Involvement of Calmodulin and Calmodulin-Dependent Myosin Light Chain Kinase in Blue Light-Dependent H Pumping by Guard Cell Protoplasts from Vicia faba L

Signal transduction processes involved in blue light-dependent proton pumping were investigated using guard cell protoplasts from Vicia faba. N-[2-(Methylamino)ethyl]-5-isoquinolinesulfonamide, an inhibitor of cyclic AMP- and cyclic GMP-dependent protein kinases, had no effect. 1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7) and calphostin C, inhibitors of protein kinase C, produced slight inhibition of the blue light-dependent proton pumping. 1-[N, O-Bis(5-isoquinolinesulfonyl)-N-methyl-l-tyrosyl] -4-phenylpiperazine, a specific inhibitor of Ca²⁺/calmodulin (CaM)-dependent protein kinase II, did not inhibit the proton pumping, but 1-(5-iodonaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine and 1-(5-chloro-naphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), inhibitors of Ca²⁺/CaM-dependent myosin light chain kinase, strongly suppressed the proton pumping. A CaM antagonist, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7), inhibited blue light-dependent proton pumping, whereas its less active structural analog, N-(6-aminohexyl)-1-naphthalenesulfonamide (W-5), had little effect on the response. Other CaM antagonists, trifluoperazine, compound 48/80, prenylamine, and 3-(2-benzothiazolyl)-4,5-dimethoxy-N-[3-(4-phenyl-piperidinyl)- propylbenzenesulfonamide inhibited the proton pumping. In accord with these results, light-induced stomatal opening in the epidermis of Commelina benghalensis ssp. was inhibited by ML-9 and W-7, but not by H-7 and W-5. Thus, it is concluded that CaM and Ca²⁺/CaM-dependent myosin light chain kinase are the components of the signal transduction process in blue light-dependent proton pumping in guard cells.     

Plasma Membrane H+-ATPase in Guard-Cell Protoplasts from Vicia faba L.

The activity of plasma membrane H⁺-ATPase was measured withmembrane fragments of guard-cell protoplasts isolated from Viciafaba L. ATP hydrolytic activity was slightly inhibited by oligomycinand ammonium molybdate, and markedly inhibited by NO₃^–and vanadate. In the presence of oligomycin, ammonium molybdateand NO₃^–, the ATP-hydrolyzing activity was strongly inhibitedby vanadate. It was also inhibited by diethylstilbestrol (DES),p-chloromercuribenzoic acid (PCMB) and Ca²⁺, but slightly stimulatedby carbonyl cyanide m-chlorophenylhydrazone (CCCP). The acitivityhad higher specificity for ATP as a substrate than other phosphoricesters such as ADP, AMP, GTP and p-nitrophenylphosphate; theK_m was 0.5 mM for ATP. The activity required Mg²⁺ but was notaffected by K⁺, and it was maximal around pH 6.8. When guard-cellprotoplasts were used instead of membrane fragments, the ATPaseactivity reached up to 800µmol Pi.(mg Chl)^–1.h^–1in the presence of lysolecithin. These results indicate thatthe guard cell has a high plasma membrane H⁺-ATPase activity. (Received December 23, 1986; Accepted April 28, 1987)     

脱落酸抑制蓝光依赖气孔开放运动的生理机制分析

以蚕豆(Vicia fabaL.)为材料,采用表皮条生物学分析技术、远红外成像技术以及电生理膜片钳技术,研究逆境信号脱落酸(ABA)和蓝光信号对蚕豆叶片表皮气孔运动及质膜K+通道的影响,以探讨ABA调节蓝光诱导的气孔开放运动的生理机制.结果表明:(1)100μmol?m-2?s-1蓝光能显著诱导气孔开放,该效应可被ABA以浓度依赖的方式抑制,并以10μmol?L-1ABA抑制效应最为明显.(2)100μmol?m-2?s-1蓝光处理能明显促使蚕豆叶面温度下降,而10μmol?L-1ABA可显著阻止蓝光诱导的蚕豆叶面温度下降.(3)100μmol?m-2?s-1蓝光可明显激活保卫细胞质膜内向K+通道,处理5 min后内向K+电流增加58%;对于1和10μmol?L-1ABA预处理蚕豆保卫细胞原生质体,在蓝光处理5 min后其内向K+电流增加值分别被抑制25%和51%,但10μmol?L-1ABA并不明显抑制壳梭孢菌素(质膜H+-ATP酶永久性激活剂)诱导的保卫细胞内向K+电流上升.研究发现,逆境信号ABA可能主要通过抑制蓝光信号转导中质膜H+-ATP酶上游位点,阻断蓝光激活的保卫细胞质膜内向K+通道,抑制蓝光诱...     

Abscisic Acid-Induced Phosphoinositide Turnover in Guard Cell Protoplasts of Vicia faba

Guard cell protoplasts of Vicia faba treated with 10 [mu]M (+)abscisic acid (ABA) in the light exhibited a 20% decrease in diameter within 1.5 h, from 24.1 to 19.6 [mu]m. Within 10 s of administration of ABA, a 90% increase in levels of inositol 1,4,5-trisphosphate was observed, provided that cells were treated with Li+, an inhibitor of inositol phosphatase activity, prior to incubation. Concomitantly, levels of 32P-labeled phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate decreased 20% compared to levels in control cells; levels of label in the membrane lipids phosphatidylcholine, phosphatidylethanolamine, and phosphatidylglycerol did not change significantly in response to ABA treatment. These results show that phosphoinositide turnover is activated in response to ABA in guard cells. We conclude that phosphoinositide signaling is likely to be a step in the biochemical cascade that couples ABA to guard cell shrinking and stomatal closure.     

Arabidopsis Mutants Lacking Blue Light-Dependent Inhibition of Hypocotyl Elongation

We have isolated a new class of photomorphogenic mutants in Arabidopsis. Hypocotyl elongation is not inhibited in the mutant seedlings by continuous blue light but is inhibited by far red light, indicating that these mutations are phenotypically different from the previously isolated long hypocotyl (hy) mutants. Complementation analysis indicated that recessive nuclear mutations at three genetic loci, designated blu1, blu2, and blu3, can result in the blu mutant phenotype and that these mutants are genetically distinct from other long hypocotyl mutants. The BLU genes appear to be important only during seedling development because the blu mutations have little effect on mature plants, whereas hypocotyl elongation and cotyledon expansion are altered in seedlings. The genetic separation of the blue and far red sensitivities of light-induced hypocotyl inhibition in the blu and hy mutants demonstrates that two photosensory systems function in this response.     

Properties of Proton Pumping in Response to Blue Light and Fusicoccin in Guard Cell Protoplasts Isolated from Adaxial Epidermis of Vicia Leaves

Guard cell protoplasts (GCPs) were isolated from the adaxial epidermis of Vicia leaves. The properties of isolated adaxial GCPs (ad GCPs) were compared with those of abaxial GCPs (ab GCPs) with respect to H+-pumping activity. A saturating pulse of blue light (200 [mu]mol m-2 s-1, 30 s) induced H+ pumping in both ad GCPs and ab GCPs under red light. The maximum rate of blue-light-dependent H+ pumping was slightly higher in ad GCPs than in ab GCPs, but the magnitude of H+ pumping in ad GCPs was 68% of that in ab GCPs. H+ pumping was responsive to the second pulse, and the rate and magnitude of the pumping increased with the time between two pulses. The periods required to achieve 50% of the maximum rate were 12 and 22 min for ad GCPs and ab GCPs, respectively. The rates of blue-light-dependent H+ pumping were saturable, with half-saturation at 630 [mu]mol m-2 (21 [mu]mol m-2 s-1, 30 s) for ad GCPs and 105 [mu]mol m-2 (3.5 [mu]mol m-2 s-1, 30s) for ab GCPs. In contrast, fusicoccin, an activator of the plasma membrane H+- ATPase, induced H+ pumping with a slightly higher rate in ad GCPs than in ab GCPs. Both types of protoplast swelled similarly in response to fusicoccin. These results suggest that ad GCPs have almost the same activity for H+ pumping as ab GCPs, whereas ad GCPs require a larger number of photons to activate the H+ pump than ab GCPs.     

Inhibition of Stomatal Opening by Analogues of Abscisic Acid

Twenty analogues of abscisic acid have been tested for theiractivity as inhibitors of stomatal opening in isolated epidermisof Commelina communis. A number of derivatives showed slightactivity but only two treatments resulted in significant stomatalclosure and this was accompanied by destruction of the guardcell membranes. Such damage is characteristic of the stomatalresponse to farnesol, another sesquiterpenoid also thought tobe involved in control of water loss. The implication of theseresults in the study of antitranspirants is considered.     

Abscisic Acid Induces Mitogen-Activated Protein Kinase Activation in Barley Aleurone Protoplasts

Abscisic acid (ABA) induces a rapid and transient mitogen-activated protein (MAP) kinase activation in barley aleurone protoplasts. MAP kinase activity, measured as myelin basic protein phosphorylation by MAP kinase immunoprecipitates, increased after 1 min, peaked after 3 min, and decreased to basal levels after ~5 min of ABA treatment in vivo. Antibodies recognizing phosphorylated tyrosine residues precipitate with myelin basic protein kinase activity that has identical ABA activation characteristics and demonstrate that tyrosine phosphorylation of MAP kinase occurs during activation. The half-maximal concentration of ABA required for MAP kinase activation, 3 x 10-7 M, is very similar to that required for ABA-induced rab16 gene expression. The tyrosine phosphatase inhibitor phenylarsine oxide can completely block ABA-induced MAP kinase activation and rab16 gene expression. These results lead us to conclude that ABA activates MAP kinase via a tyrosine phosphatase and that these steps are a prerequisite for ABA induction of rab16 gene expression.     

Competitive Inhibition of Abscisic Acid-Regulated Gene Expression by Stereoisomeric Acetylenic Analogs of Abscisic Acid

The properties of two enantiomeric synthetic acetylenic abscisic acid (ABA) analogs (PBI-51 and PBI-63) in relation to ABA-sensitive gene expression are reported. Using microspore-derived embryos of Brassica napus as the biological material and their responsiveness to ABA in the expression of genes encoding storage proteins as a quantitative bioassay, we measured the biological activity of PBI-51 and PBI-63. Assays to evaluate agonistic activity of either compound applied individually showed a dose-dependent increase in napin gene expression on application of PBI-63. Maximal activity of about 40 [mu]M indicated that PBI-63 was an agonist, although somewhat weaker than ABA. PBI-63 has a similar stereochemistry to natural ABA at the junction of the ring and side chain. In contrast, PBI-51 showed no agonistic effects until applied at 40 to 50 [mu]M. Even then, the response was fairly weak. PBI-51 has the opposite stereochemistry to natural ABA at the junction of the ring and side chain. When applied concurrently with ABA, PBI-63 and PBI-51 had distinctly different properties. PBI-63 (40 [mu]M) and ABA (5 [mu]M) combined gave results similar to the application of either compound separately with high levels of induction of napin expression. PBI-51 displayed a reversible antagonistic effect with ABA, shifting the typical ABA dose-response curve by a factor of 4 to 5. This antagonism was noted for the expression of two ABA-sensitive genes, napin and oleosin. To test whether this antagonism was at the level of ABA recognition or uptake, ABA uptake was monitored in the presence of PBI-51 or PBI-63. Neither compound decreased ABA uptake. Treatments with either PBI-51 or PBI-63 showed an effect on endogenous ABA pools by permitting increases of 5- to 7-fold. It is hypothesized that this increase occurs because of competition for ABA catabolic enzymes by both compounds. The fact that ABA pools did not decrease in the presence of PBI-51 suggests that PBI-51 must exert its antagonistic properties through direct competition with ABA at a hormone-recognition site.     

Disruption of Microtubules by Abscisic Acid in Guard Cells of Vicia faba L.

ABA disrupted cortical microtubules in guard cells, but notin epidermal cells, with concomitant closure of stomata. Otherplant growth regulators did not disrupt the microtubles in guardcells. Thus disrution of microtubules seems to be a specificeffect of ABA in guard cells but its physiological significanceremains obscure. (Received September 11, 1995; Accepted April 26, 1996)     

Changes in Lipid and Fatty Acid Metabolism of Vicia faba Mesophyll Protoplasts

The metabolism of the major polar and neutral lipids of Viciafaba protoplasts isolated from ¹⁴CO₂-fed leaves has been examined.The results show large losses in the radioactivity found inphosphatidylcholine and monogalactosyldiacylglycerol while thatof phosphatidylglycerol was stable. This loss was accountedfor by a rapid increase in the ¹⁴C content of the neutral lipids,particularly the triacylglycerols. Analysis of the fatty acidradioactivity in the lipids suggests that protoplast isolationinhibited fatty acid desaturation on phosphatidylcholine andpossibly on other lipids. These results also suggest a roleof phosphatidylcholine in the donation of fatty acids for triacylglycerolsynthesis in mesophyll protoplasts. The results are discussedin terms of the regulation of lipid metabolism and protoplastbiology. (Received April 20, 1984; Accepted August 27, 1984)     

Abscisic Acid Activates a 48-Kilodalton Protein Kinase in Guard Cell Protoplasts

A 49- and a 46-kD Ca2+-independent protein kinase and a 53-kD Ca2+-dependent protein kinase were detected in Vicia faba guard cell protoplasts (GCPs) by an in-gel protein kinase assay using myelin basic protein as a substrate. A 48-kD protein kinase designated as abscisic acid (ABA)-responsive protein kinase (ABR kinase) appeared when GCPs were treated with ABA. The activation of ABR kinase was suppressed by the protein kinase inhibitor staurosporine, indicating that a putative activator protein kinase phosphorylates and activates ABR kinase. The treatment of GCPs with 1,2-bis(o-aminophenoxy)ethan-N,N,N',N'-tetraacetic acid, a calcium chelator, suppressed the activation of ABR kinase, suggesting that an influx of extracellular Ca2+ is required for the activation. Staurosporine and K-252a inhibited both the activity of ABR kinase and the stomatal closure induced by ABA treatment of V. faba epidermal peels. These results suggest that ABR kinase and its activator kinase may consist of a protein kinase cascade in a signal transduction pathway linking ABA perception to stomatal closure. The mobility of the 53-kD Ca2+-dependent protein kinase in sodium dodecyl sulfate-polyacrylamide gel was shifted upon Ca2+ binding to the enzyme, thus exhibiting the characteristics of a Ca2+-dependent or calmodulin-like domain protein kinase. This kinase may be the activator of ABR kinase.     

Responses of Commelina communis L. Guard Cell Protoplasts to Abscisic Acid

Fitzsimons, P. J. and Weyers, J. D. B. 1987. Responses of Commelinacommunis L. guard cell protoplasts to abscisic acid.—J.exp. Bot. 38: 992–1001. Guard cell protoplasts (GCPs) isolated from the leaf epidermisof Commelina communis L. responded to abscisic acid (ABA) ina manner which was qualitatively and quantitatively similarto that of intact stomata. ABA inhibited swelling of GCPs underlow-CO₂ conditions and swollen GCPs responded to the hormoneby shrinking. Both the absolute volume decrease and the initialrate of shrinking were commensurate with the extent and ratesof solute loss computed for ABA-treated intact, open stomata.This indicates that GCPs represent a suitable experimental systemfor studies of ABA-mediated solute fluxes. A radiotracer equilibrationmethod was developed for the rapid estimation of GCP osmoticvolume changes. Using this technique it was found that, on average,82% of the reduction in solute content caused by ABA treatmentwas due to the loss of K⁺. It is envisaged that electroneutralitymight be maintained during ABA-induced shrinkage of GCPs bynet inward proton movement leading to acidification of the vacuole. Key words: Abscisic acid, Commelina communis L., guard cells, protoplasts     

Abscisic Acid Structure-Activity Relationships in Barley Aleurone Layers and Protoplasts (Biological Activity of Optically Active,Oxygenated Abscisic Acid Analogs)

Optically active forms of abscisic acid (ABA) and their oxygenated metabolites were tested for their biological activity by examining the effects of the compounds on the reversal of gibberellic acid-induced [alpha]-amylase activity in barley (Hordeum vulgare cv Himalaya) aleurone layers and the induction of gene expression in barley aleurone protoplasts transformed with a chimeric construct containing the promoter region of an albumin storage protein gene. Promotion of the albumin storage protein gene response had a more strict stereochemical requirement for elicitation of an ABA response than inhibition of [alpha]-amylase gene expression. The naturally occurring stereoisomer of ABA and its metabolites were more effective at eliciting an ABA-like response. ABA showed the highest activity, followed by 7[prime]-hydroxyABA, with phaseic acid being the least active. Racemic 8[prime]-hydroxy-2[prime],3[prime]-dihydroABA, an analog of 8[prime]-hydroxyABA, was inactive, whereas racemic 2[prime],3[prime]-dihydroABA was as effective as ABA. The differences in response of the same tissue to the ABA enantiomers lead us to conclude that there exists more than one type of ABA receptor and/or multiple signal transduction pathways in barley aleurone tissue.     

Distribution of Gibberellins and Abscisic Acid in Geotropically Stimulated Vicia faba Roots

The occurrence of gibberellins and abscisic acid (ABA) in extracts of roots of Vicia faba was demonstrated by gas-liquid chromatography (GLC) of the methylated eluates from the relevant zones of thin-layer chromatograms (TLC) of purified extracts. Quantitative determination of the hormone contents in extracts from upper and lower halves of roots which had been kept in the horizontal position for 30 min indicated a redistribution of the hormones during the geotropic stimulation. Gibberellins whose methyl esters appeared at the retention time of methylated gibberellic acid (GA₃), used as a standard, occurred in higher concentration in the upper than in the lower halves (ratio 2.08:1), whereas the concentration of ABA was highest in the lower halves (ratio 3.08:1). The ratio of the hormones in right and left halves of vertical roots was close to 1:1. Indoleacetic acid (IAA) and ABA were found to retard the elongation of roots of Vicia faba and Lepidium during the first 24 h. Additional experiments with Lepidium showed that this retardation occurs within the first hour after application. Low concentrations of GA₃, when applied to germinating seeds just after the radicles had broken the seed coat, stimulated root elongation in Vicia faba within 24 h and in Lepidium within 36 h. When applied to Lepidium seedlings with 20 mm long roots, GA₃ showed a stimulatory trend within the first 2 h, and distinct stimulation in the subsequent hours, particularly at the lowest concentrations, 0.01 and 0.001 mg/1. These results suggest the possibility of a participation of ABA and gibberellins (in addition to IAA) in the development of the positive geotropic curvature.     

Changes in the Lipid and Fatty Acid Composition of Vicia faba Mesophyll Protoplasts Induced by Isolation

The lipid and fatty acid compositions of mesophyll protoplastsof Broad bean (Vicia faba) have been analysed by gas chromatography.The results indicate that protoplast isolation triggered therapid hydrolysis of several membrane lipids, notably phosphatidylcholineand monogalactosyldiacylglycerol. The decrease of these lipidswas compensated for by an increase in the amount of the neutrallipid fraction. Analyses of the fatty acid compositions suggestthat phosphatidylcholine from the microsomes and monogalactosyldiacylglycerolfrom the chloroplast were degraded to diacylglycerol and freefatty acids and used for the biosynthesis of triacylglycerols. (Received April 20, 1984; Accepted September 27, 1984)     

Studies of the Inhibition of Stomatal Opening by Naphth-1-ylacetic Acid and Abscisic Acid

Evidence is presented which confirms the inhibitory action ofthe synthetic auxin, NAA, on stomatal opening. In contrast tothe natural auxin IAA, NAA exerts effects which, in some respects,resemble those of ABA. The mode of action of NAA on the guardcells is, however, thought to be different from that of ABA.When NAA and ABA are applied together, their combined actionresults in a greater reduction in stomatal aperture than wouldbe predicted from their separate effects. Key words: NAA, ABA, IAA Stomata, Commelina     

Stimulation of Potassium Uptake by Abscisic Acid in Potato Tuber Tissues. Relation with H+ and Ca2+ Fluxes

Previous results with potato tuber discs showed that a treatment with abscisic acid stimulated K⁺ uptake. In this investigation, we determine the relationship between increased K' uptake and H⁺extrusion, and Ca²⁺ fluxes by treating tissues with specific Ca²⁺ channel blocker (La³⁺), calmodulin (CaM) inhibitors (chlorpromazine and W₇), and with Ca²⁺ ionophore (A23187). K⁺ uptake increased with increasing external pH whether tissues were treated with ABA or not. Treatment of tissues with La³⁺ inhibited K⁺ uptake, whereas CaM inhibitors have no effect. By contrast ABA and A23187 produced a synergistic effect, suggesting that ABA may act in part, on K⁺ uptake, like a Ca²⁺ agonist, in accord with Huddart's hypothesis. This revised version was published online in July 2006 with corrections to the Cover Date.