Ebe Merilo

Arabidopsis PYR/PYL/RCAR Receptors Play a Major Role in Quantitative Regulation of Stomatal Aperture and Transcriptional Response to Abscisic Acid

A mutant lacking six abscisic acid (ABA) receptors and ABA-mediated activation of SnRK2.2/2.3/2.6 kinases shows an extreme ABA-insensitive phenotype, even though other branches for ABA perception remain functional. ABA perception through PYR/PYL/RCAR receptors plays a major role in regulating seed germination and establishment, vegetative and reproductive growth, stomatal aperture, and transcriptional response to ABA. Abscisic acid (ABA) is a key hormone for plant growth, development, and stress adaptation. Perception of ABA through four types of receptors has been reported. We show here that impairment of ABA perception through the PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR) branch reduces vegetative growth and seed production and leads to a severe open stomata and ABA-insensitive phenotype, even though other branches for ABA perception remain functional. An Arabidopsis thaliana sextuple mutant impaired in six PYR/PYL receptors, namely PYR1, PYL1, PYL2, PYL4, PYL5, and PYL8, was able to germinate and grow even on 100 μM ABA. Whole-rosette stomatal conductance (Gst) measurements revealed that leaf transpiration in the sextuple pyr/pyl mutant was higher than in the ABA-deficient aba3-1 or ABA-insensitive snrk2.6 mutants. The gradually increasing Gst values of plants lacking three, four, five, and six PYR/PYLs indicate quantitative regulation of stomatal aperture by this family of receptors. The sextuple mutant lacked ABA-mediated activation of SnRK2s, and ABA-responsive gene expression was dramatically impaired as was reported in snrk2.2/2.3/2.6. In summary, these results show that ABA perception by PYR/PYLs plays a major role in regulation of seed germination and establishment, basal ABA signaling required for vegetative and reproductive growth, stomatal aperture, and transcriptional response to the hormone.

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.

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.

The PYL4 A194T mutant uncovers a key role of PYR1-LIKE4/PROTEIN PHOSPHATASE 2CA interaction for abscisic acid signaling and plant drought resistance.

Enhanced drought resistance through mutagenesis of an ABA receptor is associated with enhanced interaction with its protein phosphatase binding partner. Because abscisic acid (ABA) is recognized as the critical hormonal regulator of plant stress physiology, elucidating its signaling pathway has raised promise for application in agriculture, for instance through genetic engineering of ABA receptors. PYRABACTIN RESISTANCE1/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS ABA receptors interact with high affinity and inhibit clade A phosphatases type-2C (PP2Cs) in an ABA-dependent manner. We generated an allele library composed of 10,000 mutant clones of Arabidopsis (Arabidopsis thaliana) PYL4 and selected mutations that promoted ABA-independent interaction with PP2CA/ABA-HYPERSENSITIVE3. In vitro protein-protein interaction assays and size exclusion chromatography confirmed that PYL4A194T was able to form stable complexes with PP2CA in the absence of ABA, in contrast to PYL4. This interaction did not lead to significant inhibition of PP2CA in the absence of ABA; however, it improved ABA-dependent inhibition of PP2CA. As a result, 35S:PYL4A194T plants showed enhanced sensitivity to ABA-mediated inhibition of germination and seedling establishment compared with 35S:PYL4 plants. Additionally, at basal endogenous ABA levels, whole-rosette gas exchange measurements revealed reduced stomatal conductance and enhanced water use efficiency compared with nontransformed or 35S:PYL4 plants and partial up-regulation of two ABA-responsive genes. Finally, 35S:PYL4A194T plants showed enhanced drought and dehydration resistance compared with nontransformed or 35S:PYL4 plants. Thus, we describe a novel approach to enhance plant drought resistance through allele library generation and engineering of a PYL4 mutation that enhances interaction with PP2CA.

Natural variation in ozone sensitivity among Arabidopsis thaliana accessions and its relation to stomatal conductance

Genetic variation between naturally occurring populations provides a unique source to unravel the complex mechanisms of stress tolerance. Here, we have analysed O(3) sensitivity of 93 natural Arabidopsis thaliana accessions together with five O(3)-sensitive mutants to acute O(3) exposure. The variation in O(3) sensitivity among the natural accessions was much higher than among the O(3)-sensitive mutants and corresponding wild types. A subset of nine accessions with major variation in their O(3) responses was studied in more detail. Among the traits assayed, stomatal conductance (g(st)) was an important factor determining O(3) sensitivity of the selected accessions. The most O(3)-sensitive accession, Cvi-0, had constitutively high g(st), leading to high initial O(3) uptake rate and dose received during the first 30 min of exposure. Analyzing O(3)-induced changes in stress hormone concentrations indicated that jasmonate (JA) concentration was also positively correlated with leaf damage. Quantitative trait loci (QTL) mapping in a Col-0 x Cvi-0 recombinant inbred line (RIL) population identified three QTLs for O(3) sensitivity, and one for high water loss of Cvi-0. The major O(3) QTL mapped to the same position as the water loss QTL further supporting the role of stomata in regulating O(3) entry and damage.

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.

The role of ABA recycling and transporter proteins in rapid stomatal responses to reduced air humidity, elevated CO2 and exogenous ABA

Guard cell ABA signaling is central for the regulation of plant stomatal conductance (gs) and responses to environmental factors (Merilo et al., 2013); however, the source of ABA is still debatable. Generally, ABA levels are regulated by (1) biosynthesis: de novo and recycling from the inactive conjugates by β-glucosidases BG1 and BG2; (2) catabolism: irreversible hydroxylation and reversible conjugation to ABA glucosyl ester (ABA-GE); and (3) transport from elsewhere in the plant. Here, we report that the stomatal responses to elevated CO2 concentration, reduced air humidity, and exogenous ABA in different mutants of ABA transport and recycling were generally intact except in abcg22 mutant.

To open or to close: species‐specific stomatal responses to simultaneously applied opposing environmental factors

Plant stomatal responses to single environmental factors are well studied; however, responses to a change in two (or more) factors - a common situation in nature - have been less frequently addressed. We studied the stomatal responses to a simultaneous application of opposing environmental factors in six evolutionarily distant mono- and dicotyledonous herbs representing different life strategies (ruderals, competitors and stress-tolerators) to clarify whether the crosstalk between opening- and closure-inducing pathways leading to stomatal response is universal or species-specific. Custom-made gas exchange devices were used to study the stomatal responses to a simultaneous application of two opposing factors: decreased/increased CO2 concentration and light availability or reduced air humidity. The studied species responded similarly to changes in single environmental factors, but showed species-specific and nonadditive responses to two simultaneously applied opposing factors. The stomata of the ruderals Arabidopsis thaliana and Thellungiella salsuginea (previously Thellungiella halophila) always opened, whereas those of competitor-ruderals either closed in all two-factor combinations (Triticum aestivum), remained relatively unchanged (Nicotiana tabacum) or showed a response dominated by reduced air humidity (Hordeum vulgare). Our results, indicating that in changing environmental conditions species-specific stomatal responses are evident that cannot be predicted from studying one factor at a time, might be interesting for stomatal modellers, too.

Mutations in the SLAC1 anion channel slow stomatal opening and severely reduce K+ uptake channel activity via enhanced cytosolic [Ca2+] and increased Ca2+ sensitivity of K+ uptake channels

The Arabidopsis guard cell anion channel SLAC1 is essential for stomatal closure in response to various endogenous and environmental stimuli. Interestingly, here we reveal an unexpected impairment of slac1 alleles on stomatal opening. We report that mutations in SLAC1 unexpectedly slow stomatal opening induced by light, low CO(2) and elevated air humidity in intact plants and that this is caused by the severely reduced activity of inward K(+) (K(+)(in)) channels in slac1 guard cells. Expression of channels and transporters involved in stomatal opening showed small but significant reductions in transcript levels in slac1 guard cells; however, this was deemed insufficient to explain the severely impaired K(+)(in) channel activity in slac1. We further examined resting cytosolic Ca(2+) concentration ([Ca(2+)](cyt)) and K(+)(in) channel sensitivity to [Ca(2+)](cyt) in slac1. These experiments showed higher resting [Ca(2+)](cyt) in slac1 guard cells and that reducing [Ca(2+)](cyt) to < 10 nM rapidly restored the activity of K(+)(in) channels in slac1 closer to wild-type levels. These findings demonstrate an unanticipated compensatory feedback control in plant stomatal regulation, which counteracts the impaired stomatal closing response of slac1, by down-regulating stomatal opening mechanisms and implicates enhanced [Ca(2+)](cyt) sensitivity priming as a mechanistic basis for the down-regulated K(+)(in) channel activity.

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

MPK4 and MPK12 inhibit the activity of protein kinase HT1, which in turn controls SLAC1 anion channel activation by OST1 and GHR1 in stomatal CO2 signaling. 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.