Direct and simultaneous determination of reduced and oxidized glutathione and homoglutathione by liquid chromatography-electrospray/mass spectrometry in plant tissue extracts

A simple, highly selective, sensitive, and reproducible liquid chromatography-electrospray ionization/mass spectrometry (time of flight) method has been developed for the direct and simultaneous determination of glutathione and related compounds such as homoglutathione in different plant tissues. These compounds are low-molecular mass antioxidants involved in cellular redox homeostasis in plants, and efforts are being made to develop methods to determine the concentrations of oxidized and reduced forms of these compounds and their ratio. Many of the methodologies developed so far, however, are time-consuming and complex; therefore, analytes can decompose and their redox status can change during the analysis process. The method we have developed allows the simultaneous determination of reduced forms (glutathione [GSH] and homoglutathione [hGSH]) and oxidized forms (glutathione disulfide [GSSG]) of these compounds and is also suitable for the determination of ascorbic acid (ASA) and S-nitrosoglutathione (GSNO). Quantification was done using isotopically labeled GSH and ASA as internal standards. All compounds were base peak resolved in less than 6 min, and limits of detection were 60 pmol for GSH, 30 pmol for hGSH, 20 pmol for GSSG, 100 pmol for ASA, and 30 pmol for GSNO. The intraday repeatability values were approximately 0.4 and 7% for retention time and peak area, respectively, whereas the interday repeatability values were approximately 0.6 and 9% for retention time and peak area, respectively. Analyte recoveries found were between 92 and 105%. The method was used to determine the concentrations of GSH, GSSG, hGSH, and ASA in extracts from several plant tissues.     

Morphology and Hormone Levels of Tobacco and Carrot Tissues Transformed by Agrobacterium tumefaciens II. IAA Biosynthetic Activity of Cultured Carrot Tissues Transformed with Wild-Type and Mutant Ti Plasmids

IAA biosynthetic activity was examined in cultured carrot tissuestransformed with Agrobacterium tumefaciens harboring wild-type,aux^– or cyt^– Ti plasmids. In vitro IAAM hydrolaseactivities in tissues transformed with wild-type, and cyt^–Ti plasmids were 3.09 and 19.82 nmol/g proteins/30 min, respectively,but not detectable when aux^– Ti plasmids were used. Theactivity of IAA biosynthesis, determined by the incorporationof radioactivity into IAA in tissues fed with [¹⁴C]-tryptophan,was 34.13, 10.92 and 32.47 pmol/g fr wt/30 min in tissues transformedwith wild type, aux^– and cyt^– Ti plasmids, respectively.The incorporation of radioactivity into the IAAM fraction wasdetected only in the tissues transformed with wild type andcyt^– Ti plasmids. These results showed that the T-DNAencoded pathway of IAA biosynthesis was active in tissues transformedwith wild-type and cyt^h Ti plasmids, and that the activity ofIAA biosynthesis in those tissues was higher than that in tissuestransformed with the aux^– Ti plasmid. (Received March 16, 1988; Accepted July 31, 1988)     

Electroporation Stimulates Plant Regeneration from Protoplasts of the Woody Medicinal Species Solarium dulcamara L.

Exposure of cell suspension protoplasts of the woody medicinalplant Solatium dulcamara L. to voltages of 250 to 1250 V cm^–1for three successive pulses, each of 10–50 us duration,stimulated growth of protoplast-derived tissues. Such tissuesexhibited increased morphogenesis and required a shorter periodin culture to exhibit this effect than tissues from untreatedprotoplasts. Regenerated shoots also rooted more readily anddeveloped more prolific root systems than shoots from untreatedprotoplasts. These observations have important implicationsfor plant genetic manipulation and may have application in therecovery and rooting of shoots from tissues of woody species,normally considered recalcitrant in culture. Key words: Electroporation, protoplasts, shoot regeneration, Solanum dulcamara (woody nightshade, bittersweet)     

The determination of glutathione, cyst(e)ine, and other thiols and disulfides in biological samples using high-performance liquid chromatography with dual electrochemical detection

A determination of glutathione, cysteine, and their disulfides using HPLC and dual electrochemical detection (HPLC-DEC) was described previously but was not validated in biological tissues for these and other important thiols and disulfides (SH/SS). Thus, our objectives were to develop this method to quantify simultaneously reduced and oxidized glutathione, cysteine, cystine, and other SH/SS in various tissues, including human blood and plasma, rat liver and hippocampus, mosquito, and spinach leaf. Optimal conditions were determined for sample processing and analysis using metaphosphoric acid and HPLC-DEC. Authentic standards of 10 common SH/SS compounds were resolved and eluted within 15 min, and all standard curves were linear from 5 to 1600 pmol. Validation was based on the following: First, tissue sample sizes were proportional to peak areas over an eightfold range. Second, recovery of SH/SS added to samples before processing was 96-101%. Finally, the results were equivalent and correlated highly with values for total SH by 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) assay (r2 = 0.996) and for total glutathione by DTNB-GSSG reductase assay (r2 = 0.998). The life span of the Au/Hg electrode was limited to 200-500 samples based on the lineal range of standard curves. On the basis of these results, we believe that this method will fill a long-time need for the simultaneous determination of SH/SS in biological tissues.     

PREPARATION OF GLUTATHIONE IMPRINTED POLYMER

Glutathione imprinted polymer was prepared using 1-vinyl imidazole and ethylene glycol dimethacrylate as the functional monomer and crosslinker, respectively, in dimethyl sulfoxide. The adsorption selectivity of glutathione-imprinted polymer was tested by reduced glutathione, oxidized glutathione, and L-Gly-Leu-Tyr in 30% phosphate buffer (0.01 M, pH 5.0)–70% acetonitrile and binding affinity values were compared. Reusability of molecularly imprinted polymer particles was also investigated. Molecularly imprinted polymer particles were found to be stable and to maintain glutathione adsorption capacity at 95% when washed with methanol–acetic acid (10%) after seven usages. Functional monomer 1-vinyl imidazole and cross linker ethylene glycol dimethacrylate-based glutathione imprinted polymer could be used as solid phase extraction material for recognition of glutathione in biological samples.     

Over-expression of chloroplast-targeted Mn superoxide dismutase in cotton (Gossypium hirsutum L., cv. Coker 312) does not alter the reduction of photosynthesis after short exposures to low temperature and high light intensity

Transgenic cotton plants from several independently-transformed lines expressing a chimeric gene encoding a chloroplast-targeted Mn superoxide dismutase (SOD) from tobacco exhibit a three-fold increase in the total leaf SOD activity, strong Mn SOD activity associated with isolated chloroplasts, and a 30% and 20% increase in ascorbate peroxidase and glutathione reductase activities, respectively. The Mn SOD plants did exhibit a slightly enhanced protection against light-mediated, paraquat-induced cellular damage but only at 0.3 µM paraquat. In addition, photosynthetic rates at 10°C and 15°C were similar to those of controls, and the immediate recovery of photosynthesis after a 35-min exposure to 5°C and full sun was only slightly better than that for wild-type plants. The recovery for longer exposure times was comparable for both genotypes as was the deactivation of the H₂O₂-sensitive, Calvin-cycle enzyme, stromal fructose 1,6-bisphosphatase (FBPase). Compared to the controls, Mn SOD plant leaves in full sun prior to chilling stress had a lower activation of FBPase, a higher ratio of oxidized to reduced forms of ascorbate, and a higher total glutathione content. After 35 min at 5°C in full sunlight, total glutathione had risen in control leaves to 88% of the Mn SOD plant values, and oxidized to reduced ascorbate ratios were higher for both genotypes. However, an 80% increase in the ratio of oxidized to reduced glutathione occurred for Mn SOD plant leaves with no change for controls. This increased demand on the ascorbate-glutathione cycle is circumstantial evidence that high Mn SOD activity in the chloroplast leads to increased H₂O₂ pools that could, in some manner, affect photosynthetic recovery after a stress period. We postulate that the pool sizes of reduced ascorbate and glutathione may restrict the ability of the ascorbate-glutathione cycle to compensate for the increased activity of SOD in cotton over-producing mitochondrial Mn SOD in chloroplasts during short-term chilling/high light stress.     

Ethylene Evolution from Bracts and Leaves of Poinsettia, Euphorbia pulcherrima Willd

Woodrow, L. and Grodzinski, B. 1987. Ethylene evolution trombracts and leaves ol Poinsettia, Euphorbia pulcherrima Willd.—J.exp. Bot. 38: 2024–2032. Ethylene release from fully expanded, red and white bracts andleaves of poinsettia, Euphorbia pulcherrima Willd., was compared.On a laminar (area) basis leaves contained about 50 times morechlorophyll and demonstrated 10 times the photosynthetic rateof the bracts. Both tissues contained starch, however, solublecarbohydrate in the bracts consisted primarily of reducing hexoseswhile the leaves contained mainly sucrose for translocation.The total free alpha-amino nitrogen content of the bract tissuewas twice that of the leaf tissue. The leaves contained moreACC (1-aminocyclopropane-1-carboxylic acid) and produced proportionallymore endogenous C²H⁴ than either the red or white bracts. ACC-stimulated₂H₄ release was also greatest from the green tissue indicatingthat the EFE (ethylene forming enzyme) was most active in theleaves. The specific activity of the ¹⁴C₂H₄/¹²C₂H₄ releasedfrom [2,3-¹⁴C]ACC confirmed ACC as the primary precursor ofC₂H₄ in this tissue. Ethylene release from the non-photosynthetic,bract tissue was not markedly affected by alterations in CO₂or light conditions. In green leaf tissue endogeneous ethylenerelease increased from 1·5 to 6·0 pmol C₂H₄ cm^–2h^–1 while ACC-stimulated ethylene release increased from10 to 35 pmol C₂H₄ cm^2– h^1– as the CO₂ partial pressureincreased from 100 to 1 200 µbar. Key words: Poinsettia, ethylene, bracts     

Cyclic AMP in Rhizobia and Symbiotic Nodules

Cyclic adenosine 3',5'-monophosphate (cAMP) content of variouscultured rhizobia strains and tissues of legumes and non-leguminousplants was measured by enzyme immunoassays. Most rhizobia, culturedfor 44 to 165 h, contained cAMP ranging from 0.6 to 5 pmol mg^-1proteinexcept forAzorhizobium caulinodansORS571. The culture mediaalso contained varying amounts of cAMP depending on the strainof rhizobia.Azorhizobiumcells and their media contained no detectablecAMP. Nodules from most legumes and non-legumes had cAMP contentsranging from 2–70 pmol g^-1f.wt. However, nodules fromSesbaniarostrata,Crotalaria spectabilisandParasponia andersoniishowedundetectable cAMP levels, and those fromGlycine maxandVignaangularisoccasionally showed levels below the detection limit.The leaves of non-legumes mostly had cAMP levels below detectionlimit (approx. 1.0 pmol g^-1 f.wt), while the leaves ofa few legumes occasionally had detectable cAMP. The possiblerole of cAMP as a symbiotic signal is discussed. cAMP; legumes; modules; rhizobia; symbiosis     

SHORT COMMUNICATION Cyclic Nucleotides inFrankiaand Symbiotic Nodules

Cyclic adenosine 3',5'-monophosphate (cAMP) and cyclic guanosine3',5'-monophosphate (cGMP) contents of cultured cells ofFrankiastrainsoriginally isolated from nodules ofAlnus sieboldiana, MyricarubraandElaeagnus macrophyllawere measured by enzyme immunoassays(EIA).Frankiacells, cultured for 59–121 d, had cAMP contentsranging from 2.9 to 76.1 pmol mg^-1protein and cGMP contentsranging from 0.9 to 5.2 pmol mg^-1protein. FollowingFrankiaculture,the media contained extremely large quantities of cAMP and significantlevels of cGMP. The nature of accumulation and secretion ofcyclic nucleotides by slow-growingFrankiacells was comparableto that by a fast-growing actinomyceteStreptomyces lividansTK24,suggesting that secretion of cAMP byFrankiacells may occur throughthe cell membrane but not by cell lysis. cAMP and cGMP contentsin the symbiotic nodules, leaves and roots of actinorrhizalplants and leaves of non-actinorrhizal trees were also measured.The nodules of actinorrhizal woody plants(A. sieboldiana, E.macrophylla, E. umbellata, E. pungensandM. rubra)had cAMP contentsranging from 4 to 258 pmol g^-1f. wt and cGMP contents rangingfrom 1.1 to 5.2 pmol mg^-1protein. Most leaves and some rootsof actinorrhizal plants and all the leaves of non-actinorrhizalwoody plants examined contained small but significant amountsof cAMP and cGMP. This is the first report of significant contentsof cAMP and cGMP in culturedFrankiacells andFrankia-infectednodules. Possible roles of cyclic nucleotides as symbiotic signalsare discussed.Copyright 1998 Annals of Botany Company cAMP, cGMP, actinorrhizal plants, nodules,Frankia,symbiosis.     

Reduction and uptake of methylene blue by human erythrocytes

A thiazine dye reductase has been described in endothelial cells that reduces methylene blue (MB), allowing its uptake into cells. Because a different mechanism of MB uptake in human erythrocytes has been proposed, we measured MB uptake and reduction in this cell type. Oxidized MB (MB⁺) stimulated reduction of extracellular ferricyanide in a time- and concentration-dependent manner, reflecting extracellular reduction of the dye. Reduced MB was then taken up by the cells and partially oxidized to MB⁺. Both forms were retained against a concentration gradient, and their redox cycling induced an oxidant stress in the cells. Whereas concentrations of MB⁺ <5 µM selectively oxidized NAD(P)H, higher concentrations also oxidized both glutathione (GSH) and ascorbate, especially in the absence of D-glucose. MB⁺-stimulated ferricyanide reduction was inhibited by thiol reagents with different mechanisms of action. Phenylarsine oxide, which is selective for vicinal dithiols in proteins, inhibited MB⁺-dependent ferricyanide reduction more strongly than it decreased cell GSH and pentose phosphate cycle activity, and it did not affect cellular NADPH. Open erythrocyte ghost membranes facilitated saturable NAD(P)H oxidation by MB⁺, which was abolished by pretreating ghosts with low concentrations of trypsin and phenylarsine oxide. These results show that erythrocytes sequentially reduce and take up MB⁺, that both reduced and oxidized forms of the dye are concentrated in cells, and that the thiazine dye reductase activity initially responsible for MB⁺ reduction may correspond to MB⁺-dependent NAD(P)H reductase activity in erythrocyte ghosts. thiazine dyes; ascorbic acid; ferricyanide; phenylarsine oxide; oxidant stress; redox cycling     

Some properties of shikimate: NADP oxidoreductase produced in sweet potato root tissue after slicing

The activity of shikimate: NADP oxidoreductase [EC 1. 1. 1.25] in sweet potato root tissue increased soon after slicing.Enzyme preparations obtained from both sliced tissue and fromfresh tissue probably contained a single enzyme component, andthey showed identical chromatographical behaviour. K_m values of the enzyme for NADP and shikimate were 1.0x10^–4Mand 1.3 x 10^–3M, respectively. Enzyme activity was potentlyinhibited by SH-inhibitors such as p-chloromercuribenzoate andoxidized glutathione. Enzyme activity was affected neither by mononucleotides suchas ATP, ADP and AMP, divalent cations, Mg⁺⁺, Ca⁺⁺ and Mn⁺⁺,nor by metabolites such as tryptophan, phenylalanine, tyrosineand t-cinnamic acid which are involved in aromatic compoundsyntheses. The enzyme rapidly lost its activity. This inactivation reactionshowed a time course consisting of two steps of the first-orderreaction. The inactivated enzyme preparation was not reactivatedby thiol compounds such as cysteine, 2-mercaptoethanol and glutathione,although these reagents, to a certain extent, protected theenzyme from inactivation. The results suggest that denaturationof the enzyme protein was involved in inactivation of the enzyme. ¹Part 74 of the phytopathological chemistry of sweet potatowith black rot and injury. ²Present address: Department of Biology, Faculty of Science,Tokyo Metropolitan University, Setagaya-ku, Tokyo. (Received August 5, 1968; )     

Some Properties of an Acid Phosphatase Isolated from the Seed Coats of Developing Pea Seeds

An acid phosphatase (EC 3 1 3 2)isolated from the seed-coatsof developing pea seeds was estimated to have MW 30000 by gelfiltration on Sephadex G-75 It was shown to act on a broad spectrumof physiological substrates, the most preferred being ß-glycerophosphate,3-phosphoglycerate and ADP, wich all showed rates of about halfthe maximum rate shown with p-nitrophenyl phosphate (p-NPP)Another model substrate frequently used in enzyme localizationstudies, -naphthyl acid phosphate, was hydrolysed at about 30% of the rate shown with p-NPP This acid phosphatase was enhancedor stabilized by the chelators EDTA and 1, 10-phenanthrolme,unaffected by Mg²⁺ and N-ethyl maleimide, but strongly inhibitedby Zn²⁺ and F^– Both oxidized and reduced glutathione werewithout effect at low concentration and slightly inhibitoryat high concentration (15 mm) Thiol groups are clearly not involvedin regulating the activity of this acid phosphatase, a featurewhich distinguishes it from acid phosphatases from several otherplant species. Pisum sativum L, pea, acid phosphatase, seed-coats, seed development     

1962—2007年北京地区木本植物秋季物候动态

根据中国物候观测网络北京观测站点的物候资料及气候资料,分析了1962—2007年北京地区20种主要木本植物秋季物候对气候变化的响应情况.结果表明：1962—2007年间,北京地区秋季物候开始日期基本保持不变,但结束日期有所推迟,推迟的幅度为3.2 d·10 a^-1,导致该区秋季延长了约14 d;研究期间,北京地区木本植物秋季叶始变色期均表现为推迟趋势,平均推迟幅度为4.9 d·10 a^-1;平均最低气温是影响北京地区木本植物叶始变色期早晚的主要气候因子.气候增暖可能是导致近40年北京地区木本植物秋季物候期推迟的主要原因.     

Light affects the flux of Ca2+ from excised tomato leaves within four seconds

The flux of Ca²⁺ from excised tomato leaves, conditioned in100 mM KCI for 60 min, was shown to be affected by turning anincandescent light (32 µmol m^–2 s^–1) on oroff. Calcium concentrations were measured with a single junctioncombination electrode connected to a high impedence electrometeramplifier interfaced with a microcomputer. Net Ca²⁺ fluxes fromexcised leaves 30 s prior to and 30 s after turning on the lightwere 68 and 122pmol g ^–1 dry weight s ^–1 respectively.The Ca²⁺ fluxes for the 30 s prior to and 30 s after turningoff the light were 113 and 51 pmol g^–1 dry weight s^–1respectively. Close examination of the first 10 s after thelight was turned off showed that there was a 4 s delay in theflux of Ca²⁺ . The heat given off by the incandescent bulb hadno effect on Ca²⁺ flux during these short time periods. Theeffect of light on the Ca²⁺ flux was evident for at least 2h after the initial treatment. Key words: Ca²⁺, signalling, light, tomato     

Effects of Continuous SO2 Fumigation on SH-containing Compounds in Two Wheat Cultivars of Different Sensitivities

Two cultivars (Mec and Chiarano) of wheat (Triticum aestivum)were exposed to constant low levels of SO₂ (35, 75 and 120 nll^–1) over a period of 4 months. In previous studies Mechas been shown to be more sensitive to SO₂ and this has beenconfirmed in the present study where Mec showed a greater tendencythan Chiarano to accumulate soluble non-protein SH compounds,principally glutathione and cysteine. The reduced glutathione to oxidized glutathione ratio (GSH/GSSG)increased significantly in Mec with SO₂ treatment, but no changein the activity of glutathione reductase was observed. Key words: Wheat, SO₂ fumigation, SH-compounds     

Endogenous and induced monooxygenase activity in gypsy moth larvae feeding on natural and artificial diets

NADPH oxidase activity was measured in third to sixth instar gypsy moth larvae fed oak or pine foliage. Activity levels ranged from 400 to 1,900 pmol NADPH oxidized/min/mg microsomal protein, but enzyme activity was not correlated with host plant ingested. Similarly, activity levels in larvae fed diets containing inducers, such as the terpenoid α-pinene or pentamethylbenzene, ranged from 700 to 1,500 pmol NADPH oxidized/min/mg protein, levels that were comparable to those measured for larvae fed control diets. O-demethylase activity in older instar gypsy moth larvae fed pine averaged 109 pmol p-nitrophenol/min/mg protein, and activity levels in those fed diet containing α-pinene ranged from 22 to 55 pmol/min/mg protein. Although statistically significant, these induced O-demethylase levels are well below those observed for Heliothis zea larvae. Our findings indicate that monooxygenases play a minor, if any, role in the ability of later instar gypsy moth larvae to develop successfully on pine foliage.     

Sialic acid concentrations in plants are in the range of inadvertent contamination

The long held but challenged view that plants do not synthesize sialic acids was re-evaluated using two different procedures to isolate putative sialic acid containing material from plant tissues and cells. The extracts were reacted with 1,2-diamino-4,5-methylene dioxybenzene and the fluorescently labelled 2-keto sugar acids analysed by reversed phase and normal phase HPLC and by HPLC–electrospray tandem mass spectrometry. No N-glycolylneuraminic acid was found in the protein fraction from Arabidopsis thaliana MM2d cells. However, we did detect 3-deoxy-d-manno-octulosonic acid and trace amounts (3–18 pmol/g fresh weight) of a compound indistinguishable from N-acetylneuraminic acid by its retention time and its mass spectral fragmentation pattern. Thus, plant cells and tissues contain five orders of magnitude less sialic acid than mammalian tissues such as porcine liver. Similar or lower amounts of N-acetylneuraminic acid were detected in tobacco cells, mung bean sprouts, apple and banana. Yet even yeast and buffer blanks, when subjected to the same isolation procedures, apparently contained the equivalent of 5 pmol of sialic acid per gram of material. Thus, we conclude that it is not possible to demonstrate unequivocally that plants synthesize sialic acids because the amounts of these sugars detected in plant cells and tissues are so small that they may originate from extraneous contaminants.     

In Vitro Floral Development of Oilseed Rape (Brassica napus L.): The Effects of pH and Plant Growth Regulators

The effects of various plant growth regulators and that of pHon the in vitro growth and development of young inflorescencesof Brassica napus L. cv. Westar were examined. A cytokinin wasrequired for normal maturation of floral buds, including thecompletion of microsporogenesis, and it stimulated the initiationof additional buds on the inflorescence axis. Benzylaminopurine(BAP) was the most effective of the cytokinins tested. Gibberellicacid (GA₃) and naphthaleneacetic acid (NAA) alone were ineffective.In combination with BAP, both reduced the positive influenceof the cytokinin but GA₃ was more inhibitory than NAA. At alow initial pH (3.9–4.6), the percentage of cultures whichproduced normal buds was significantly higher, especially inthe presence of 10^-7 M or 5 ? 10^-7 M BAP, in comparison to cultureswith a pH of 5.3-6.0, the standard range for plant tissue culture.     

Development and Characterization of a Monoclonal Antibody to Phytosiderophores

Mugineic acid-family phytosiderophores (MAs) are low molecularweight chelators that are secreted by graminaceous plants, formcomplexes with soil Fe(III) and are essential for plant growth.Methods to detect MAs which include HPLC and radio-immunoassaywith polyclonal antibody require sophisticated equipment orradio-labelled MAs which are difficult to synthesize. Our objectivewas to develop a detection and quantitation system for MAs basedon monoclonal antibody specificity and technology. A monoclonalantibody was produced which reacts with nicotianamine (NA),deoxymugineic acid (DMA), mugineic acid (MA) and epi-hydroxymugineicacid (epi-HMA) in a competitive ELISA. Azetidine-2-carboxylicacid (A-2-C) was not reactive while N-(3-amino-3-carboxypropyl)azetidine-2-carboxylic acid (A-2-C dimer) was partially reactive.The range of detection using the competitive ELISA is from 2x 10^–6 to 2 x 10^–7 M MAs. Besides detection andquantification of MAs, the potential uses for the monoclonalantibody are numerous and include affinity chromatography andimmunocytochemistry. (Received September 26, 1991; Accepted December 16, 1991)     

Effects of Chemical Antitranspirants on Transpiration and Growth of Grass

The effects of foliar sprays of the metabolic inhibitors dodecenylsuccinicacid (DSA) and phenylmercuric acetate (PMA), as antitranspirants,were tested on grass grown outdoors (in lysimeters), but moreaccurate tests were made with PMA in growth rooms, using smallweighable transpirometers. Concentrations of PMA which wereweaker than 10^-3.8 M resulted in only slight reductions in transpiration,whereas concentrations stronger than 10^-3.2 M were phytotoxic,though water losses were reduced by about 30 per cent. PMA at10^-3.5 M gave the greatest decrease in transpiration (about20 per cent) without reducing growth, but its effectivenessdepended on the amount applied per unit area of vegetation.The effects of PMA also differed with plant species and withenvironment, being greatest under conditions of low soil moisturestress and temperature. The antitranspirant reduced stomatalapertures and increased leaf temperatures.