Responses of Stomata in Epidermal Strips of Vicia fabato Carbon Dioxide and Growth Hormones when Incubated on Potassium Chloride and Potassium Iminodiacetate

The effect of various K⁺ levels in combination with Cl^− or iminodiacetate (IDA^{& minus;}) on stomatal responsesin isolated epidermal strips of Vicia faba L. were examinedin order to determine the role of malate during guard cell movements.Responses of guard cells to ABA, kinetin, and varying CO₂ levelswere similar when epidermal strips were floated on KCL or KIDAat 10 mM; such responses were typical in that ABA caused closure,kinetin stimulated opening in ambient air, and apertures weregreater in CO₂-free than ambient air. Maximal stomatal openingwas observed in both ambient and CO₂-free air with KCL at 100mM. The transfer of epidermal strips from 100 mM KCL to solutionsof 100 mM KCL supplemented with ABA or kinetin did not bringabout changes in stomatal aperture. KCL at 100 mM supporteda greater degree of stomatal opening than did 100 mM KIDA irrespectiveof the CO₂ content of the air. In CO₂-free air transfer of epidermalstrips from 100 mM KIDA to solutions containing 100 mM KIDAsupplemented with ABA or kinetin caused little change in stomatalaperture, whereas, in ambient air, the same treatments resultedin stomatal opening. The results are discussed in relation tothe role of malate during guard cell movements.     

Stimulation of Net Photosynthesis in Cucumber Leaf Discs: Effect of K-Glyoxylate and KCL

Incubation of leaf discs of Cucumis sativus in 15 mol m^–3K-glyoxylate (pH 4.6) doubled the rate of net photosynthesisat limiting CO₂ or HCO^–₃ compared with discs floated ondistilled water. When both control and treated discs were incubatedin Mes-TMAOH buffer at pH 5.0, K-glyoxylate still increasednet photosynthesis by as much as 70%. The tetramethylammoniumsalt (TMA-glyoxylate) was without effect but KCl enhanced netphotosynthesis. Both KCl and K-glyoxylate increased stomatalaperture at pH 5.0. At pH 7.5 (Mops-TMAOH), neither stomatalaperture or photosynthesis was altered by K-glyoxylate, KClor TMA-glyoxylate. None of these salts was found to stimulatephotosynthesis in isolated cucumber mesophyll cells over a rangeof pH values although the cells incorporated as much ¹⁴C-glyoxylateas did leaf discs. The data suggest that enhanced photosynthesisin leaf discs is not due directly to a stimulation of mesophyllcell photosynthesis but rather is a consequence of increasedCO₂ availability and decreased stomatal resistance at low pHin the presence of potassium. Key words: Cucumber, Photosynthesis, Potassium glyoxylate     

Retardation of Leaf Senescence by Ascorbic Acid

Leaf discs of Solatium melongena were floated on various concentrationsof ascorbic acid (AA), gibberellic acid (GA₃), and kinetin inorder to study their effect on senescence. AA was highly effectivein retarding senescence as shown by the arrest of the fall inlevels of chlorophyll, DNA, RNA, and proteins. AA was effectiveat a lower concentration than that of GA₃ or kinetin.     

Effects of Plant Growth Regulators on Grain-filling and Yield of Rice

Effects of three phytohormones (IAA₁, GA₃ and kinetin) on grain-fillingand the pattern of ³²P translocation from individual leavesto grains were studied at intervals of 7 days during the progressof reproductive development of rice (Oryza saliva L. cv. Jaya).The plants were sprayed with 100 µg ml^–1 aqueoussolutions of the hormones at 100 days, when the plants wereentering the reproductive stage. Kinetin produced a pronouncedeffect on grain-filling as well as on ³²P mobilization fromindividual leaf to grains and increased yield, possibly by increasingleaf longevity. GA₃ and IAA also increased the grain-fillingand ³²P mobilization significantly over control but the effectswere less marked than those of kinetin. Oryza sativa L., rice, grain yield, translocation, growth regulators, gibberellic acid, indol-3-yl acetic acid, kinetin     

Kinetin Reversal of NaCl Effects

Leaf discs of Nicotiana rustica L. were floated on NaCl in the presence of kinetin or abscisic acid. On the 5th day ¹⁴CO₂ fixation, [³H]leucine incorporation, stomatal conductance, and chlorophyll content were determined. Kinetin either partially or completely reversed the inhibitory effects of NaCl while ABA had no effect.     

Photosynthesis and Diffusion Conductance of the Valencia Orange Fruit under Field Conditions

CO₂ uptake and diffusion conductance of Valencia orange fruits(Citrus sinensis L. Osbeck) were measured in the field duringthe growing season of 1977/78 to ascertain if, as in the leaf,stomata control photosynthesis and transpiration under changingenvironmental conditions. Measurements were made on 15 yearold trees grown in a sandy loam soil and receiving either adry or a wet treatment. Fruit diffusive conductance was measuredwith a modified water vapour diffusion conductance meter andgross photosynthesis was measured with a ¹⁴CO₂ uptake meter.Photosynthetically active radiation (PAR) was measured witha quantum sensor. Fruits exposed to light assimilated CO₂ ata rate which was 25–50% of that assimilated by leaves.The uptake was dependent on fruit size, PAR, chlorophyll content,and on diffusive conductance of the fruit epidermis. Epidermalconductance showed a diurnal trend which was similar in shapeto that of the leaf except in the late afternoon. Cuticularconductance of the fruit was calculated and ranged between 0.22and 0.30 mm s^–1. It was speculated that the CO₂ uptakeby the fruit could support the growth of flavedo cell layerswhen exposed to light. Dry soil caused an increase in the ¹⁴CO₂uptake by fruit possibly caused by the increased potential areaof the stomatal opening per unit of fruit surface area.     

Comparison of Palaeobotanical Observations with Experimental Data on the Leaf Anatomy of Durmast Oak [Quercus petraea(Fagaceae)] in Response to Environmental Change

To test whether stomatal density measurements on oak leaf remainsare reliable tools for assessing palaeoatmospheric carbon dioxideconcentration [CO₂], under changing Late Miocene palaeoenvironmentalconditions, young seedings of oak (Quercus petraea,Liebl.) weregrown at elevatedvs.ambient atmospheric [CO₂] and at high humiditycombined with an increased air temperature. The leaf anatomyof the young oaks was compared with that of fossil leaves ofthe same species. In the experiments, stomatal density and stomatalindex were significantly decreased at elevated [CO₂] in comparisonto ambient [CO₂]. Elevated [CO₂] induced leaf cell expansionand reduced the intercellular air space by 35%. Leaf cell sizeor length were also stimulated at high air humidity and temperature.Regardless of a temperate or subtropical palaeoclimate, leafcell size in fossil oak was not enhanced, since neither epidermalcell density nor length of the stomatal apparatus changed. Theabsence of these effects may be attributed to the phenologicalresponse of trees to climatic changes that balanced temporalchanges in environmental variables to maintain leaf growth underoptimal and stable conditions.Quercus petraea,which evolvedunder recurring depletions in the palaeoatmospheric [CO₂], maypossess sufficient phenotypic plasticity to alter stomatal frequencyin hypostomatous leaves allowing high maximum stomatal conductanceand high assimilation rates during these phases of low [CO₂].Copyright1998 Annals of Botany Company Atmospheric CO₂, high humidity, elevated temperature,Quercus petraea,durmast oak, Late Miocene, palaeoclimates, leaf anatomy, stomatal density, stomatal index     

The Effects of Cytokinins and ABA on Stomatal Behaviour of Maize and Commelina

Epidermal strips and leaf fragments of Commelina and leaf fragmentsof maize were incubated on solutions containing naturally-occurringor synthetic cytokinins and/or ABA. The effects of these treatmentson stomatal behaviour were assessed. Cytokinins alone did notpromote stomatal opening in either species but concentrationsof both zeatin and kinetin from 10^–3 to 10^–1 molm^–3 caused some reversal of ABA-stimulated closure ofmaize stomata. The reversal of the ABA effect increased withincreasing cytokinin concentration. Cytokinins had no effecton ABA-stimulated closure of Commelina stomata. When appliedalone, at high concentration (10^–1 mol m^–3), toCommelina epidermis or leaf pieces both zeatin and kinetin restrictedstomatal opening. Key words: ABA, Cytokinins, Stomata, Maize, Commelina     

Auxin-gibberellin relationships in their effects on hypocotyl elongation of light-grown cucumber seedlings VI. Promotive and inhibitory effects of kinetin

The interaction of kinetin with IAA and GA₃ on the elongationof hypocotyl sections of Cucumis sativus L. cv. National Picklingwas studied. Kinetin in the concentration range of 10^–7M to 10^–4 M markedly inhibited IAA-induced elongation,while in a lower range from 10^–10 M to 10^–8 M, itsynergistically enhanced IAA-induced elongation. Kinetin alonein this range had no effect. A 5-to 15-min pulse treatment seemsenough to induce the maximum effect for both inhibition andpromotion. Since the magnitude of the maximum inhibition dependedon the concentration and not on the duration of treatment, thereaction in the cell caused by kinetin seemed to be completedwithin a short period. Washing of the sections with distilledwater after kinetin treatment (30 min) did not significantlyeliminate the kinetin effect. This probably indicates that thebinding of kinetin molecules to a supposed acceptor is not reversible.Interaction of kinetin with GA₃ in their pretreatment effectson IAA-induced elongation shows that in the inhibitory concentrationrange, the kinetin effect was partly overcome by GA₃, and thatin the promotive range, the magnitude of the enhancement wasdetermined by kinetin regardless of the presence of GA₃. Theeffect of kinetin seems to dominate over that of GA₃ indicatingthat the modes of their pretreatment effects differ from oneanother. (Received June 24, 1977; )     

Effect of the Mesophyll on Stomatal Opening in Commelina communis

The effect of a number of factors on the opening of stomatain the intact leaf and in the isolated leaf epidermis of Commelinacommunishas been investigated. Stomata in the intact leaf opened widein the light and closed rapidly on transfer to the dark. Theywere also sensitive to CO₂. In contrast, stomata in isolatedepidermis floated on an incubation solution containing 100 molm^–3KCl responded neither to light nor CO₂. They opened as widelyas those in the intact leaf when treated with fusicoccin. Stomata in isolated epidermis opened almost as wide as thosein the intact leaf when they were incubated with isolatedmesophyllcells in the light. The solution in which the mesophyll cellswere incubated was separated by centrifugation. Themedium fromcells previously incubated in the light caused the stomata inisolated epidermis to open but that from cells kept inthe darkhad no effect. A similar effect was observed when isolated chloroplastswere incubated with the isolated epidermis.However, the supernatantfrom the chloroplast suspension had no significant effect onstomatal opening. These results indicate that the mesophyll plays an importantrole in stomatal opening in the light. The mesophyll appearstoproduce in the light, but not in the dark, a soluble compoundwhich moves to the guard cells to bring about stomatal opening.Theexperiments with isolated chloroplasts suggest that this substanceis a product of photosynthesis. Key words: Commelina communis, stomata, light, mesophyll     

Flag Leaf Photosynthesis of Triticum aestivum and Related Diploid and Tetraploid Species

Rates of net photosynthesis of the flag leaves of 15 genotypesof wheat and related species were measured throughout theirlife, using intact leaves on plants grown in the field. At thestage when rates were maximal, they were in general highestfor the diploid species, intermediate for the tetraploidspeciesand lowest for Triticum aestivum (means of 38, 32 and 28 mgCO₂ dm^–2 h^–1 respectively). Rates were stronglynegatively correlated with leaf area, leaf width and the meanplan area per mesophyll cell and positvely correlated with stomatalfrequency and number of veins per mm of leaf width. The differencesamong species in these attributes were mainly related to ploidylevel. It was not possible to determine the relative importanceof each anatomical feature, though the changes in stomatal frequencyhad only slight effects on stomatal conductance and the observeddifferences in rates of photosynthesis were much greater thanwould be expected from those in stomatal conductance alone. There was genetic variation in rates of light dependent oxygenevolution of isolated protoplasts and intact chloroplasts butno difference attributable to ploidy. The mean rate, 91 µmolO₂ mg^–1 chlorophyll h^–1, equivalent to 3.9 mg CO₂mg^-1chlorophyll h^-1 was considerably less than the rate of photosynthesisin comparable intact leaves, which was 7.2 mg CO₂ mg^–1chlorophyll h^–1. The total above-ground dry matter yields were least for thewild diploids T. urartu and T. thauodar and the wild tetraploidT. dicoccoides, but the other wild diploids produced as muchdry matter as the hexaploids. The prospects of exploiting differences in photosynthetic ratein the breeding of higher yielding varieties are discussed. Triticum aestivum L., wheat, Aegilops spp, photosynthesis, stomatal conductance, stomatal frequency, polyploidy     

Stomatal Responses and the Senescence of Leaves

Guard cell responses were examined in green and senescing leavesof Victa faba using detached epidermal strips to eliminate influencesfrom the mesophyll. Stomatal opening was greater in epidermalstrips from mature leaves than from senescing leaves althoughthe latter still retained the ability to respond to CO₂ andto kinetin. It was concluded that the decline in stomatal activityduring senescence is an independent but parallel process tochanges occurring in the mesophyll. Vicia faba, leaf senescence, stomata, kinetin     

Acclimation in Seedlings of a Tropical Tree, Bischofia javanica, Following a Stepwise Reduction in Light

Acclimation of fully developed leaves of Bischofia javanicaBlume to shadelight was examined. Seedlings were grown undersimulated daylight (1000 µmol m^–2 s^–1), thentransferred to a simulated shadelight (40 µmol m^–2s^–1). When a high-light leaf was transferred to low light, large negativenet photosynthetic rates (P_m) were recorded. This decrease wasrapid, but within 7 d the rate increased and became equal tothe low-light control leaf. These changes in photosynthesisdid not follow the pattern of changes in stomatal conductance(g_s). Transfer to the low light resulted in a dramatic decreasein leaf weight per unit area (L_w), and most of the decreasesin L_w occurred within 3 d of transfer when the P_m of the transferredleaf was well below that of the low-light control leaf. There was a significant decrease in chlorophyll a in the transferredleaf without an appreciable change in chlorophyll b resultingin a large decrease in the chlorophyll a to chlorophyll b ratio.Leaf chlorophylls per unit area were higher in the transferredleaf than the low-light control leaf. Maximum photosyntheticrate in the transferred leaf was decreased by 40% compared tothat for the high-control leaf, but was almost at the same extenthigher than the low-light control leaf The results are discussedin the context of carbon gain capacity of its seedlings underlight-limiting forest understorey habitats. Bischofia, chlorophylls, light, photosynthesis, shade acclimation, tree seedlings, tropical tree     

Studies on the Mechanisms of Action of the Antitranspirant Phenylmercuric Acetate, and its Penetration into the Mesophyll

Experiments have examined the effect of phenylmercuric acetate(PMA) on the guard cells of Commelina communis. In one series,PMA was supplied to the leaf surface; after different time intervalsthe epidermis was removed and the ability of the stomata toopen was determined. In the other series, different concentrationsof PMA were included in the medium used for inccubating epidermalstrips with which ion-stimulated stomatal opening was assayed.At concentrations of 10^-54 M and above the effect of PMA wassevere and the structural integrity of the guard cells was affected;they were unable to accumulate neutral red. At concentrationsarpound 10^-6 M the guard cells were less affected and PMA broughtabout a transient stimulation of stomatal opening by releasingsubsidiary-cell turgor pressure. A solution of 5 x 10^-4 M PMA applied to leaves reduced by halfthe photosynthetic ¹⁴CO₂ incorporation into C. communis mesophyll.In Zea mays it increased the CO₂ compensation point and alsothe resistance to diffusion in the gas phase (R_A, but therewas a proportionately greater increase in the apparent liquidphase resistance (R_t). This direct inhibition of mesophyll photosynthesisundermines one of the major objectives of applying anatitranspirants,and for this reason it is suggested that PMA is unsuitable forgeneral application to crops.     

Kinetin and carbohydrate metabolism in chinese cabbage

The effects of kinetin on starch and sugar levels and on ¹⁴CO₂ and ³²P-orthophosphate labeling patterns of floated Chinese cabbage (Brassica pekinensis) leaf discs were investigated. Kinetin caused gross starch degradation. Neutral sugars were depressed by 30 to 40% in leaf tissue treated with kinetin for 24 hours. ¹⁴CO₂ labeling of leaf discs pretreated with kinetin for 24 hours showed increased radioactivity in chloroform-soluble material and most sugar phosphates, and a 35 to 40% decrease in radioactivity in the neutral sugars, glucose, sucrose, and fructose. Incorporation into ATP was increased by 40% by kinetin. ³²P-Orthophosphate uptake was inhibited 30% by kinetin. When corrected for uptake, kinetin stimulated incorporation into chloroform-soluble material but had little effect on other cell fractions. These results indicate that kinetin mobilizes starch reserves and increases the flow of sugars required for the synthesis of lipids and structural materials in floated discs.     

After-effect of Water Stress on Stomatal Opening Potential: II. POSSIBLE CAUSES

Experiments were carried out to elucidate the temporary after-effectof a period of water stress on the opening potential of stomataof tobacco (Nicotiana tabaccum) and beans (Vicia faba). Stomatalaperture was measured on floating leaf discs or epidermal stripsafter a period of time in light under controlled conditions;direct effects of leaf-water deficit were thus avoided. Theresponses of the two species were not qualitatively different. Only a minor part of the post-stress damage was located in themesophyll in beans, the major part (approximately two-thirds)being located in the guard cells themselves. The CO¹ compensationpoint for tobacco leaves showed no after-effect of stress. However,the response of stomata to normal and CO¹-free air in both speciessuggested that a minor part of the after-effect on stomatalopening, presumably that part arising in the mesophyll, maybe explained by elevated CO¹ concentrations in the stomatalcavity. The nature of that part of the after-effect arising within theguard cells themselves was not clarified. Guard-cell starchcontent was apparently unaffected by stress in both species;in tobacco, epidermal-cell starch content was reduced in thestress and post-stress condition. Stomatal responses to theshort-term addition of energy-supplying metabolites (ATP, glycolate,and glucose-1-phosphate) showed no interaction with the post-stresscondition. Similarly responses to the addition of stomatal inhibitors(sodium azide, dodecenylsuccinic acid, atrazine, and phenylmercuricacetate) did not indicate the exact nature of the after-effect,although there was a marked increase in the sensitivity to atrazinein post-stress leaf discs. In tobacco leaf discs an examination of the process of eliminationof this after-effect, as distinct from studies directly on itsnature, suggested that complete recovery involved at least twosteps, together lasting 2 to 3 days. One step required timeand was independent of light; the other required light of atleast 1 klx for a day or more and did not occur in CO²-freeair. Stomata recovered almost completely in 2 days without lightwhen discs were floated on 005 M glucose-1-phosphate.     

Effect of Elevated CO2 on Stomatal Density and Distribution in a C4 Grass and a C3 Forb under Field Conditions

Two common tallgrass prairie species, Andropogon gerardii, thedominant C₄ grass in this North American grassland, and Salviapitcheri, a C₃ forb, were exposed to ambient and elevated (twiceambient) CO₂ within open-top chambers throughout the 1993 growingseason. After full canopy development, stomatal density on abaxialand adaxial surfaces, guard cell length and specific leaf mass(SLM; mg cm^-2) were determined for plants in the chambers aswell as in adjacent unchambered plots. Record high rainfallamounts during the 1993 growing season minimized water stressin these plants (leaf xylem pressure potential was usually >-1·5 MPa in A. gerardii) and also minimized differencesin water status among treatments. In A. gerardii, stomatal densitywas significantly higher (190 ± 7 mm^-2; mean ±s.e.) in plants grown outside of the chambers compared to plantsthat developed inside the ambient CO₂ chambers (161 ±5 mm^-2). Thus, there was a significant 'chamber effect' on stomataldensity. At elevated levels of CO₂, stomatal density was evenlower (P < 0·05; 121 ± 5 mm^-2). Most stomatawere on abaxial leaf surfaces in this grass, but the ratio ofadaxial to abaxial stomatal density was greater at elevatedlevels of CO₂. In S. pitcheri, stomatal density was also significantlylower when plants were grown in the open-top chambers (235 ±10 mm^-2 outside vs. 140 ± 6 mm^-2 in the ambient CO₂ chamber).However, stomatal density was greater at elevated CO₂ (218 ±12 mm^-2) compared to plants from the ambient CO₂ chamber. Theratio of stomata on adaxial vs. abaxial surfaces did not varysignificantly in this herb. Guard cell lengths were not significantlyaffected by growth in the chambers or by elevated CO₂ for eitherspecies. Growth within the chambers resulted in lower SLM inS. pitcheri, but CO₂ concentration had no effect. In A. gerardii,SLM was lower at elevated CO₂. These results indicate that stomataland leaf responses to elevated CO₂ are species specific, andreinforce the need to assess chamber effects along with treatmenteffects (CO₂) when using open-top chambers.Copyright 1994, 1999Academic Press Andropogon gerardii, elevated CO₂, Salvia pitcheri, stomatal density, tallgrass prairie     

The Effects of CO2 Enrichment and Nutrient Supply on Growth Morphology and Anatomy of Phaseolus vulgaris L. Seedlings

Plants of Phaseolus vulgaris were grown from seed in open-topgrowth chambers at the present (P, 350 µmol mol^–1)atmospheric CO₂ concentration and at an elevated (E, 700 µmolmol^–1) CO₂ concentration, and at low (L, without additionalnutrient solution) and high (H, with additional nutrient solution)nutrient supply for 28 d The effects of CO₂ and nutrient availabilitywere examined on growth, morphological and biochemical characteristics Leaf area and dry mass were significantly increased by CO₂ enrichmentand by high nutrient supply Stomatal density, stomatal indexand epidermal cell density were not affected by elevated CO₂concentration or by nutrient supply Leaf thickness respondedpositively to CO₂ increasing particularly in mesophyll areaas a result of cell enlargement Intercellular air spaces inthe mesophyll decreased slightly in plants grown in elevatedCO₂ Leaf chlorophyll content per unit area or dry mass was significantlylower in elevated CO₂ grown plants and increased significantlywith increasing nutrient availability The content of reducingcarbohydrates of leaves, stem, and roots was not affected byCO₂ but was significantly increased by nutrient addition inall plant parts Starch content in leaves and stem was significantlyincreased by elevated CO₂ concentration and by high nutrientsupply Phaseolus vulgaris, elevated atmospheric CO₂, CO₂-nutrient interaction, stomatal density, leaf anatomy, chlorophyll, carbohydrates, starch     

Tentoxin Suppresses Stomatal Opening by Inhibiting Photophosphorylation

Tentoxin and, to a lesser extent, dihydrotentoxin (both at 10mmol m^–3) reduce stomatal opening in epidermal stripsof Commelina communis in the light but not in darkness. Thiseffect was significantly greater in normal air than in CO₂-freeair. Fusicoccin overcame the tentoxin effect. However, tentoxindid not inhibit stomatal opening in the light in epidermal stripsof Paphiopedilum harrisianum, a species which lacks guard cellchloroplasts. It is concluded that tentoxin exerts its actionon stomata not by an ionophorous effect in the plasmalemma ofguard cells but by the inhibition of photophosphorylation intheir chloroplasts. The effects of DCMU and tentoxin on guardcells are discussed in terms of their effects on chloroplastsand the extent to which energy is supplied from this organelleduring stomatal opening in the light. The results indicate thatneither photophosphorylation nor non-cyclic electron transportin guard cell chloroplasts are essential for stomatal opening. Key words: Commelina, epidermal strips, Paphiopedilum, photophosphorylation, stomata, tentoxin     

Acclimation of Lolium temulentum to enhanced carbon dioxide concentration

Acclimation of single plants of Lolium temulentum to changing[CO₂] was studied on plants grown in controlled environmentsat 20°C with an 8 h photoperiod. In the first experimentplants were grown at 135 µ;mol m^–2 s^–1 photosyntheticphoton flux density (PPFD) at 415µl l^–1 or 550µll^–1 [CO₂] with some plants transferred from the lowerto the higher [CO₂] at emergence of leaf 4. In the second experimentplants were grown at 135 and 500 µmol m^–2 s^–1PPFD at 345 and 575 µl l^–1 [CO₂]. High [CO₂] during growth had little effect on stomatal density,total soluble proteins, chlorophyll a content, amount of Rubiscoor cytochrome f. However, increasing [CO₂] during measurementincreased photosynthetic rates, particularly in high light.Plants grown in the higher [CO₂] had greater leaf extension,leaf and plant growth rates in low but not in high light. Theresults are discussed in relation to the limitation of growthby sink capacity and the modifications in the plant which allowthe storage of extra assimilates at high [CO₂]. Key words: Lolium, carbon dioxide, photosynthesis, growth, stomatal density