Hanna Hõrak

The Breakdown of Stored Triacylglycerols Is Required during Light-Induced Stomatal Opening

Summary Stomata regulate the uptake of CO2 and the loss of water vapor [1] and contribute to the control of water-use efficiency [2] in plants. Although the guard-cell-signaling pathway coupling blue light perception to ion channel activity is relatively well understood [3], we know less about the sources of ATP required to drive K+ uptake [3, 4, 5, 6]. Here, we show that triacylglycerols (TAGs), present in Arabidopsis guard cells as lipid droplets (LDs), are involved in light-induced stomatal opening. Illumination induces reductions in LD abundance, and this involves the PHOT1 and PHOT2 blue light receptors [3]. Light also induces decreases in specific TAG molecular species. We hypothesized that TAG-derived fatty acids are metabolized by peroxisomal β-oxidation to produce ATP required for stomatal opening. In silico analysis revealed that guard cells express all the genes required for β-oxidation, and we showed that light-induced stomatal opening is delayed in three TAG catabolism mutants (sdp1, pxa1, and cgi-58) and in stomata treated with a TAG breakdown inhibitor. We reasoned that, if ATP supply was delaying light-induced stomatal opening, then the activity of the plasma membrane H+-ATPase should be reduced at this time. Monitoring changes in apoplastic pH in the mutants showed that this was the case. Together, our results reveal a new role for TAGs in vegetative tissue and show that PHOT1 and PHOT2 are involved in reductions in LD abundance. Reductions in LD abundance in guard cells of the lycophyte Selaginella suggest that TAG breakdown may represent an evolutionarily conserved mechanism in light-induced stomatal opening.

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.

Abscisic Acid Transport and Homeostasis in the Context of Stomatal Regulation

The discovery of cytosolic ABA receptors is an important breakthrough in stomatal research; signaling via these receptors is involved in determining the basal stomatal conductance and stomatal responsiveness. However, the source of ABA in guard cells is still not fully understood. The level of ABA increases in guard cells by de novo synthesis, recycling from inactive conjugates via β-glucosidases BG1 and BG2 and by import, whereas it decreases by hydroxylation, conjugation, and export. ABA importers include the NRT1/PTR family protein AIT1, ATP-binding cassette protein ABCG40, and possibly ABCG22, whereas the DTX family member DTX50 and ABCG25 function as ABA exporters. Here, we review the proteins involved in ABA transport and homeostasis and their physiological role in stomatal regulation. Recent experiments suggest that functional redundancy probably exists among ABA transporters between vasculature and guard cells and ABA recycling proteins, as stomatal functioning remained intact in abcg22, abcg25, abcg40, ait1, and bg1bg2 mutants. Only the initial response to reduced air humidity was significantly delayed in abcg22. Considering the reports showing autonomous ABA synthesis in guard cells, we discuss that rapid stomatal responses to atmospheric factors might depend primarily on guard cell-synthesized ABA, whereas in the case of long-term soil water deficit, ABA synthesized in the vasculature might have a significant role.

ERD15—An attenuator of plant ABA responses and stomatal aperture

Plants are continuously challenged by abiotic and biotic stress factors and need to mount appropriate responses to ensure optimal growth and survival. We have identified ERD15 as a central component in several stress responses in Arabidopsis thaliana. Comparative genomics demonstrates that ERD15 is a member of a small but highly conserved protein family ubiquitous but specific to the plant kingdom. The origin of ERD15 family of proteins can be traced to the time of emergence of land plants. The presence of the conserved PAM2 motif in ERD15 proteins is indicative of a possible interaction with poly(A) binding proteins and could suggest a role in posttranscriptional regulation of gene expression. The function of the other highly conserved motifs in ERD15 remains to be elucidated. The biological role of all ERD15 family members studied so far appears associated to stress responses and stress adaptation. Studies in Arabidopsis demonstrate a role in abiotic stress tolerance where ERD15 is a negative regulator of ABA signaling. The role in ABA signaling may also explain how ERD15 regulates stomatal aperture and consequently controls plant water relations.

Fern stomatal responses to ABA and CO2 depend on species and growth conditions

Growth conditions and species affect fern stomatal responsiveness to ABA and CO2. Changing atmospheric CO2 levels, climate, and air humidity affect plant gas exchange that is controlled by stomata, small pores on plant leaves and stems formed by guard cells. Evolution has shaped the morphology and regulatory mechanisms governing stomatal movements to correspond to the needs of various land plant groups over the past 400 million years. Stomata close in response to the plant hormone abscisic acid (ABA), elevated CO2 concentration, and reduced air humidity. Whether the active regulatory mechanisms that control stomatal closure in response to these stimuli are present already in mosses, the oldest plant group with stomata, or were acquired more recently in angiosperms remains controversial. It has been suggested that the stomata of the basal vascular plants, such as ferns and lycophytes, close solely hydropassively. On the other hand, active stomatal closure in response to ABA and CO2 was found in several moss, lycophyte, and fern species. Here, we show that the stomata of two temperate fern species respond to ABA and CO2 and that an active mechanism of stomatal regulation in response to reduced air humidity is present in some ferns. Importantly, fern stomatal responses depend on growth conditions. The data indicate that the stomatal behavior of ferns is more complex than anticipated before, and active stomatal regulation is present in some ferns and has possibly been lost in others. Further analysis that takes into account fern species, life history, evolutionary age, and growth conditions is required to gain insight into the evolution of land plant stomatal responses.

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.

Quantitative trait loci mapping and transcriptome analysis reveal candidate genes regulating the response to ozone in Arabidopsis thaliana

As multifaceted molecules, reactive oxygen species (ROS) are known to accumulate in response to various stresses. Ozone (O3 ) is an air pollutant with detrimental effect on plants and O3 can also be used as a tool to study the role of ROS in signalling. Genetic variation of O3 sensitivity in different Arabidopsis accessions highlights the complex genetic architecture of plant responses to ROS. To investigate the genetic basis of O3 sensitivity, a recombinant inbred line (RIL) population between two Arabidopsis accessions with distinct O3 sensitivity, C24 (O3 tolerant) and Te (O3 sensitive) was used for quantitative trait loci (QTL) mapping. Through analysis of QTL mapping combined with transcriptome changes in response to O3 , we identified three causal QTLs and several potential candidate genes regulating the response to O3 . Based on gene expression data, water loss and stomatal conductance measurement, we found that a combination of relatively low stomatal conductance and constitutive activation of salicylic acid (SA)-mediated defence signalling were responsible for the O3 tolerance in C24. Application of exogenous SA prior to O3 exposure can mimic the constitutive SA signalling in C24 and could attenuate O3 -induced leaf damage in the sensitive Arabidopsis accessions Te and Cvi-0.

Natural Variation in Arabidopsis Cvi-0 Accession Uncovers Regulation of Guard Cell CO2 Signaling by MPK12

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 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 HT1, a central node in guard cell CO2 signaling, and that MPK12 can function 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.

The receptor-like pseudokinase GHR1 is required for stomatal closure

GHR1 is a receptor-like pseudokinase that activates SLOW ANION CHANNEL1 and acts in stomatal closure through its scaffolding functions rather than by directly phosphorylating its target proteins. Guard cells control the aperture of stomatal pores to balance photosynthetic carbon dioxide uptake with evaporative water loss. Stomatal closure is triggered by several stimuli that initiate complex signaling networks to govern the activity of ion channels. Activation of SLOW ANION CHANNEL1 (SLAC1) is central to the process of stomatal closure and requires the leucine-rich repeat receptor-like kinase (LRR-RLK) GUARD CELL HYDROGEN PEROXIDE-RESISTANT1 (GHR1), among other signaling components. Here, based on functional analysis of nine Arabidopsis thaliana ghr1 mutant alleles identified in two independent forward-genetic ozone-sensitivity screens, we found that GHR1 is required for stomatal responses to apoplastic reactive oxygen species, abscisic acid, high CO2 concentrations, and diurnal light/dark transitions. Furthermore, we show that the amino acid residues of GHR1 involved in ATP binding are not required for stomatal closure in Arabidopsis or the activation of SLAC1 anion currents in Xenopus laevis oocytes and present supporting in silico and in vitro evidence suggesting that GHR1 is an inactive pseudokinase. Biochemical analyses suggested that GHR1-mediated activation of SLAC1 occurs via interacting proteins and that CALCIUM-DEPENDENT PROTEIN KINASE3 interacts with GHR1. We propose that GHR1 acts in stomatal closure as a scaffolding component.