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.

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.

A role for calcium-dependent protein kinases in differential CO2 - and ABA-controlled stomatal closing and low CO2 -induced stomatal opening in Arabidopsis.

Low concentrations of CO2 cause stomatal opening, whereas [CO2 ] elevation leads to stomatal closure. Classical studies have suggested a role for Ca2+ and protein phosphorylation in CO2 -induced stomatal closing. Calcium-dependent protein kinases (CPKs) and calcineurin-B-like proteins (CBLs) can sense and translate cytosolic elevation of the second messenger Ca2+ into specific phosphorylation events. However, Ca2+ -binding proteins that function in the stomatal CO2 response remain unknown. Time-resolved stomatal conductance measurements using intact plants, and guard cell patch-clamp experiments were performed. We isolated cpk quintuple mutants and analyzed stomatal movements in response to CO2 , light and abscisic acid (ABA). Interestingly, we found that cpk3/5/6/11/23 quintuple mutant plants, but not other analyzed cpk quadruple/quintuple mutants, were defective in high CO2 -induced stomatal closure and, unexpectedly, also in low CO2 -induced stomatal opening. Furthermore, K+ -uptake-channel activities were reduced in cpk3/5/6/11/23 quintuple mutants, in correlation with the stomatal opening phenotype. However, light-mediated stomatal opening remained unaffected, and ABA responses showed slowing in some experiments. By contrast, CO2 -regulated stomatal movement kinetics were not clearly affected in plasma membrane-targeted cbl1/4/5/8/9 quintuple mutant plants. Our findings describe combinatorial cpk mutants that function in CO2 control of stomatal movements and support the results of classical studies showing a role for Ca2+ in this response.

Calcium signals in guard cells enhance the efficiency by which abscisic acid triggers stomatal closure.

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.

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.

Guard cell SLAC1-type anion channels mediate flagellin-induced stomatal closure.

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.

Closing gaps: linking elements that control stomatal movement.

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(+), Ca(2+), 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.

Rapid Responses to Abiotic Stress: Priming the Landscape for the Signal Transduction Network.

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 Ca2+ 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.