Shaowu Xue

Carbonic anhydrases are upstream regulators of CO2-controlled stomatal movements in guard cells

The continuing rise in atmospheric CO2 causes stomatal pores in leaves to close and thus globally affects CO2 influx into plants, water use efficiency and leaf heat stress. However, the CO2-binding proteins that control this response remain unknown. Moreover, which cell type responds to CO2, mesophyll or guard cells, and whether photosynthesis mediates this response are matters of debate. We demonstrate that Arabidopsis thaliana double-mutant plants in the β-carbonic anhydrases βCA1 and βCA4 show impaired CO2-regulation of stomatal movements and increased stomatal density, but retain functional abscisic-acid and blue-light responses. βCA-mediated CO2-triggered stomatal movements are not, in first-order, linked to whole leaf photosynthesis and can function in guard cells. Furthermore, guard cell βca-overexpressing plants exhibit instantaneous enhanced water use efficiency. Guard cell expression of mammalian αCAII complements the reduced sensitivity of ca1 ca4 plants, showing that carbonic anhydrase-mediated catalysis is an important mechanism for βCA-mediated CO2-induced stomatal closure and patch clamp analyses indicate that CO2/HCO3− transfers the signal to anion channel regulation. These findings, together with ht1-2 (ref. 9) epistasis analysis demonstrate that carbonic anhydrases function early in the CO2 signalling pathway, which controls gas-exchange between plants and the atmosphere.

Reconstitution of abscisic acid activation of SLAC1 anion channel by CPK6 and OST1 kinases and branched ABI1 PP2C phosphatase action

The plant hormone abscisic acid (ABA) is produced in response to abiotic stresses and mediates stomatal closure in response to drought via recently identified ABA receptors (pyrabactin resistance/regulatory component of ABA receptor; PYR/RCAR). SLAC1 encodes a central guard cell S-type anion channel that mediates ABA-induced stomatal closure. Coexpression of the calcium-dependent protein kinase 21 (CPK21), CPK23, or the Open Stomata 1 kinase (OST1) activates SLAC1 anion currents. However, reconstitution of ABA activation of any plant ion channel has not yet been attained. Whether the known core ABA signaling components are sufficient for ABA activation of SLAC1 anion channels or whether additional components are required remains unknown. The Ca2+-dependent protein kinase CPK6 is known to function in vivo in ABA-induced stomatal closure. Here we show that CPK6 robustly activates SLAC1-mediated currents and phosphorylates the SLAC1 N terminus. A phosphorylation site (S59) in SLAC1, crucial for CPK6 activation, was identified. The group A PP2Cs ABI1, ABI2, and PP2CA down-regulated CPK6-mediated SLAC1 activity in oocytes. Unexpectedly, ABI1 directly dephosphorylated the N terminus of SLAC1, indicating an alternate branched early ABA signaling core in which ABI1 targets SLAC1 directly (down-regulation). Furthermore, here we have successfully reconstituted ABA-induced activation of SLAC1 channels in oocytes using the ABA receptor pyrabactin resistant 1 (PYR1) and PP2C phosphatases with two alternate signaling cores including either CPK6 or OST1. Point mutations in ABI1 disrupting PYR1–ABI1 interaction abolished ABA signal transduction. Moreover, by addition of CPK6, a functional ABA signal transduction core from ABA receptors to ion channel activation was reconstituted without a SnRK2 kinase.

Central functions of bicarbonate in S-type anion channel activation and OST1 protein kinase in CO2 signal transduction in guard cell

Plants respond to elevated CO2 via carbonic anhydrases that mediate stomatal closing, but little is known about the early signalling mechanisms following the initial CO2 response. It remains unclear whether CO2, HCO3− or a combination activates downstream signalling. Here, we demonstrate that bicarbonate functions as a small‐molecule activator of SLAC1 anion channels in guard cells. Elevated intracellular [HCO3−]i with low [CO2] and [H+] activated S‐type anion currents, whereas low [HCO3−]i at high [CO2] and [H+] did not. Bicarbonate enhanced the intracellular Ca2+ sensitivity of S‐type anion channel activation in wild‐type and ht1‐2 kinase mutant guard cells. ht1‐2 mutant guard cells exhibited enhanced bicarbonate sensitivity of S‐type anion channel activation. The OST1 protein kinase has been reported not to affect CO2 signalling. Unexpectedly, OST1 loss‐of‐function alleles showed strongly impaired CO2‐induced stomatal closing and HCO3− activation of anion channels. Moreover, PYR/RCAR abscisic acid (ABA) receptor mutants slowed but did not abolish CO2/HCO3− signalling, redefining the convergence point of CO2 and ABA signalling. A new working model of the sequence of CO2 signalling events in gas exchange regulation is presented.

Calcium elevation‐dependent and attenuated resting calcium‐dependent abscisic acid induction of stomatal closure and abscisic acid‐induced enhancement of calcium sensitivities of S‐type anion and inward‐rectifying K+ channels in Arabidopsis guard cells

Stomatal closure in response to abscisic acid depends on mechanisms that are mediated by intracellular [Ca2+] ([Ca2+]i), and also on mechanisms that are independent of [Ca2+]i in guard cells. In this study, we addressed three important questions with respect to these two predicted pathways in Arabidopsis thaliana. (i) How large is the relative abscisic acid (ABA)-induced stomatal closure response in the [Ca2+]i-elevation-independent pathway? (ii) How do ABA-insensitive mutants affect the [Ca2+]i-elevation-independent pathway? (iii) Does ABA enhance (prime) the Ca2+ sensitivity of anion and inward-rectifying K+ channel regulation? We monitored stomatal responses to ABA while experimentally inhibiting [Ca2+]i elevations and clamping [Ca2+]i to resting levels. The absence of [Ca2+]i elevations was confirmed by ratiometric [Ca2+]i imaging experiments. ABA-induced stomatal closure in the absence of [Ca2+]i elevations above the physiological resting [Ca2+]i showed only approximately 30% of the normal stomatal closure response, and was greatly slowed compared to the response in the presence of [Ca2+]i elevations. The ABA-insensitive mutants ost1-2, abi2-1 and gca2 showed partial stomatal closure responses that correlate with [Ca2+]i-dependent ABA signaling. Interestingly, patch-clamp experiments showed that exposure of guard cells to ABA greatly enhances the ability of cytosolic Ca2+ to activate S-type anion channels and down-regulate inward-rectifying K+ channels, providing strong evidence for a Ca2+ sensitivity priming hypothesis. The present study demonstrates and quantifies an attenuated and slowed ABA response when [Ca2+]i elevations are directly inhibited in guard cells. A minimal model is discussed, in which ABA enhances (primes) the [Ca2+]i sensitivity of stomatal closure mechanisms.

PYR/RCAR Receptors Contribute to Ozone-, Reduced Air Humidity-, Darkness-, and CO2-Induced Stomatal Regulation

Signaling through abscisic acid PYR/RCAR receptors plays a fundamental role in controlling whole-plant stomatal conductance and affects stomatal closure in response to low air humidity, darkness, O3, and elevated CO2. Rapid stomatal closure induced by changes in the environment, such as elevation of CO2, reduction of air humidity, darkness, and pulses of the air pollutant ozone (O3), involves the SLOW ANION CHANNEL1 (SLAC1). SLAC1 is activated by OPEN STOMATA1 (OST1) and Ca2+-dependent protein kinases. OST1 activation is controlled through abscisic acid (ABA)-induced inhibition of type 2 protein phosphatases (PP2C) by PYRABACTIN RESISTANCE/REGULATORY COMPONENTS OF ABA RECEPTOR (PYR/RCAR) receptor proteins. To address the role of signaling through PYR/RCARs for whole-plant steady-state stomatal conductance and stomatal closure induced by environmental factors, we used a set of Arabidopsis (Arabidopsis thaliana) mutants defective in ABA metabolism/signaling. The stomatal conductance values varied severalfold among the studied mutants, indicating that basal ABA signaling through PYR/RCAR receptors plays a fundamental role in controlling whole-plant water loss through stomata. PYR/RCAR-dependent inhibition of PP2Cs was clearly required for rapid stomatal regulation in response to darkness, reduced air humidity, and O3. Furthermore, PYR/RCAR proteins seem to function in a dose-dependent manner, and there is a functional diversity among them. Although a rapid stomatal response to elevated CO2 was evident in all but slac1 and ost1 mutants, the bicarbonate-induced activation of S-type anion channels was reduced in the dominant active PP2C mutants abi1-1 and abi2-1. Further experiments with a wider range of CO2 concentrations and analyses of stomatal response kinetics suggested that the ABA signalosome partially affects the CO2-induced stomatal response. Thus, we show that PYR/RCAR receptors play an important role for the whole-plant stomatal adjustments and responses to low humidity, darkness, and O3 and are involved in responses to elevated CO2.

Differential Sodium and Potassium Transport Selectivities of the Rice OsHKT2;1 and OsHKT2;2 Transporters in Plant Cells

Na+ and K+ homeostasis are crucial for plant growth and development. Two HKT transporter/channel classes have been characterized that mediate either Na+ transport or Na+ and K+ transport when expressed in Xenopus laevis oocytes and yeast. However, the Na+/K+ selectivities of the K+-permeable HKT transporters have not yet been studied in plant cells. One study expressing 5′ untranslated region-modified HKT constructs in yeast has questioned the relevance of cation selectivities found in heterologous systems for selectivity predictions in plant cells. Therefore, here we analyze two highly homologous rice (Oryza sativa) HKT transporters in plant cells, OsHKT2;1 and OsHKT2;2, that show differential K+ permeabilities in heterologous systems. Upon stable expression in cultured tobacco (Nicotiana tabacum) Bright-Yellow 2 cells, OsHKT2;1 mediated Na+ uptake, but little Rb+ uptake, consistent with earlier studies and new findings presented here in oocytes. In contrast, OsHKT2;2 mediated Na+-K+ cotransport in plant cells such that extracellular K+ stimulated OsHKT2;2-mediated Na+ influx and vice versa. Furthermore, at millimolar Na+ concentrations, OsHKT2;2 mediated Na+ influx into plant cells without adding extracellular K+. This study shows that the Na+/K+ selectivities of these HKT transporters in plant cells coincide closely with the selectivities in oocytes and yeast. In addition, the presence of external K+ and Ca2+ down-regulated OsHKT2;1-mediated Na+ influx in two plant systems, Bright-Yellow 2 cells and intact rice roots, and also in Xenopus oocytes. Moreover, OsHKT transporter selectivities in plant cells are shown to depend on the imposed cationic conditions, supporting the model that HKT transporters are multi-ion pores.

Chemical genetics reveals negative regulation of abscisic acid signaling by a plant immune response pathway.

Coordinated regulation of protection mechanisms against environmental abiotic stress and pathogen attack is essential for plant adaptation and survival. Initial abiotic stress can interfere with disease-resistance signaling [1-6]. Conversely, initial plant immune signaling may interrupt subsequent abscisic acid (ABA) signal transduction [7, 8]. However, the processes involved in this crosstalk between these signaling networks have not been determined. By screening a 9600-compound chemical library, we identified a small molecule [5-(3,4-dichlorophenyl)furan-2-yl]-piperidine-1-ylmethanethione (DFPM) that rapidly downregulates ABA-dependent gene expression and also inhibits ABA-induced stomatal closure. Transcriptome analyses show that DFPM also stimulates expression of plant defense-related genes. Major early regulators of pathogen-resistance responses, including EDS1, PAD4, RAR1, and SGT1b, are required for DFPM-and notably also for Pseudomonas-interference with ABA signal transduction, whereas salicylic acid, EDS16, and NPR1 are not necessary. Although DFPM does not interfere with early ABA perception by PYR/RCAR receptors or ABA activation of SnRK2 kinases, it disrupts cytosolic Ca(2+) signaling and downstream anion channel activation in a PAD4-dependent manner. Our findings provide evidence that activation of EDS1/PAD4-dependent plant immune responses rapidly disrupts ABA signal transduction and that this occurs at the level of Ca(2+) signaling, illuminating how the initial biotic stress pathway interferes with ABA signaling.

AtHKT1;1 mediates nernstian sodium channel transport properties in Arabidopsis root stelar cells.

The Arabidopsis AtHKT1;1 protein was identified as a sodium (Na+) transporter by heterologous expression in Xenopus laevis oocytes and Saccharomyces cerevisiae. However, direct comparative in vivo electrophysiological analyses of a plant HKT transporter in wild-type and hkt loss-of-function mutants has not yet been reported and it has been recently argued that heterologous expression systems may alter properties of plant transporters, including HKT transporters. In this report, we analyze several key functions of AtHKT1;1-mediated ion currents in their native root stelar cells, including Na+ and K+ conductances, AtHKT1;1-mediated outward currents, and shifts in reversal potentials in the presence of defined intracellular and extracellular salt concentrations. Enhancer trap Arabidopsis plants with GFP-labeled root stelar cells were used to investigate AtHKT1;1-dependent ion transport properties using patch clamp electrophysiology in wild-type and athkt1;1 mutant plants. AtHKT1;1-dependent currents were carried by sodium ions and these currents were not observed in athkt1;1 mutant stelar cells. However, K+ currents in wild-type and athkt1;1 root stelar cell protoplasts were indistinguishable correlating with the Na+ over K+ selectivity of AtHKT1;1-mediated transport. Moreover, AtHKT1;1-mediated currents did not show a strong voltage dependence in vivo. Unexpectedly, removal of extracellular Na+ caused a reduction in AtHKT1;1-mediated outward currents in Columbia root stelar cells and Xenopus oocytes, indicating a role for external Na+ in regulation of AtHKT1;1 activity. Shifting the NaCl gradient in root stelar cells showed a Nernstian shift in the reversal potential providing biophysical evidence for the model that AtHKT1;1 mediates passive Na+ channel transport properties.

The BIG protein distinguishes the process of CO2‐induced stomatal closure from the inhibition of stomatal opening by CO2

Summary We conducted an infrared thermal imaging‐based genetic screen to identify Arabidopsis mutants displaying aberrant stomatal behavior in response to elevated concentrations of CO 2. This approach resulted in the isolation of a novel allele of the Arabidopsis BIG locus (At3g02260) that we have called CO 2 insensitive 1 (cis1). BIG mutants are compromised in elevated CO 2‐induced stomatal closure and bicarbonate activation of S‐type anion channel currents. In contrast with the wild‐type, they fail to exhibit reductions in stomatal density and index when grown in elevated CO 2. However, like the wild‐type, BIG mutants display inhibition of stomatal opening when exposed to elevated CO 2. BIG mutants also display wild‐type stomatal aperture responses to the closure‐inducing stimulus abscisic acid (ABA). Our results indicate that BIG is a signaling component involved in the elevated CO 2‐mediated control of stomatal development. In the control of stomatal aperture by CO 2, BIG is only required in elevated CO 2‐induced closure and not in the inhibition of stomatal opening by this environmental signal. These data show that, at the molecular level, the CO 2‐mediated inhibition of opening and promotion of stomatal closure signaling pathways are separable and BIG represents a distinguishing element in these two CO 2‐mediated responses.