ABA Signaling in Arabidopsis Stomatal Guard Cells
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ABA Signaling in Arabidopsis Stomatal Guard Cells

Plants have pores, Stomata, on their leaf surfaces that allow CO2 (Carbon Dioxide) in for photosynthesis and through which water evaporates. The specific cells that border and define these pores are Guard Cells. Guard Cells literally guard the size of the pore by alternately swelling, which opens the pore, or shrinking, which closes the pore. Plants must respond to a variety of environmental cues and regulate their Stomata accordingly so that enough CO2 gets in, but not so much water escapes that the plant dries out. This balance between opening and closure of Stomata is essential in determining a plant’s survival and how much it will grow and yield. The swelling and shrinking of Guard Cells occurs through the coordinated movement of water and Ions across the Guard Cell Membrane. Ions move through regulated proteinaceous pores in the membrane called Ion Channels. The activity of Ion Channels is regulated by many signals and proteins. These include environmental signals such as O3 (Ozone), Plant hormones such as ABA (Abscisic Acid), Ca2+cyt (Calcium Ion levels), GTP-binding proteins, Kinase and Phosphatase enzymes (Ref.1).

ABA is synthesized in almost all cells, but its transport from roots to shoots, and the recirculation of ABA in both Xylem and Phloem are important aspects of its physiological role. The most extensively investigated developmental and physiological effects of ABA are those involved in Seed Maturation and Dormancy and in the Regulation of Stomata. These diverse functions of ABA involve complex regulatory mechanisms that control its Production, Degradation, Signal Perception, and Transduction. Upon arrival at Guard cells, ABA can be perceived either Intra- or Extracellularly. ABA initiates its effects on cells by binding to a Receptor protein, the identity of which is unknown and the location of which is uncertain. Evidence suggests that extracellular perception prevents Stomatal Opening, while intracellular ABA can induce Stomatal Closure. Recently, evidence for Transmembrane Receptor has also been found. Activation of the putative Receptor causes a chain of events that results in rapid changes in Ion channels and slower changes in the pattern of gene transcription. Many individual components of this chain of events have been identified, but a complete picture has not been obtained yet. Since ABA is involved in responses to Stress, there is considerable overlap between the signaling processes induced by ABA and those altered directly by Stress (Ref.2).

A great number of components have been revealed to be involved in ABA signaling in the Guard cell. Early events in ABA signaling involve participation of GTP binding proteins, Phospholipases, Protein Kinases and Phosphatases. In the case of Heterotrimeric G-proteins, Arabidopsis thaliana genome encodes single prototypical G-alpha GPA1 (Guanine Nucleotide-binding Protein Alpha-1 subunit) and G-beta AGB1 (Guanine Nucleotide-binding Protein Beta subunit-1) subunits, and two probable G-gamma subunits AGG1 (Heterotrimeric G protein Gamma-subunit-1) and AGG2 (Guanine Nucleotide-binding Protein Beta subunit-2). Arabidopsis has only one or two isoforms of each G-protein subunit and loss of function for GPA1 gene disrupts aspects of ABA signaling. One Arabidopsis gene, GCR1 (Putative G-protein-Coupled Receptor-1), encodes a protein with significant sequence similarity to nonplant GPCRs (G-Protein Coupled Receptors) and a predicted 7-transmembrane domain structure characteristic of GPCRs. GCR1 binds to GPA1 and is believed to act as a negative regulator of GPA1-mediated ABA responses in Guard cells. Another major class of GTPase molecular switches in plants is a plant-specific branch of the RAS superfamily, the monomeric Rops (Rho/Rac-related GTPases from plants). The 11 Arabidopsis members of this subfamily, also known as Aracs or AtRacs represent four distinct groups. ABA-triggered inactivation of the Arabidopsis thaliana Rac1 (AtRac1) is essential for Stomatal closing and mediates cytoskeletal disruption induced by ABA. Inactivation of the AtRac1 GTPase is inhibited in the ABI1-1 (Abscissic Acid Insensitive-1) mutant, which also interferes with expression of ABA-induced genes. The AtRac1 protein is believed to interact with PAK (p21-Activated Kinase)-type Protein Kinases, although these have not yet been identified in plants. PAK activation is inhibited by pertussis toxin, linking it to G-protein signaling (Ref.3 & 4).

Secondary messengers in ABA signaling regulating Stomatal function and gene expression include IP3 (Inositol triphosphate) and PA (Phosphatidic Acid), produced by PLC (Phospholipase-C) and PLD-Alpha (Phospholipase-D-Alpha), respectively. Arabidopsis contains 6 PLC genes; ABA induces expression of only one of these, AtPLC1AtPLC1 is required, but not sufficient, for ABA effects on germination, growth and vegetative gene expression. In addition, the more highly phosphorylated Inositide IP6 contributes to ABA-inhibition of Stomatal Opening. Stimulation of PLD activity is mediated by G-protein activity following perception of ABA at the Plasmamembrane. Many Kinases representing multiple gene families have been implicated in ABA signaling affecting Stomatal regulation and/or Gene expression. ABA also regulates Ion channel activity in Guard cells through protein phosphorylation and dephosphorylation. Guard cells have multiple types of K+ (GORK (Guard cell Outward Rectifying K(+) channel), Kat1), Anion (Chloride Channels), and Ca2+ channels in the Plasma and Vacuolar membranes, many of which carry Ions in only one direction. A 48-kD AAPK (ABA-Activated Serine-Threonine Protein Kinase) interferes specifically with ABA-induced Stomatal Closure, but not Stomatal Opening, and it interferes with activation of slow Anion Channels by ABA. A Guard cell-specific Kinase of the same size phosphorylates a peptide from the COOH-terminus of the Kat1 inward-rectifying K+ Channel protein. A CDPK (Ca2+-Dependent Protein Kinase) has also phosphorylates the Kat1 K+ Channel protein. ABA induces oscillating increases in Cytosolic Ca2+ via: production of ROS (Reactive Oxygen Species) that contribute to opening of Plasmamembrane Ca2+ in channels-release from internal stores through three types of Ca2+ channels regulated by IP3, cADPR (cyclic ADP Ribose), and Ca2+ itself. The increased Ca2+ -inhibits Plasmamembrane H+ pumps -inhibits K+ in channels, and activates Cl- out (Anion) channels, resulting in depolarization of the membrane. Depolarization activates K+ out and further inhibits K+ in channels (Ref.5 and 6).

ABA induced PLD-mediated PA production inactivates K+ in channels. ABA causes an increase in cytosolic pH, which activates K+out channels and inhibits H+ pump activity by depleting the substrate.  ABA stimulates NO (Nitric Oxide) synthesis in Guard Cells, which induces Stomatal Closure in a cADPR and cGMP-dependent manner, indicating that NO is an even earlier secondary messenger in this response pathway. Yet another calcium-mobilizing molecule in plants, S1P (Sphingosine-1-Phosphate), was recently implicated in linking drought-induced ABA signaling to Stomatal Closure. Furthermore, increased external pH decreases K+ in channel activity and increases activation of a Guard cell localized K+ out channel. Increasing pH may also be a feedback mechanism for ABA desensitization via activation of ABI1, a negative regulator of ABA response. For short-term regulation of anion channel activity in guard cells, ABA-sensitive but Ca2+-independent signaling components such as the Protein Kinases Ost1 or AAPK, or the Protein Phosphatases ABI 1-2¸ represents good candidates. Ost1 acts in the interval between ABA perception and ROS production. ROS and NO (Nitric Oxide) collaborate to mediate ABA-induced stomata closure. NO synthesis and stomata closure in response to ABA are severely reduced in the NADPH oxidase double mutant AtrbohD AtrbohF, suggesting that endogenous H2O2 production elicited by ABA is required for NO synthesis. (Ref.6).

Several Protein Phosphatases have also been shown to affect ABA signaling. PP2Cs are likely to act as negative regulators of ABA response. PP2C (Protein Phosphatase-2C) family contains 69 members, 25 of which contain two conserved G residues correlated with ABA signaling. In addition to ABI1 and ABI2, two other family members (AtPP2C and AtPP2C-HA) have been shown to repress ABA response when over-expressed. The ABI PP2Cs and GCA2 (Guard Cell Associated Protein-2) all act upstream of the Ca2+ oscillations, with ABI2 and GCA2 apparently mediating response to H2O2, and ABI1 affecting ROS production. Inhibition of PP1/PP2A phosphatases by OKA (Okadaic Acid) also alters both ABA-induced gene expression and Stomatal closure. The effect of OKA on ABA response varies among species; in Arabidopsis, OKA partially inhibits ABA activation of S-type anion channels and Stomatal closure. In addition, the PP2B class of calcineurin-like Ca2+ binding proteins are potential Ca2+ sensors; one of these, AtCBL1, is induced by drought. Evidence is accumulating that membrane Vesicle trafficking and fusion are central to ABA signaling. The Syr1 (Syntaxin) gene product, a Syntaxin-family protein believed to function in Vesicle trafficking, mediates ABA-triggered changes in ion channel activity (Ref.7 & 8).

The signaling mechanisms involved in transcriptional and posttranscriptional regulation by ABA have not been explored as extensively as have the mechanisms of signaling to membrane channels in guard cells. In addition, several transcription factors have been identified whose loss confers an ABA-insensitive phenotype, giving rise to the ABI3 , ABI4, and ABI5 mutant alleles. One route of ABA and Stress signal transmission to the transcriptional machinery is through a MAPK (Mitogen-Activated Protein Kinase) cascade. MAPK cascades comprise an arrangement of three consecutive protein kinases, each of which activates the next enzyme by phosphorylation on two residues. These are, in reverse order of phosphorylation, a MAPK, a MAPKK (Mitogen-Activated Protein Kinase Kinase), and a MAPKKK (Mitogen-Activated Protein Kinase Kinase Kinase). In Arabidopsis ABA activation results in the activation of two MAPKs, AtMPK3 and AtMPK6 . These MAPKs receive signals from the ANP1 family of MAPKKKs (ANP1 , ANP2 , ANP3) and probably transmit the signal through AtMKK4 and AtMKK5.  The signaling components connecting the ABA-activated MAPK cascade to ABA reception have not yet been identified. However, there are several likely links. PAK is believed to be one of them. PAK protein downstream targets include a subunit of the NADPH Oxidase and MAPK cascade members. Hence, PAKs may be MAPKKKKs and represent the links between G protein signaling, NADPH oxidase and Kinase cascades, as well. In addition to the MAPK cascade, CDPK is also implicated in transmitting the ABA signal to the transcriptional machinery. ABA stimulates ABI5 transcription factor activation of ABA-inducible promoters. Co-expression of ABI5 and ABI3 transcription factors, which interact physically, synergistically activated several promoters in the presence of ABA. The ABI5 transcription factor is subject to posttranslational regulation by Proteasome-dependent proteolysis. An RNA binding protein with specificity for dsRNA (double-stranded RNA) is encoded by the Arabidopsis HYL1 gene. The HYL1 mutant is hypersensitive to ABA in seed germination and root growth (Ref.5, 9 and 10).

Although much information has been gathered about ABA signaling, still the view of the relevant pathways is fragmented. Some of the remaining unknowns include the identities of the Receptors, the substrates of the various Kinases and Phosphatases, which of the known ABA response loci interact directly or indirectly, and the identities of additional signaling elements linking the known elements into complete pathways or networks. However, the genes cloned to date can be used for construction of transgenic plants with conditionally altered hormone synthesis or response. Given the efficacy of ABA in regulating such basic processes as seed development, dormancy vs. germination, transpiration and stress responses, these could have important biotechnological applications (Ref.11).