Roles of the different components of magnesium chelatase in abscisic acid signal transduction

The H subunit of Mg-chelatase (CHLH) was shown to regulate abscisic acid (ABA) signaling and the I subunit (CHLI) was also reported to modulate ABA signaling in guard cells. However, it remains essentially unknown whether and how the Mg-chelatase-catalyzed Mg-protoporphyrin IX-production differs from ABA signaling. Using a newly-developed surface plasmon resonance system, we showed that ABA binds to CHLH, but not to the other Mg-chelatase components/subunits CHLI, CHLD (D subunit) and GUN4. A new rtl1 mutant allele of the CHLH gene in Arabidopsis thaliana showed ABA-insensitive phenotypes in both stomatal movement and seed germination. Upregulation of CHLI1 resulted in ABA hypersensitivity in seed germination, while downregulation of CHLI conferred ABA insensitivity in stomatal response in Arabidopsis. We showed that CHLH and CHLI, but not CHLD, regulate stomatal sensitivity to ABA in tobacco (Nicotiana benthamiana). The overexpression lines of the CHLD gene showed wild-type ABA sensitivity in Arabidopsis. Both the GUN4-RNA interference and overexpression lines of Arabidopsis showed wild-type phenotypes in the major ABA responses. These findings provide clear evidence that the Mg-chelatase-catalyzed Mg-ProtoIX production is distinct from ABA signaling, giving information to understand the mechanism by which the two cellular processes differs at the molecular level.     

Methyl jasmonate signaling and signal crosstalk between methyl jasmonate and abscisic acid in guard cells

Plants tightly control stomatal aperture in response to various environmental changes. A drought-inducible phytohormone, abscisic acid (ABA), triggers stomatal closure and ABA signaling pathway in guard cells has been well studied. Similar to ABA, methyl jasmonate (MeJA) induces stomatal closure in various plant species but MeJA signaling pathway is still far from clear. Recently we found that Arabidopsis calcium dependent protein kinase CPK6 functions as a positive regulator in guard cell MeJA signaling and provided new insights into cytosolic Ca²⁺-dependent MeJA signaling. Here we discuss the MeJA signaling and also signal crosstalk between MeJA and ABA pathways in guard cells.Key words: methyl jasmonate, abscisic acid, guard cell, reactive oxygen species, nitric oxide, calciumStomata, which are formed by pairs of specialized cells called guard cells, control gas exchanges and transpirational water loss. Guard cells can shrink and swell in response to various physiological stimuli, resulting in stomatal closing and opening.^1,2 To optimize growth under various environmental conditions, plants have developed fine-tuned signal pathway in guard cells. Abscisic acid (ABA) is synthesized under drought stress and induces stomatal closure to reduce transpirational water loss.² ABA signal transduction in guard cells has been widely studied. ABA induces increases of various second messengers such as cytosolic Ca²⁺, reactive oxygen species (ROS) and nitric oxide (NO) in guard cells. These early signal components finally evoke ion efflux through plasma membrane ion channels, resulting in reduction of guard cell turgor pressure.Jasmonates are plant hormones synthesized via the octadecanoid pathway and regulate various physiological processes in plants such as pollen maturation, tendril coiling, senescence and responses to wounding and pathogen attacks.³ Similar to ABA, jasmonates also trigger stomatal closure and the response is conserved among various plant species including Arabidopsis thaliana,⁴ Hordeum vulgare,⁵ Commelina benghalensis,⁶ Vicia faba,⁷ Nicotiana glauca,⁸ Paphiopedilum Supersuk⁹ and Paphiopedilum tonsum.⁹ A volatile methyl ester of jasmonic acid (JA), methy jasmonate (MeJA), has been widely used for studying jasmonate signaling pathway. To date, pharmacological and reverse genetic approaches have revealed many important signal components involved in MeJA-induced stomatal closure and suggest a signal crosstalk between MeJA and ABA in guard cells. In this review, we mainly focus on the three important second messengers, ROS, NO and cytosolic Ca²⁺ and discuss recent advance about MeJA signaling and signal interaction between MeJA and ABA in guard cells.     

The control of specificity in guard cell signal transduction

Stomatal guard cells have proven to be an attractive system for dissecting the mechanisms of stimulus-response coupling in plants. In this review we focus on the intracellular signal transduction pathways by which extracellular signals bring about closure and opening of the stomatal pore. It is proposed that guard cell signal transduction pathways may be organized into functional arrays or signalling cassettes that contain elements common to a number of converging signalling pathways. The purpose of these signalling cassettes may be to funnel extracellular signals down onto the ion transporters that control the fluxes of ions that underlie stomatal movements. Evidence is emerging that specificity in guard cell signalling may be, in part, encoded in complex spatio-temporal patterns of increases in the concentration of cytosolic-free calcium ([Ca²⁺]_cyt). It is suggested that oscillations in [Ca²⁺]_cyt may generate calcium signatures that encode information concerning the stimulus type and strength. New evidence is presented that suggests that these calcium signatures may integrate information when many stimuli are present.     

Pathways of the abscisic acid hormonal signal transduction across the plant cell plasma membrane

Signal transduction pathways of a phytohormone abscisic acid (ABA) in the plant cell plasma membrane are reviewed. The mechanisms of ABA perception; ABA-induced alterations of Ca2+-, K+-, and anion channel activity, the ABA effects on intracellular pH and membrane-bound enzyme activity, as well as the phytohormone interaction with the lipid phase of plasma membrane are considered, and the role of the membrane protein phosphorylation is discussed. The available data suggest that the regulatory signal of ABA can be transmitted via a ramified chain of reactions. In different tissues, functioning of various branches of this chain can be independent or interrelated.     

Convergence of the abscisic acid, CO2, and extracellular calcium signal transduction pathways in stomatal guard cells.

We show that guard cells from Arabidopsis thaliana plants carrying the abscisic acid-insensitive mutations abi1 and abi2 fail to respond to CO2 and extracellular calcium. This demonstrates that the signal transduction pathways for all three stimuli converge on, or close to, the ABI1 and ABI2 gene products.     

Calcium ions as second messengers in guard cell signal transduction

Ca²⁺ is a ubiquitous second messenger in plant cell signalling. In this review we consider the role of Ca²⁺-based signal transduction in stomatal guard cells focusing on three important areas: (1) the regulation of guard cell turgor relations and the control of gene expression in guard cells, (2) the control of specificity in Ca²⁺ signalling, (3) emerging technologies and new approaches for studying intracellular signalling. Stomatal apertures alter in response to a wide array of environmental stimuli as a result of changes in guard cell turgor. For example, the plant hormone abscisic acid (ABA) stimulates a reduction in stomatal aperture through a decrease in guard cell turgor. Furthermore, guard cells have been shown to be competent to relay an ABA signal from its site of perception to the nucleus. An increase in the concentration of cytosolic free Ca²⁺ ([Ca²⁺]₁) is central to the mechanisms underlying ABA-induced changes in guard cell turgor. We describe a possible model of Ca²⁺-based ABA signal transduction during stomatal closure and discuss recent evidence which suggests that Ca²⁺ is also involved in ABA nuclear signal transduction. Many other environmental stimuli which affect stomatal apertures, in addition to ABA, induce an increase in guard cell [Ca²⁺]₁) This raises questions regarding how increases in [Ca²⁺]₁) can be a common component in the signal transduction pathways by which stimuli cause both stomatal opening and closure. We discuss several mechanisms of increasing the amount of information contained within the Ca²⁺ signal, including encoding information in a stimulus-specific Ca²⁺ signal or Ca²⁺ signature', the concept of the ‘physiological address’ of the cell, and the use of other second messengers. We conclude by addressing the emerging technologies and new approaches which can be used in conjunction with guard cells to dissect further the molecular mechanisms of Ca²⁺-mediated signalling in plants.     

Roles of ion channels and transporters in guard cell signal transduction

Stomatal complexes consist of pairs of guard cells and the pore they enclose. Reversible changes in guard cell volume alter the aperture of the pore and provide the major regulatory mechanism for control of gas exchange between the plant and the environment. Stomatal movement is facilitated by the activity of ion channels and ion transporters found in the plasma membrane and vacuolar membrane of guard cells. Progress in recent years has elucidated the molecular identities of many guard cell transport proteins, and described their modulation by various cellular signal transduction components during stomatal opening and closure prompted by environmental and endogenous stimuli.     

The roles of CATALASE2 in abscisic acid signaling in Arabidopsis guard cells

We investigated the roles of catalase (CAT) in abscisic acid (ABA)-induced stomatal closure using a cat2 mutant and an inhibitor of CAT, 3-aminotriazole (AT). Constitutive reactive oxygen species (ROS) accumulation due to the CAT2 mutation and AT treatment did not affect stomatal aperture in the absence of ABA, whereas ABA-induced stomatal closure, ROS production, and [Ca(2+)](cyt) oscillation were enhanced.     

Nitric oxide functions in both methyl jasmonate signaling and abscisic acid signaling in Arabidopsis guard cells

Intracellular components in methyl jasmonate (MeJA) signaling remain largely unknown, to compare those in well-understood abscisic acid (ABA) signaling. We have reported that nitric oxide (NO) is a signaling component in MeJA-induced stomatal closure, as well as ABA-induced stomatal closure in the previous study. To gain further information about the role of NO in the guard cell signaling, NO production was examined in an ABA- and MeJA-insensitive Arabidopsis mutant, rcn1. Neither MeJA nor ABA induced NO production in rcn1 guard cells. Our data suggest that NO functions downstream of the branch point of MeJA and ABA signaling in Arabidopsis guard cells.Key words: abscisic acid, Arabidopsis thaliana, guard cells, methyl jasmonate, nitric oxideStomatal pores that are formed by pairs of guard cells respond to various environmental stimuli including plant hormones. Some signal components commonly function in MeJA- and ABA-induced stomatal closing signals,¹ such as cytosolic alkalization, ROS generation and cytosolic free calcium ion elevation. Recently, we demonstrated that NO functions in MeJA signaling, as well as ABA signaling in guard cells.²NO production by nitric oxide synthase (NOS) and nitrate reductase (NR) plays important roles in physiological processes in plants.^3,4 It has been shown that NO functions downstream of ROS production in ABA signaling in guard cells.⁵ NO mediates elevation of cytosolic free Ca²⁺ concentration ([Ca²⁺]_cyt), inactivation of inward-rectifying K⁺ channels and activation of S-type anion channels,⁶ which are known to be key factors in MeJA- and ABA-induced stomatal closure.^2,7–9It has been reported that ROS was not induced by MeJA and ABA in the MeJA- and ABA-insensitive mutant, rcn1 in which the regulatory subunit A of protein phosphatase 2A, RCN1, is impaired.^7,10 We examined NO production induced by MeJA and ABA in rcn1 guard cells (Fig. 1). NO production by MeJA and ABA was impaired in rcn1 mutant (p = 0.87 and 0.25 for MeJA and ABA, respectively) in contrast to wild type. On the other hand, the NO donor, SNP induced stomatal closure both in wild type and rcn1 mutant (data not shown). These results are consistent with our previous results, i.e., NO is involved in both MeJA- and ABA-induced stomatal closure and functions downstream of the branching point of MeJA and ABA signaling in Arabidopsis guard cells.⁷ Our finding implies that protein phosphatase 2A might positively regulate NO levels in guard cells (Fig. 2).Open in a separate windowFigure 1Impairment of MeJA- and ABA-induced NO production in rcn1 guard cells. (A) Effects of MeJA (n = 10) and ABA (n = 9) on NO production in wild-type guard cells. (B) Effects of MeJA (n = 7) and ABA (n = 7) on NO production in rcn1 guard cells. The vertical scale represents the percentage of diaminofluorescein-2 diacetate (DAF-2 DA) fluorescent levels when fluorescent intensities of MeJA- or ABA-treated cells are normalized to control value taken as 100% for each experiment. Each datum was obtained from at least 30 guard cells. Error bars represent standard errors. Significance of differences between data sets was assessed by Student''s t-test analysis in this paper. We regarded differences at the level of p < 0.05 as significant.Open in a separate windowFigure 2A model of signal interaction in MeJA-induced and ABA-induced stomatal closure. Neither MeJA nor ABA induces ROS production, NO production, I_Kin and stomatal closure in rcn1 mutant. These results suggest that NO functions downstream of the branch point of MeJA signaling and ABA signaling in Arabidopsis guard cells.     

FIA functions as an early signal component of abscisic acid signal cascade in Vicia faba guard cells

An abscisic acid (ABA)-insensitive Vicia faba mutant, fia (fava bean impaired in ABA-induced stomatal closure) had previously been isolated. In this study, it was investigated how FIA functions in ABA signalling in guard cells of Vicia faba. Unlike ABA, methyl jasmonate (MeJA), H(2)O(2), and nitric oxide (NO) induced stomatal closure in the fia mutant. ABA did not induce production of either reactive oxygen species or NO in the mutant. Moreover, ABA did not suppress inward-rectifying K(+) (K(in)) currents or activate ABA-activated protein kinase (AAPK) in mutant guard cells. These results suggest that FIA functions as an early signal component upstream of AAPK activation in ABA signalling but does not function in MeJA signalling in guard cells of Vicia faba.     

Roles of intracellular hydrogen peroxide accumulation in abscisic acid signaling in Arabidopsis guard cells

Reactive oxygen species (ROS), including hydrogen peroxide (H₂O₂), are among the important second messengers in abscisic acid (ABA) signaling in guard cells. In this study, to investigate specific roles of H₂O₂ in ABA signaling in guard cells, we examined the effects of mutations in the guard cell-expressed catalase (CAT) genes, CAT1 and CAT3, and of the CAT inhibitor 3-aminotriazole (AT) on stomatal movement. The cat3 and cat1 cat3 mutations significantly reduced CAT activities, leading to higher basal level of H₂O₂ in guard cells, when assessed by 2′,7′-dichlorodihydrofluorescein, whereas they did not affect stomatal aperture size under non-stressed condition. In addition, AT-treatment at concentrations that abolish CAT activities, showed trivial affect on stomatal aperture size, while basal H₂O₂ level increased extensively. In contrast, cat mutations and AT-treatment potentiated ABA-induced stomatal closure. Inducible ROS production triggered by ABA was observed in these mutants and wild type as well as in AT-treated guard cells. These results suggest that ABA-inducible cytosolic H₂O₂ elevation functions in ABA-induced stomatal closure, while constitutive increase of H₂O₂ do not cause stomatal closure.     

Elementary signaling modes predict the essentiality of signal transduction network components

Background  

Understanding how signals propagate through signaling pathways and networks is a central goal in systems biology. Quantitative dynamic models help to achieve this understanding, but are difficult to construct and validate because of the scarcity of known mechanistic details and kinetic parameters. Structural and qualitative analysis is emerging as a feasible and useful alternative for interpreting signal transduction.     

Convergence of calcium signaling pathways of pathogenic elicitors and abscisic acid in Arabidopsis guard cells

A variety of stimuli, such as abscisic acid (ABA), reactive oxygen species (ROS), and elicitors of plant defense reactions, have been shown to induce stomatal closure. Our study addresses commonalities in the signaling pathways that these stimuli trigger. A recent report showed that both ABA and ROS stimulate an NADPH-dependent, hyperpolarization-activated Ca(2+) influx current in Arabidopsis guard cells termed "I(Ca)" (Z.M. Pei, Y. Murata, G. Benning, S. Thomine, B. Klüsener, G.J. Allen, E. Grill, J.I. Schroeder, Nature [2002] 406: 731-734). We found that yeast (Saccharomyces cerevisiae) elicitor and chitosan, both elicitors of plant defense responses, also activate this current and activation requires cytosolic NAD(P)H. These elicitors also induced elevations in the concentration of free cytosolic calcium ([Ca(2+)](cyt)) and stomatal closure in guard cells. ABA and ROS elicited [Ca(2+)](cyt) oscillations in guard cells only when extracellular Ca(2+) was present. In a 5 mM KCl extracellular buffer, 45% of guard cells exhibited spontaneous [Ca(2+)](cyt) oscillations that differed in their kinetic properties from ABA-induced Ca(2+) increases. These spontaneous [Ca(2+)](cyt) oscillations also required the availability of extracellular Ca(2+) and depended on the extracellular potassium concentration. Interestingly, when ABA was applied to spontaneously oscillating cells, ABA caused cessation of [Ca(2+)](cyt) elevations in 62 of 101 cells, revealing a new mode of ABA signaling. These data show that fungal elicitors activate a shared branch with ABA in the stress signal transduction pathway in guard cells that activates plasma membrane I(Ca) channels and support a requirement for extracellular Ca(2+) for elicitor and ABA signaling, as well as for cellular [Ca(2+)](cyt) oscillation maintenance.