The Metabolism of Abscisic Acid

The light-catalysed isomerization of (+)-abscisic acid (ABA)to its trans isomer during isolation from leaves was monitoredby the addition of (±)-[2-¹⁴C]ABA to the extraction medium.(+)Trans-abscisic acid (t-ABA) was found to occur naturallyin rose (Rosa arvensis) leaves at 20µg/kg fresh weight;(+)-ABA was present at 594µg/kg. (±)-[2-¹⁴D]Trans-abscisicacid was not isomerized enzymically to ABA in tomato shoots. (±)-Abscisic acid was converted by tomato shoots to awater-soluble neutral product, ‘Metabolite B’, whichwas identified as abscisyl-ß-D-glucopyranoside. When(±)-[2-¹⁴C]trans-abscisic acid in an equimolar mixturewith (±)-[2-¹⁴C}ABA was fed to tomato shoots it was convertedto its glucose ester 10 times faster than was ABA. Trans-abscisyl-ß-D-glucopyrano8ide only was formedfrom (±)-[2-¹⁴C]t-ABA in experiments lasting up to 30h. Glucosyl abscisate was formed slowly from ABA and the freeacid fraction contained an excess of the unnatural (–).ABAas did the ABA released from abscisyl-ß-D-glucopyranosideby alkaline hydrolysis. The (+).ABA appeared to be the solesource of the acidic ‘Metabolite C" previously noted. The concentrations of endogenous (+)-, (+)-[2-¹⁴C]-, and (–)-[2-¹⁴C]ABAremaining as free acid, and also in the hydrolysate of abscisyl-ß-D-glucopyranoside,were measured by the ORD, UV absorption, and scintillation spectrometryof highly purified extracts of ABA from tomato shoots whichhad been supplied with racemic [2-^l4C]ABA.     

Effects of Atmospheric O3 on Azolla-Anabaena Symbiosis

Cultures of water fern Azolla pinnata R. Br. exposed for 1 weekto either 30, 50 or 80 nl l^-1 O₃ showed significant reductionsin rates of growth and N₂ fixation, and had fewer heterocysts.Although the levels of glutamine synthetase (GS) and glutamatedehydrogenase (GDH) activity were decreased by low concentrationsof O₃ exposures (30 or 50 nl l^-1), significant increases inlevels of the same enzymes were caused by higher concentrationsof O₃ (80 nl l^-1). Increased levels of total protein, polyamines(putrescine and spermidine), and the xanthophyll-cycle precursorof abscisic acid (ABA), violaxanthin, were also found with higherlevels of O₃ (80 nl l^-1). Levels of ABA itself were significantlyincreased by low level O₃ fumigation (30 nl l^-1) but significantlydecreased by exposure to 80 nl l^-1 O₃. This may indicate thathigher levels of atmospheric O₃ inhibit the final stages ofABA biosynthesis from violaxanthin.Copyright 1994, 1999 AcademicPress Abscisic acid, nitrogen assimilation, nitrogen fixation, ozone pollution, polyamines, violaxanthin     

The Specificity of the Carrier-Mediated Uptake of ABA by Root Segments of Phaseolus coccineus L.

Earlier work has established that the saturable component ofuptake of RS-[2¹⁴C]ABA by bean (Phaseolus coccineus L. cv. Prizewinner)root segments can be attributed to the action of a carrier.We now show that the carrier-mediated uptake is unaffected byRS-2-trans-ABA and lunularic acid and the unnatural R-ABA alsoappears to be ineffective. The specificity for S-ABA requiresthe halving of the K_m value for ABA determined previously (2.6mmol m_-3 for RS-; 1.3 mmol m_-3 for S-ABA). The RS-1', 4'-cis-dioland RS-1'-deoxy ABA reduce the uptake of RS-[2-₁₄C]ABA aboutas strongly as does unlabelled ABA, the K¹ for 1'-deoxy ABAwas similar to the K_m for ABA. The K₁ for RS-1', 4'-trans-diolwas 15.7 mmol m_-3. Consideration of the stereochemistry of thesecompounds suggests that the face of the ring of ABA away fromthe 1'-hydroxyl group interacts with the carrier site. Labelled material diffused out of undamaged root surfaces whichhad absorbed RS-[³H]ABA through an apical cut, suggesting thatABA is present in the apoplast. A simplified hypothesis is presented that can account for polartransport of ABA based on a gradient of a carrier in a tissuebut where the carrier is distributed uniformly on the apicaland basal ends of each cell. Key words: Uptake carrier, Abscisic acid, 1', 4'-Diol, Lunularic acid, Phaseolus coccineus, Polar-transport, Deoxyabscisic acid     

Characterization of an abscisic acid carrier in suspension-cultured barley cells

The uptake of [³H]-abscisic acid in barley (Hordeum vulgareL. cv. Heartland) cell cultures was found to be mediated throughboth non-saturable and saturable components. The kinetic parametersof the saturable component, determined at pH 4.5 and 21 °C,showed a K_m for natural or (+ )-ABA of 1.3±0.7µMand an apparent V_mex of 7.0 ± 2.8 nmol g^–1 cellsh^–1. The carrier showed a strong preference for the naturalenantiomer of ABA as compared to the unnatural one. Other substancestested, e.g. amino acids, organic acids, and other growth regulators,did not appear to interfere with the carrier-mediated uptakeof ABA. At low external concentrations of ABA (below 2.0 µM),the saturable component was greater than the diffusion component.Similarly, between pH 4.0 and 6.0, the saturable uptake wasresponsible for more than 50% of the total uptake. The carriermay be important in vivo for mediating uptake when endogenouslevels of ABA are low (c. 1 µM). The carrier specificity was evident in inhibition experimentsdone with ABA analogues. Our data showed that the carrier couldaccommodate small modifications in the ABA structure. Four analogueswere able to compete efficiently with ( + )-ABA for the bindingsite of the carrier. Three of these competitors were of the(+)-series. Only one ( –)-analogue, (–)-ABA, wasable to inhibit markedly the saturable uptake of ( + )-ABA.The induction of the ABA-respons-ive gene WCS120 (Houde et al.,1992) presented stricter requirements for the ABA molecule thanthe carrier, although with a similar preference for the ( +)-analogues. Besides ( + )-ABA itself, only two of the analoguestested, both ( + )-series, were able to induce the WCS120 geneafter a 24 h incubation period. The absence of correlation betweenthe activity of the analogues as ABA inhibitors in the carriersystem, and their capacity to induce the WCS120 gene tend tosuggest that the carrier is not directly involved in the signaltransduction pathway leading to the induction of this specificgene. Key words: Abscisic acid, barley, gene induction, Hordeum vulgare, uptake carrier     

Normalizing Development of Cultured Triticum aestivum L. Embryos: I. LOW OXYGEN TENSIONS AND EXOGENOUS ABA

Wheat (Triticum aestivum L.) embryos form in dynamically-regulatedovular environments. Our objectives were to improve developmentof cultured immature wheat embryos by simulating, in vitro,abscisic acid (ABA) levels and O₂ tensions as found in wheatovules during zygotic embryogenesis. We characterized from intactwheat kernels embryo respiration, embryo morphology and embryoand endosperm + ABA levels at 13, 19 and 25 d post-anthesis(DPA). Young (13 DPA) embryos were then excised and culturedin vitro, where they were exposed to 0·2 or 2·Ommol m^–3 ±ABA and 2.·1, 2·5 or 7·4mol m^–3 (6, 7 and 21%, respectively) gaseous O₂. At 6and 12 d in culture, + ABA levels, embryo respiration and embryomorphology were characterized by treatment. Thirteen-day-oldembryos from two different plant populations differed by 17-foldin initial ABA content. However, this difference did not affectprecocious germination in vitro, nor did it affect the amountof exogenous ABA required to reduce precocious germination by40%. In this respect, embryos from both populations were equallysensitive to exogenous ABA. Cavity sap O₂ levels (2·1to 2·5 mol m^–3) were much more effective in preventingprecocious germination of cultured embryos than were cavitysap levels of ABA (0·2 to 2·0 mmol m^–3).The combination of physiological levels of both ABA and O₂ largelynormalized DW accumulation and embryo morphology without alteringendogenous + ABA levels. Residual respiration of cultured embryoswas higher than that of embryos grown in situ, and was not influencedby the exogenous O₂ and ABA treatments Key words: Abscisic acid, embryo development, oxygen tensions, respiration, wheat     

Hydrogen peroxide generated by copper amine oxidase is involved in abscisic acid-induced stomatal closure in Vicia faba

H₂O₂ is an essential signal in absicic acid (ABA)-induced stomatalclosure. It can be synthesized by several enzymes in plants.In this study, the roles of copper amine oxidase (CuAO) in H₂O₂production and stomatal closure were investigated. ExogenousABA stimulated apoplast CuAO activity, increased H₂O₂ productionand [Ca²⁺]_cyt levels in Vicia faba guard cells, and inducedstomatal closure. These processes were impaired by CuAO inhibitor(s).In the metabolized products of CuAO, only H₂O₂ could inducestomatal closure. By the analysis of enzyme kinetics and polyaminecontents in leaves, putrescine was regarded as a substrate ofCuAO. Putrescine showed similar effects with ABA on the regulationof H₂O₂ production, [Ca²⁺]_cyt levels, as well as stomatal closure.The results suggest that CuAO in V. faba guard cells is an essentialenzymatic source for H₂O₂ production in ABA-induced stomatalclosure via the degradation of putrescine. Calcium messengeris an important intermediate in this process. Key words: Abscisic acid, calcium, copper amine oxidase, hydrogen peroxide, putrescine, stomatal closure, Vicia faba Received 13 October 2007; Revised 16 December 2007 Accepted 20 December 2007     

Hydrogen peroxide is involved in the acclimation of the Mediterranean shrub, Cistus albidus L., to summer drought

This study evaluated the possible role of hydrogen peroxide(H₂O₂) in the acclimation of a Mediterranean shrub, Cistus albidusL., to summer drought growing under Mediterranean field conditions.For this purpose, changes in H₂O₂ concentrations and localizationthroughout a year were analysed. H₂O₂ changes in response toenvironmental conditions in parallel with changes in abscisicacid (ABA) and oxidative stress markers, together with ligninaccumulation, xylem and sclerenchyma differentiation, and leafarea were also investigated. During the summer drought, leafH₂O₂ concentrations increased 11-fold, reaching values of 10µmol g^–1 dry weight (DW). This increase occurredmainly in mesophyll cell walls, xylem vessels, and sclerenchymacells in the differentiation stage. An increase in ABA levelspreceded that of H₂O₂, but both peaked at the same time in conditionsof prolonged stress. C. albidus plants tolerated high concentrationsof H₂O₂ because of its localization in the apoplast of mesophyllcells, xylem vessels, and in differentiating sclerenchyma cells.The increase in ABA, and consequently of H₂O₂, in plants subjectedto drought stress might induce a 3.5-fold increase in ascorbicacid (AA), which maintained and even decreased its oxidativestatus, thus protecting plants from oxidative damage. Afterrecovery from drought following late-summer and autumn rainfall,a decrease in ABA, H₂O₂, and AA to their basal levels (60 pmolg^–1 DW, 1 µmol g^–1 DW, and 20 µmol g^–1DW) was observed. Key words: Abscisic acid, ascorbate, ascorbate oxidative status, Cistus albidus, hydrogen peroxide, leaf plasticity, lignin, Mediterranean shrubs, oxidative markers, summer drought Received 29 July 2008; Revised 15 September 2008 Accepted 8 October 2008     

Separation and Quantification of Abscisic Acid and its Metabolites by High-performance Liquid Chromatography

A technique for the separation and quantitative estimation ofabscisic acid and its metabolic products, phaseic acid and dihydrophaseicacid by high performance liquid chromatography (HPLC) is described.The efficiency of two HPLC columns, LiChrosorb RP-18 and LiChrosorbNH₂ is compared. The method has been tested using extracts fromtomato plants and some of the results are presented. Abscisic acid, dihydrophaseic acid, phaseic acid, chromatography, tomato, Lycopersicon esculentum, Mill     

Regulation of IBA synthetase from maize (Zea mays L.) by drought stress and ABA

The rate of indole-3-butyric acid (IBA) synthesis in maize seedlingsis dependent on the culture conditions of the plants. When theseedlings were grown on filter paper soaked with different amountsof water, the activity of IBA synthetase differed strongly.High amounts of water (150 and 200 ml per bowl) inhibited IBAsynthesis completely in vitro, whereas 30 and 50 ml water perbowl increased the activity dramatically. Under conditions whereIBA synthetase was inhibited (150 ml H₂O), an increase of enzymeactivity was observed when abscisic acid (ABA) was exogenouslyadded in concentrations between 510^–4 to 510^–7M. Under ‘drought’ conditions (50 ml H₂O per bowl)the same ABA concentrations were inhibitory. Jasmonic acid andsalicylic acid also enhanced IBA synthetase activity to someextent, whereas indole-3-acetic acid (IAA) and kinetin had noeffect. Activity could also be enhanced by osmotic stress (NaCIand sorbitol), but not under temperature stress. In accompanyinginvestigations the endogenous contents of IAA, IBA, and ABAunder the different culture conditions have been determinedas well as the energy charge of the seedlings. Similar observationshave been made with Amaranthus, wheat and pea seedlings Key words: Abscisic acid, Amaranthus paniculatus, drought stress, inole-3-butyric acid biosynthesis, Pisum sativum, Triticum aestivum, Zea mays     

Studies on cutin-esterase I. Preparation of cutin and its fatty acid component from tomato fruit peel

Examination was made of the fatty acid component of tomato cutinvia gas-liquid chromatography and thin layer chromatography.Dihydroxyeicosanoic acid was identified as a major componentof tomato cutinic acid in contrast with the results of BAKERand MARTIN (1) who recognized 10,16-dihydroxyhexadecanoic acidas the dominant acid of cutin in all plants tested. On the thinlayer chromatograms we found more than nine kinds of fatty acidsin the cutin hydrolysate which was saponified with ethanol-potashsolution. The gas-liquid chromatogram for trimethylsilyl etherderivatives of methyl cutinate showed somewhat different results,i.e., unsaturated decanoic, t_R 1.4, unsaturated stearic, t_R4.2 and unsaturated octadecanedioic acid, t_R 16.0 as unsaturatedfatty acids. Two more than C₂₂-hydroxyfatty acids were recognizedas minor components. Beside these components, octanoic, t_R 0.9,hydroxydecanoic, t_R 7.0 and cis-epoxy-hydroxyoctadecanoic acid,t_R 18.7 were identified. The biosynthesis of cutin is positednot to be fulfilled or to be delayed due to less lipoxidaseactivity in tomato fruit. ¹Biological Laboratory, Research Department, Nihon Noyaku Co.Ltd., Kawachinagano, Osaka, Japan (Received December 8, 1969; )     

Abscisic Acid and Apical Dominance in Phaseolus coccineus L. The Role of Tissue Age

Abscisic acid (ABA) applied to the decapitated second internodeof runner bean plants enhanced outgrowth of lateral buds onlywhen internode stumps were no longer elongating. Applied toelongating internodes of slightly younger plants, ABA causesinhibition of bud outgrowth. Together with 10^–4 M indol-3-ylacetic acid (IAA), ABA stimulated internode elongation and interactedadditively in the inhibition of bud outgrowth. A mixture of10^–5 M ABA and 10^–6 M gibberellic acid (GA₃ ) causedsimilar effects on internode growth as IAA + ABA, but was mutuallyantagonistic in effect on growth of the lateral buds. Abscisic acid, apical dominance, gibberellic acid, indol-3yl acetic acid, Phaseolus coccineus, bean     

The Metabolism of cis ABA-aldehyde by the Wilty Mutants of Potato, Pea and Arabidopsis thaliana

RS-[²H₁] cis ABA-aldehyde was fed to ABA-deficient mutants ofpotato (droopy), pea (wilty) and Arabidopsis thaliana (aba¹)along with appropriate non-mutant controls. Both the wilty andaba¹ mutants readily oxidized the monodeuterated ABA-aldehydeto ABA. The incorporation of label into ABA by these two mutantswas indistinguishable from that detected in the non-mutant controls.In contrast, the droopy mutants poorly incorporated the labelledprecursor into ABA. Instead they reduced and isomerized RS-[²H₁] cis ABA-aldehyde to a mixture of 2, cis and 2, trans ABA-alcohols.Thus the droopy mutant affects the last step in ABA biosynthesis,a position it shares with the tomato mutants, flacca and sitiens.Genetic evidence suggesting that droopy and sitiens may be correspondinggene loci is discussed. Key words: ABA metabolism, wilty mutants, pea, potato, Arabidopsis     

Glucosamine inhibits angiotensin II-induced cytoplasmic Ca2+ elevation in neonatal cardiomyocytes via protein-associated O-linked N-acetylglucosamine

We previously reported that glucosamine and hyperglycemia attenuate the response of cardiomyocytes to inositol 1,4,5-trisphosphate-generating agonists such as ANG II. This appears to be related to an increase in flux through the hexosamine biosynthesis pathway (HBP) and decreased Ca²⁺ entry into the cells; however, a direct link between HBP and intracellular Ca²⁺ homeostasis has not been established. Therefore, using neonatal rat ventricular myocytes, we investigated the relationship between glucosamine treatment; the concentration of UDP-N-acetylglucosamine (UDP-GlcNAc), an end product of the HBP; and the level of protein O-linked N-acetylglucosamine (O-GlcNAc) on ANG II-mediated changes in intracellular free Ca²⁺ concentration ([Ca²⁺]_i). We found that glucosamine blocked ANG II-induced [Ca²⁺]_i increase and that this phenomenon was associated with a significant increase in UDP-GlcNAc and O-GlcNAc levels. O-(2-acetamido-2-deoxy-D-glucopyranosylidene)-amino-N-phenylcarbamate, an inhibitor of O-GlcNAcase that increased O-GlcNAc levels without changing UDP-GlcNAc concentrations, mimicked the effect of glucosamine on the ANG II-induced increase in [Ca²⁺]_i. An inhibitor of O-GlcNAc-transferase, alloxan, prevented the glucosamine-induced increase in O-GlcNAc but not the increase in UDP-GlcNAc; however, alloxan abrogated the inhibition of the ANG II-induced increase in [Ca²⁺]_i. These data support the notion that changes in O-GlcNAc levels mediated via increased HBP flux may be involved in the regulation of [Ca²⁺]_i homeostasis in the heart. hypertrophy; left ventricle; calcium channels; calcium signaling     

Effects of Abscisic Acid on the Growth Pattern of the Shoot Apical Meristem and on Flowering in Chenopodium rubrum L.

Abscisic acid (ABA) at 1 x 10^–4 M or 3 x 10^–4 Mwas applied to the apical buds of Chenopodium rubrum plantsexposed to different photoperiodic treatments and showing differentpatterns of floral differentiation. Stimulation of growth inwidth of the apical meristem of the shoot and/or inhibitionof growth in length was obtained under all photoperiodic treatments.This change of growth pattern was followed by different effectson flowering. In non-induced plants grown under continuous light ABA stimulatedpericlinal divisions in the peripheral zone and the initiationof leaves as well as the growth in width of bud primordia. Inplants induced by two short days reduced growth of the meristemcoincided with ABA application. Longitudinal growth of the meristemwas inhibited in this case and only a temporary stimulationof inflorescence formation took place. In plants induced ata very early stage, ABA exerted a strong inhibitory effect onflowering. A permanent and reproducible stimulatory effect onflowering was obtained in plants induced by three sub-criticalphotoperiodic cycles if ABA was applied to apices released fromapical dominance. In this case formation of lateral organs andinternodes was promoted by ABA and was followed by stimulatedinflorescence formation. Gibberellic acid (GA2) at 1x 10^–4M or 3 x 10^–4 M brought about a similar effect on floweringas ABA, although the primary growth effect was different, i.e.GA₂ stimulated longitudinal growth. The effects of ABA and GA₂ on floral differentiation have beencompared with earlier results obtained from auxin and kinetinapplications. These growth hormones are believed to regulateflowering by changing cellular growth within the shoot apex.Depending on the actual state of the meristem identical growthresponses may result in different patterns of organogenesisand even in opposite effects on flowering. Shoot apex, flowering, photoperiodic induction, abscisic acid, gibberellic acid, Chenopodium rubrum L.     

Incorporation of 2H from 2H2O into ABA in Tomato Shoots: Evidence for a Large Pool of Precursors

Tomato shoots were supplied simultaneously with S-[¹⁴C]abscisicacid and ²H₂O for 4 h. After a further 6 h incubation no deuteriumhad been incorporated into abscisic acid (ABA) extracted fromwilted or turgid shoots although up to 93% of the ABA had beensynthesized in the presence of ²H₂O as shown by the dilutionof [¹⁴C]ABA. This provides substantial evidence for a largepool of ABA precursors. When tomato shoots were incubated for6d in 80% ²H₂O between 20% and 32% of ABA molecules became labelledwith from 1 to 14 deuterium atoms. From this data the minimumsize of the precursor pool was calculated to be approximately35 times that of ABA. In addition, ABA from wilted plants containedsignificantly less deuterium than that from turgid plants suggestinga second or an enlarged pool of precursor in wilted plants. Key words: Abscisic acid, precursor pool, ²H incorporation     

Inhibition of Ion Transport in Excised Barley Roots by Abscisic Acid; Relation to Water Permeability of the Roots

Previous papers have shown that abscisic acid can inhibit transportof ions across the root to the xylem vessels, resulting in reducedexudation from excised roots or inhibiting guttation from intactplants. However, it has not been established whether the inhibitionwas due to a reduction in salt transport (J_s) or in permeabilityof the roots to water (L_p). This paper investigates the effectof ABA on L_p and J_s separately. It is shown that L_p increasedin ABA and then fell, but was about the same as in control rootswhen transport was inhibited. The effect of ABA on exudationtherefore appeared to be mainly due to reduction in J_s. Inhibitionof J_s was also present in intact, transpiring plants and sowas not due to reduced water flow. The inhibition of ion releaseto the xylem affected Na⁺, Mg²⁺, Ca²⁺, and phosphate as wellas the major ion in the exudate, K⁺. It is concluded that ABAinhibits salt transport to the shoot by acting on ion transportinto the xylem, and not by reducing water flow coupled withsalt transport.     

Abscisic alcohol is an intermediate in abscisic Acid biosynthesis in a shunt pathway from abscisic aldehyde

It has previously been shown that the abscisic acid (ABA)-deficient flacca and sitiens mutants of tomato are impaired in ABA-aldehyde oxidation and accumulate trans-ABA-alcohol as a result of the biosynthetic block (IB Taylor, RST Linforth, RJ Al-Naieb, WR Bowman, BA Marples [1988] Plant Cell Environ 11: 739-745). Here we report that the flacca and sitiens mutants accumulate trans-ABA and trans-ABA glucose ester and that this accumulation is due to trans-ABA biosynthesis. ¹⁸O labeling of water-stressed wild-type and mutant tomato leaves and analysis of [¹⁸O]ABA by tandem mass spectrometry show that the tomato mutants synthesize a significant percentage of their ABA and trans-ABA as [¹⁸O]ABA with two ¹⁸O atoms in the carboxyl group. We further show, by feeding experiments with [²H₆]ABA-alcohol and ¹⁸O₂, that this doubly-carboxyl-labeled ABA is synthesized from [¹⁸O]ABA-alcohol with incorporation of molecular oxygen. In vivo inhibition of [²H₆]ABA-alcohol oxidation by carbon monoxide establishes the involvement of a P-450 monooxygenase. Likewise, carbon monoxide inhibits the synthesis of doubly-carboxyl-labeled ABA in ¹⁸O-labeling experiments. This minor shunt pathway from ABA-aldehyde to ABA-alcohol to ABA operates in all plants examined. For the ABA-deficient mutants impaired in ABA-aldehyde oxidation, this shunt pathway is an important source of ABA and is physiologically significant.     

Gene expression patterns, and uptake and fate of fed ABA in white spruce somatic embryo tissues2

Changes in cellular protein accumulation and in in vivo andin vitro protein synthesis, in somatic embryo tissues of whitespruce during a 42 d maturation period were followed by two-dimensionalsodium dodecyl sulphate-polyacrylamide gel electrophoresis (2-DSDS-PAGE). These investigations were complemented by an analysisof uptake and fate of fed abscisic acid (ABA) in somatic embryotissues grown on maturation medium. When Stage 1 somatic embryoswere cultured on ABA-containing maturation medium, many changeswere observed in patterns of gene expression and in proteinsynthesis and accumulation which could be associated with embryodevelopment. The polypeptides observed could be categorizedas constitutive, embryo-abundant, embryo maturation-relatedand embryo stage-related, as well as those with non-specificchanges. Accumulation of label from fed ³H-(+)-ABA in embryotissues reached a plateau 3 d after Stage 1 somatic embryoswere placed on maturation medium. ABA taken into tissues wasrapidly metabolized, and 40% of radioactivity in tissues after1 d of culture resulted from ABA metabolites. This value increasedto 90% after 3 weeks culture. Conjugated ABA and oxidized ABA(phaseic acid and dihydrophaseic acid) were major forms of ABAmetabolites in spruce embryo tissues. Using a single 42 d cultureperiod following transfer to medium with ABA, the conditionsthat stimulate the sequence of developmental changes of somaticembryo maturation during the first 21 d do not reoccur duringthe second 21 d. Unless greater synchronization of Stage 1 culturescan be achieved, it is therefore unlikely that yields of maturesomatic embryos will be increased by this method. Key words: Abscisic acid, gene expression, Picea glauca (Moench) Voss, protein synthesis, somatic embryo maturation     

Nitric oxide induces [Ca2+]i oscillations in pituitary GH3 cells: involvement of IDR and ERG K+ currents

The role of nitric oxide (NO) in the occurrence of intracellular Ca²⁺ concentration ([Ca²⁺]_i) oscillations in pituitary GH₃ cells was evaluated by studying the effect of increasing or decreasing endogenous NO synthesis with L-arginine and nitro-L-arginine methyl ester (L-NAME), respectively. When NO synthesis was blocked with L-NAME (1 mM) [Ca²⁺]_i, oscillations disappeared in 68% of spontaneously active cells, whereas 41% of the quiescent cells showed [Ca²⁺]_i oscillations in response to the NO synthase (NOS) substrate L-arginine (10 mM). This effect was reproduced by the NO donors NOC-18 and S-nitroso-N-acetylpenicillamine (SNAP). NOC-18 was ineffective in the presence of the L-type voltage-dependent Ca²⁺ channels (VDCC) blocker nimodipine (1 µM) or in Ca²⁺-free medium. Conversely, its effect was preserved when Ca²⁺ release from intracellular Ca²⁺ stores was inhibited either with the ryanodine-receptor blocker ryanodine (500 µM) or with the inositol 1,4,5-trisphosphate receptor blocker xestospongin C (3 µM). These results suggest that NO induces the appearance of [Ca²⁺]_i oscillations by determining Ca²⁺ influx. Patch-clamp experiments excluded that NO acted directly on VDCC but suggested that NO determined membrane depolarization because of the inhibition of voltage-gated K⁺ channels. NOC-18 and SNAP caused a decrease in the amplitude of slow-inactivating (I_DR) and ether-à-go-go-related gene (ERG) hyperpolarization-evoked, deactivating K⁺ currents. Similar results were obtained when GH₃ cells were treated with L-arginine. The present study suggests that in GH₃ cells, endogenous NO plays a permissive role for the occurrence of spontaneous [Ca²⁺]_i oscillations through an inhibitory effect on I_DR and on I_ERG. voltage-gated potassium channels; ether-à-go-go-related gene potassium channels; slow-inactivating outward currents; fast-inactivating outward currents     

Stomatal Functioning of In Vitro and Greenhouse Apple Leaves in Darkness, Mannitol, ABA, and CO2

Leaves from in vitro and greenhouse cultured plants of Malusdomestica (Borkh.) cv. Mark were subjected to 4 h of darkness;4 h of 1 M mannitol induced water stress; 1 h of 10^–4M to 10^–7 M cis-trans abscisic acid (ABA) treatment; 1h of 0.12% atmospheric CO₂. Stomatal closure was determinedby microscopic examination of leaf imprints. In all treatments,less than 5% of the stomata from leaves of in vitro culturedplants were closed. The diameter of open stomata on leaves fromin vitro culture remained at 8 µm. In contrast, an averageof 96% of the stomata on leaves of greenhouse grown plants wereclosed after 4 h in darkness; 56% after 4 h of mannitol inducedwater stress; 90% after 1 h of 10^–4 M ABA treatment; 61%after 1 h in an atmosphere of 0.12% CO₂. Stomata of in vitroapple leaves did not seem to have a closure mechanism, but acquiredone during acclimatization to the greenhouse environment. Thelack of stomatal closure in in vitro plants was the main causeof rapid water loss during transfer to low relative humidity.