Plants grow and reproduce within a highly dynamic environment that can see abrupt changes in conditions, such as light intensity, temperature, humidity, or interactions with biotic agents. Recent studies revealed that plants can respond within seconds to some of these conditions, engaging many different metabolic and molecular networks, as well as rapidly altering their stomatal aperture. Some of these rapid responses were further shown to propagate throughout the entire plant via waves of reactive oxygen species (ROS) and Ca 2+ that are possibly mediated through the plant vascular system. Here, we propose that the integration of these signals is mediated through pulses of gene expression that are coordinated throughout the plant in a systemic manner by the ROS/Ca +2 waves. The Dynamic Environment of PlantsWhile growing within their natural habitat, or in a manmade field environment, plants are subjected to many different changes in their physical and biological surroundings. These can be gradual, such as the slow decrease in soil water content over time during summer, or rapid, such as changes in light intensity occurring as a result of sun flecks during a cloudy day [1-9] (Figure 1, Key Figure). Some of these rapid changes can occur simultaneously, or on the background of other more persistent stress conditions, such as a prolonged drought or a heat wave, essentially representing a state of stress combination [4,5,10]. In addition, due to the placement of the plant within its environment, for example its position within a row of plants or plot in the field, or its position in nature in close proximity to a tree cover, not all parts or tissues of the plant may experience the rapid change in environmental conditions simultaneously [4,11]. Under such conditions, the rapid responses (see Glossary) at the affected tissue could trigger systemic signaling pathways, such as the ROS [12] and calcium waves [13], electric signals [14], and/or hydraulic waves [15]. The occurrence of rapid changes in the physical and/ or biological environment of the plant, coupled with the discovery of rapid systemic signaling pathways activated by many of these conditions [12][13][14][15], and the discovery of rapid transcriptional and metabolic responses to stress [16,17], support a hypothesis that plants evolved sensing and acclimation mechanisms that may function [ 3 9 0 _ T D $ D I F F ] within the seconds to minute timescale and are important for the overall fitness of the plant and its ability to survive rapid changes within its environment (Figure 1). Here, we examine the molecular networks, and physiological and metabolic changes that occur in plants during rapid responses to stress, and propose that the integration of these responses is mediated through pulses of gene expression that are coordinated throughout the plant in a systemic manner by [ 3 9 1 _ T D $ D I F F ] the ROS/Ca +2 waves. HighlightsRecent studies reveal that plants respond within the seconds to minutes time-scale to different biotic and/or abiotic stimuli.The ra...

Contents 44I.44II.45III.46IV.53V.5657References57 Summary Stomata are an attractive experimental system in plant biology, because the responses of guard cells to environmental signals can be directly linked to changes in the aperture of stomatal pores. In this review, the mechanics of stomatal movement are discussed in relation to ion transport in guard cells. Emphasis is placed on the ion pumps, transporters, and channels in the plasma membrane, as well as in the vacuolar membrane. The biophysical properties of transport proteins for H+, K+, Ca2+, and anions are discussed and related to their function in guard cells during stomatal movements. Guard cell signaling pathways for ABA, CO2, ozone, microbe‐associated molecular patterns (MAMPs) and blue light are presented. Special attention is given to the regulation of the slow anion channel (SLAC) and SLAC homolog (SLAH)‐type anion channels by the ABA signalosome. Over the last decade, several knowledge gaps in the regulation of ion transport in guard cells have been closed. The current state of knowledge is an excellent starting point for tackling important open questions concerning stress tolerance in plants.

Activation of the guard cell S-type anion channel SLAC1 is important for stomatal closure in response to diverse stimuli, including elevated CO 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 CO-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 CO responses, in an ozone sensitivity screen of Arabidopsis thaliana The mutation caused constitutively open stomata and impaired stomatal CO 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 CO-induced stomatal closure.

Summary During infection plants recognize microbe‐associated molecular patterns (MAMPs), and this leads to stomatal closure. This study analyzes the molecular mechanisms underlying this MAMP response and its interrelation with ABA signaling.Stomata in intact Arabidopsis thaliana plants were stimulated with the bacterial MAMP flg22, or the stress hormone ABA, by using the noninvasive nanoinfusion technique. Intracellular double‐barreled microelectrodes were applied to measure the activity of plasma membrane ion channels.Flg22 induced rapid stomatal closure and stimulated the SLAC1 and SLAH3 anion channels in guard cells. Loss of both channels resulted in cells that lacked flg22‐induced anion channel activity and stomata that did not close in response to flg22 or ABA. Rapid flg22‐dependent stomatal closure was impaired in plants that were flagellin receptor (FLS2)‐deficient, as well as in the ost1‐2 (Open Stomata 1) mutant, which lacks a key ABA‐signaling protein kinase. By contrast, stomata of the ABA protein phosphatase mutant abi1‐1 (ABscisic acid Insensitive 1) remained flg22‐responsive.These data suggest that the initial steps in flg22 and ABA signaling are different, but that the pathways merge at the level of OST1 and lead to activation of SLAC1 and SLAH3 anion channels.

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.

Summary During drought, abscisic acid (ABA) induces closure of stomata via a signaling pathway that involves the calcium (Ca2+)‐independent protein kinase OST1, as well as Ca2+‐dependent protein kinases. However, the interconnection between OST1 and Ca2+ signaling in ABA‐induced stomatal closure has not been fully resolved. ABA‐induced Ca2+ signals were monitored in intact Arabidopsis leaves, which express the ratiometric Ca2+ reporter R‐GECO1‐mTurquoise and the Ca2+‐dependent activation of S‐type anion channels was recorded with intracellular double‐barreled microelectrodes. ABA triggered Ca2+ signals that occurred during the initiation period, as well as in the acceleration phase of stomatal closure. However, a subset of stomata closed in the absence of Ca2+ signals. On average, stomata closed faster if Ca2+ signals were elicited during the ABA response. Loss of OST1 prevented ABA‐induced stomatal closure and repressed Ca2+ signals, whereas elevation of the cytosolic Ca2+ concentration caused a rapid activation of SLAC1 and SLAH3 anion channels. Our data show that the majority of Ca2+ signals are evoked during the acceleration phase of stomatal closure, which is initiated by OST1. These Ca2+ signals are likely to activate Ca2+‐dependent protein kinases, which enhance the activity of S‐type anion channels and boost stomatal closure.

29Plant gas exchange is regulated by guard cells that form stomatal pores. Stomatal adjustments are crucial 30 for plant survival; they regulate uptake of CO2 for photosynthesis, loss of water and entrance of air 31 pollutants such as ozone. We mapped ozone hypersensitivity, more open stomata and stomatal CO2-32 insensitivity phenotypes of the Arabidopsis thaliana accession Cvi-0 to a single amino acid substitution in 33 MAP kinase 12 (MPK12). In parallel we showed that stomatal CO2-insensitivity phenotypes of a mutant cis 34 (CO2-insensitive) were caused by a deletion of MPK12. Lack of MPK12 impaired bicarbonate-induced 35 activation of S-type anion channels. We demonstrated that MPK12 interacted with the protein kinase HT1, 36 a central node in guard cell CO2 signaling, and that MPK12 can function as an inhibitor of HT1. These data 37 provide a new function for plant MPKs as protein kinase inhibitors and suggest a mechanism through which 38 guard cell CO2 signaling controls plant water management. 39 40 Important components of Arabidopsis thaliana guard cell CO2 signaling are carbonic anhydrases (CA1 and 60 CA4) that convert CO2 to bicarbonate and the protein kinase HT1 (HIGH LEAF TEMPERATURE 1) that has 61 been suggested to function as a negative regulator of CO2-induced stomatal movements [8, 9]. Ultimately 62 for stomata to close, the signal has to activate protein kinases such as OST1 (OPEN STOMATA1) that in turn 63 activate plasma membrane anion channels, including SLAC1 (SLOW ANION CHANNEL 1) followed by 64 extrusion of ions and water that causes stomatal closure [10][11][12][13]. Bicarbonate-induced activation of SLAC1 65Our initial QTL mapping of ozone sensitivity in Cvi-0 placed the two major contributing loci on the lower 87 ends of chromosomes 2 and 3 [18]. To identify the causative loci related to the extreme ozone sensitivity 88 and more open stomata of Cvi-0, we created a near isogenic line (NIL) termed Col-S (for Col-0 ozone 89 insert in cas-2 was removed, thereby generating the mutant cis (CO2 insensitive). Both cis and Col-S2 had 107 impaired responses to high CO2 (800 ppm) leading to longer half-response times, but a residual CO2 108 response could still be observed ( Fig 1D and S1E Fig). 109Mapping and whole genome sequencing of cis × C24 populations revealed a complete deletion of the 110 MPK12 gene and its neighbor BYPASS2 in cis (Fig 1E and S1D Fig). Thus, cis was renamed to mpk12-4. A 111 second mutant (gdsl3-1) from the GABI collection (GABI-492D11) contained an identical deletion of 112 BYPASS2 and MPK12 (S2 Fig). We also identified a line with a T-DNA insert in exon 2 of MPK12 from the 113 SAIL collection (Fig 1E), which was recently named mpk12-3 [24]. No full-length transcript was found in 114 MPK12 functions in guard cell CO2 signaling 132Reduction of CO2 levels inside the leaf [25] is a signal that indicates shortage of substrate for 133 photosynthesis and triggers stomatal opening. The rate of stomatal opening in response to low CO2 was 134 severely impaired in mpk12 and Col-S2 ( ...

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 (ALMT12)/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.