V. darrowii and Vaccinium corymbosum. This conserved genome structure may explain the high fertility observed during crossbreeding of V. darrowii with other blueberry cultivars. Analysis of gene expansion and tandem duplication indicated possible roles for defense- and flowering-associated genes in the adaptation of V. darrowii to the subtropics. Putative SOC1 genes in V. darrowii were identified based on phylogeny and expression analysis. Blueberries are covered in a thick cuticle layer and contain anthocyanins, which confer their powdery blue color. Using RNA sequencing, we delineated the cuticle biosynthesis pathways of Vaccinium species in V. darrowii. This result can serve as a reference for breeding berries whose colors are appealing to customers. The V. darrowii reference genome, together with the unique traits of this species, including its diploid genome, short vegetative phase, and high compatibility in hybridization with other blueberries, make V. darrowii a potential research model for blueberry species.

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.

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).

Genotype-Specific Expression and NLR Repertoire Contribute to Phenotypic Resistance Diversity in Plantago lanceolata.

High levels of phenotypic variation in resistance appears to be nearly ubiquitous across natural host populations. Molecular processes contributing to this variation in nature are still poorly known, although theory predicts resistance to evolve at specific loci driven by pathogen-imposed selection. Nucleotide-binding leucine-rich repeat (NLR) genes play an important role in pathogen recognition, downstream defense responses and defense signaling. Identifying the natural variation in NLRs has the potential to increase our understanding of how NLR diversity is generated and maintained, and how to manage disease resistance. Here, we sequenced the transcriptomes of five different Plantago lanceolata genotypes when inoculated by the same strain of obligate fungal pathogen Podosphaera plantaginis. A de novo transcriptome assembly of RNA-sequencing data yielded 24,332 gene models with N50 value of 1,329 base pairs and gene space completeness of 66.5%. The gene expression data showed highly varying responses where each plant genotype demonstrated a unique expression profile in response to the pathogen, regardless of the resistance phenotype. Analysis on the conserved NB-ARC domain demonstrated a diverse NLR repertoire in P. lanceolata consistent with the high phenotypic resistance diversity in this species. We find evidence of selection generating diversity at some of the NLR loci. Jointly, our results demonstrate that phenotypic resistance diversity results from a crosstalk between different defense mechanisms. In conclusion, characterizing the architecture of resistance in natural host populations may shed unprecedented light on the potential of evolution to generate variation.

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.

Evolutionary conservation and post-translational control of S-adenosyl-L-homocysteine hydrolase in land plants.

Trans-methylation reactions are intrinsic to cellular metabolism in all living organisms. In land plants, a range of substrate-specific methyltransferases catalyze the methylation of DNA, RNA, proteins, cell wall components and numerous species-specific metabolites, thereby providing means for growth and acclimation in various terrestrial habitats. Trans-methylation reactions consume vast amounts of S-adenosyl-L-methionine (SAM) as a methyl donor in several cellular compartments. The inhibitory reaction by-product, S-adenosyl-L-homocysteine (SAH), is continuously removed by SAH hydrolase (SAHH), which essentially maintains trans-methylation reactions in all living cells. Here we report on the evolutionary conservation and post-translational control of SAHH in land plants. We provide evidence suggesting that SAHH forms oligomeric protein complexes in phylogenetically divergent land plants and that the predominant protein complex is composed by a tetramer of the enzyme. Analysis of light-stress-induced adjustments of SAHH in Arabidopsis thaliana and Physcomitrella patens further suggests that regulatory actions may take place on the levels of protein complex formation and phosphorylation of this metabolically central enzyme. Collectively, these data suggest that plant adaptation to terrestrial environments involved evolution of regulatory mechanisms that adjust the trans-methylation machinery in response to environmental cues.

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.

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.

Differential role of MAX2 and strigolactones in pathogen, ozone, and stomatal responses.

Strigolactones are a group of phytohormones that control developmental processes including shoot branching and various plant-environment interactions in plants. We previously showed that the strigolactone perception mutant more axillary branches 2 (max2) has increased susceptibility to plant pathogenic bacteria. Here we show that both strigolactone biosynthesis (max3 and max4) and perception mutants (max2 and dwarf14) are significantly more sensitive to Pseudomonas syringae DC3000. Moreover, in response to P. syringae infection, high levels of SA accumulated in max2 and this mutant was ozone sensitive. Further analysis of gene expression revealed no major role for strigolactone in regulation of defense gene expression. In contrast, guard cell function was clearly impaired in max2 and depending on the assay used, also in max3, max4, and d14 mutants. We analyzed stomatal responses to stimuli that cause stomatal closure. While the response to abscisic acid (ABA) was not impaired in any of the mutants, the response to darkness and high CO2 was impaired in max2 and d14-1 mutants, and to CO2 also in strigolactone synthesis (max3, max4) mutants. To position the role of MAX2 in the guard cell signaling network, max2 was crossed with mutants defective in ABA biosynthesis or signaling. This revealed that MAX2 acts in a signaling pathway that functions in parallel to the guard cell ABA signaling pathway. We propose that the impaired defense responses of max2 are related to higher stomatal conductance that allows increased entry of bacteria or air pollutants like ozone. Furthermore, as MAX2 appears to act in a specific branch of guard cell signaling (related to CO2 signaling), this protein could be one of the components that allow guard cells to distinguish between different environmental conditions.

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.

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.