Juntaro Negi

CO2 regulator SLAC1 and its homologues are essential for anion homeostasis in plant cells

The continuing rise in atmospheric [CO2] is predicted to have diverse and dramatic effects on the productivity of agriculture, plant ecosystems and gas exchange. Stomatal pores in the epidermis provide gates for the exchange of CO2 and water between plants and the atmosphere, processes vital to plant life. Increased [CO2] has been shown to enhance anion channel activity proposed to mediate efflux of osmoregulatory anions (Cl– and malate2–) from guard cells during stomatal closure. However, the genes encoding anion efflux channels in plant plasma membranes remain unknown. Here we report the isolation of an Arabidopsis gene, SLAC1 (SLOW ANION CHANNEL-ASSOCIATED 1, At1g12480), which mediates CO2 sensitivity in regulation of plant gas exchange. The SLAC1 protein is a distant homologue of bacterial and fungal C4-dicarboxylate transporters, and is localized specifically to the plasma membrane of guard cells. It belongs to a protein family that in Arabidopsis consists of four structurally related members that are common in their plasma membrane localization, but show distinct tissue-specific expression patterns. The loss-of-function mutation in SLAC1 was accompanied by an over-accumulation of the osmoregulatory anions in guard cell protoplasts. Guard-cell-specific expression of SLAC1 or its family members resulted in restoration of the wild-type stomatal responses, including CO2 sensitivity, and also in the dissipation of the over-accumulated anions. These results suggest that SLAC1-family proteins have an evolutionarily conserved function that is required for the maintenance of organic/inorganic anion homeostasis on the cellular level.

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.

Arabidopsis HT1 kinase controls stomatal movements in response to CO2.

Guard cells, which form stomata in leaf epidermes, sense a multitude of environmental signals and integrate this information to regulate stomatal movements. Compared with the advanced understanding of light and water stress responses in guard cells, the molecular mechanisms that underlie stomatal CO2 signalling have remained relatively obscure. With a high-throughput leaf thermal imaging CO2 screen, we report the isolation of two allelic Arabidopsis mutants (high leaf temperature 1; ht1-1 and ht1-2) that are altered in their ability to control stomatal movements in response to CO2. The strong allele, ht1-2, exhibits a markedly impaired CO2 response but shows functional responses to blue light, fusicoccin and abscisic acid (ABA), indicating a role for HT1 in stomatal CO2 signalling. HT1 encodes a protein kinase that is expressed mainly in guard cells. Phosphorylation assays demonstrate that the activity of the HT1 protein carrying the ht1-1 or ht1-2 mutation is greatly impaired or abolished, respectively. Furthermore, dominant-negative HT1(K113W) transgenic plants, which lack HT1 kinase activity, show a disrupted CO2 response. These findings indicate that the HT1 kinase is important for regulation of stomatal movements and its function is more pronounced in response to CO2 than it is to ABA or light.

CO2 Sensing and CO2 Regulation of Stomatal Conductance: Advances and Open Questions

Guard cells form epidermal stomatal gas-exchange valves in plants and regulate the aperture of stomatal pores in response to changes in the carbon dioxide (CO2) concentration ([CO2]) in leaves. Moreover, the development of stomata is repressed by elevated CO2 in diverse plant species. Evidence suggests that plants can sense [CO2] changes via guard cells and via mesophyll tissues in mediating stomatal movements. We review new discoveries and open questions on mechanisms mediating CO2-regulated stomatal movements and CO2 modulation of stomatal development, which together function in the CO2 regulation of stomatal conductance and gas exchange in plants. Research in this area is timely in light of the necessity of selecting and developing crop cultivars that perform better in a shifting climate.

A dof transcription factor, SCAP1, is essential for the development of functional stomata in arabidopsis

Stomata are highly specialized organs that consist of pairs of guard cells and regulate gas and water vapor exchange in plants [1-3]. Although early stages of guard cell differentiation have been described [4-10] and were interpreted in analogy to processes of cell type differentiation in animals [11], the downstream development of functional stomatal guard cells remains poorly understood. We have isolated an Arabidopsis mutant, stomatal carpenter 1 (scap1), that develops irregularly shaped guard cells and lacks the ability to control stomatal aperture, including CO2-induced stomatal closing and light-induced stomatal opening. SCAP1 was identified as a plant-specific Dof-type transcription factor expressed in maturing guard cells, but not in guard mother cells. SCAP1 regulates the expression of genes encoding key elements of stomatal functioning and morphogenesis, such as K(+) channel protein, MYB60 transcription factor, and pectin methylesterase. Consequently, ion homeostasis was disturbed in scap1 guard cells, and esterification of extracellular pectins was impaired so that the cell walls lining the pores did not mature normally. We conclude that SCAP1 regulates essential processes of stomatal guard cell maturation and functions as a key transcription factor regulating the final stages of guard cell differentiation.

A Munc13-like protein in Arabidopsis mediates H+-ATPase translocation that is essential for stomatal responses

Plants control CO2 uptake and water loss by modulating the aperture of stomata located in the epidermis. Stomatal opening is initiated by the activation of H+-ATPases in the guard-cell plasma membrane. In contrast to regulation of H+-ATPase activity, little is known about the translocation of the guard cell H+-ATPase to the plasma membrane. Here we describe the isolation of an Arabidopsis gene, PATROL1, that controls the translocation of a major H+-ATPase, AHA1, to the plasma membrane. PATROL1 encodes a protein with a MUN domain, known to mediate synaptic priming in neuronal exocytosis in animals. Environmental stimuli change the localization of plasma membrane-associated PATROL1 to an intracellular compartment. Plasma membrane localization of AHA1 and stomatal opening require the association of PATROL1 with AHA1. Increased stomatal opening responses in plants overexpressing PATROL1 enhance the CO2 assimilation rate, promoting plant growth.

New approaches to the biology of stomatal guard cells

CO2 acts as an environmental signal that regulates stomatal movements. High CO2 concentrations reduce stomatal aperture, whereas low concentrations trigger stomatal opening. In contrast to our advanced understanding of light and drought stress responses in guard cells, the molecular mechanisms underlying stomatal CO2 sensing and signaling are largely unknown. Leaf temperature provides a convenient indicator of transpiration, and can be used to detect mutants with altered stomatal control. To identify genes that function in CO2 responses in guard cells, CO2-insensitive mutants were isolated through high-throughput leaf thermal imaging. The isolated mutants are categorized into three groups according to their phenotypes: (i) impaired in stomatal opening under low CO2 concentrations; (ii) impaired in stomatal closing under high CO2 concentrations; and (iii) impaired in stomatal development. Characterization of these mutants has begun to yield insights into the mechanisms of stomatal CO2 responses. In this review, we summarize the current status of the field and discuss future prospects.

Environmental regulation of stomatal response in the Arabidopsis Cvi-0 ecotype

The Arabidopsis Cape Verde Islands (Cvi-0) ecotype is known to differ from other ecotypes with respect to environmental stress responses. We analyzed the stomatal behavior of Cvi-0 plants, in response to environmental signals. We investigated the responses of stomatal conductance and aperture to high [CO2] in the Cvi-0 and Col-0 ecotypes. Cvi-0 showed constitutively higher stomatal conductance and more stomatal opening than Col-0. Cvi-0 stomata opened in response to light, but the response was slow. Under low humidity, stomatal opening was increased in Cvi-0 compared to Col-0. We then assessed whether low humidity affects endogenous ABA levels in Cvi-0. In response to low humidity, Cvi-0 had much higher ABA levels than Col-0. However, epidermal peels experiments showed that Cvi-0 stomata were insensitive to ABA. Measurements of organic and inorganic ions in Cvi-0 guard cell protoplasts indicated an over-accumulation of osmoregulatory anions (malate and Cl−). This irregular anion homeostasis in the guard cells may explain the constitutive stomatal opening phenotypes of the Cvi-0 ecotype, which lacks high [CO2]-induced and low humidity-induced stomatal closure.

The Transmembrane Region of Guard Cell SLAC1 Channels Perceives CO2 Signals via an ABA-Independent Pathway in Arabidopsis.

A transmembrane region of the guard cell SLAC1 anion channel contains sites of CO2 signal perception, which could be different from the sites of ABA signal perception. The guard cell S-type anion channel, SLOW ANION CHANNEL1 (SLAC1), a key component in the control of stomatal movements, is activated in response to CO2 and abscisic acid (ABA). Several amino acids existing in the N-terminal region of SLAC1 are involved in regulating its activity via phosphorylation in the ABA response. However, little is known about sites involved in CO2 signal perception. To dissect sites that are necessary for the stomatal CO2 response, we performed slac1 complementation experiments using transgenic plants expressing truncated SLAC1 proteins. Measurements of gas exchange and stomatal apertures in the truncated transgenic lines in response to CO2 and ABA revealed that sites involved in the stomatal CO2 response exist in the transmembrane region and do not require the SLAC1 N and C termini. CO2 and ABA regulation of S-type anion channel activity in guard cells of the transgenic lines confirmed these results. In vivo site-directed mutagenesis experiments targeted to amino acids within the transmembrane region of SLAC1 raise the possibility that two tyrosine residues exposed on the membrane are involved in the stomatal CO2 response.

Dominant and recessive mutations in the Raf-like kinase HT1 gene completely disrupt stomatal responses to CO2 in Arabidopsis

Highlight Loss-of-function and gain-of-function ht1 Arabidopsis mutants have completely disrupted CO2 responses due to reduced and enhanced kinase activities, respectively.