Apoplastic pH of intact leaves of Vicia faba as influenced by light

The fluorochrome FITC-dextran was used to measure the effectof light on the apoplastic pH of intact Vicia faba leaves withthe ratio imaging technique. In darkadapted leaves the apoplasticpH varied depending on the leaf between 5.2 and 5.9. Red light(660 nm, 4–12 W m^–2) leads to multiphasic responses:in the first seconds an alkalinization ({small tilde}0.3 pHunits), and thereafter an acidification of the leaf apoplast({small tilde}0.4 pH units) were observed. Both effects couldbe inhibited by DCMU. While variation of CO₂ concentration revealedno effect on light-induced apoplastic pH changes, a decreasein O₂ concentration decreased the effect. On the basis of ourdata it is suggested that the influence of photosynthesis onplasmalemma H⁺ ATPase is responsible for the observed effects,rather than altered CO₂ uptake. Key words: Leaf apoplast, apoplastic pH, light, ratio imaging, pH-sensitive fluorescent dye, Vicia fab     

Inverse Correlation of Thiol Proteinase with Nitrate Reductase Activities in Barley Leaves

Experiments conducted to determine the effects of leupeptin,a specific inhibitor of thiol proteinase, on extractable nitratereductase (NR) activity in leaves of Hordeum distichum duringdarkness revealed that leupeptin (0.01 mg.ml^–1) appliedto detached leaves significantly reduced the loss of NR activity.At the same time it also reduced the formation of small cytochromec reductase species, which is a degradation product of NR complex,Upon nitrate induction, extractable NR activity increased butthe content of thiol proteinase decreased. This inverse correlationwas also observed upon transfer of nitrate-grown barley seedlingsto nitrate-free nutrient solution. Furthermore, cycloheximide(0.1 mg.ml^–1) treatment of barley seedlings reduced thecontent of thiol proteinase and retarded the loss of NR activityunder noninducing conditions. These results suggest that invivo changes in NR content in leaves of Hordeum distichum arethe result of proteolysis by an endogenous thiol proteinase. (Received May 16, 1985; Accepted July 22, 1985)     

Measurement of the Apoplastic Activity of K+ and Cl- in the Leaf Epidermis of Commelina communis in Relation to Stomatal Activity

Bowling, D. J. F. 1987. Measurement of the apoplastic activityof K⁺ and Cl^– in the leaf epidermis of Commelina communisin relation to stomatal activity.–J. exp. Bot. 38: 1351–1355. Ionic activity of K⁺ and Cl^– in the apoplast of the lowerepidermis of the leaf of Commelina communis was measured usingion selective micro-electrodes. Large gradients across the stomatalcomplex were observed which were related to stomatal aperture.On stomatal closure the activity of K⁺ and Cl^– in theapoplast of the guard cell rose from 3·0 mol m^–3to 100 mol m^–3 and 33 mol m^–3 respectively. It wasconcluded that the apoplast is an important pathway for iontransport between the cells. Key words: Stomata, ionic activity, leaves, apoplast     

Apoplastic Solute Concentrations of Organic Acids and Mineral Nutrients in the Leaves of Several Fagaceae

Ion chromatographic methods determined organic acids and mainnutrient minerals in the apoplastic solution from leaves ofseveral Fagaceae (Quercus ilex L., Quercus cerris L., Quercusvirgiliana (Ten.) Ten, and Fagus sylvatica L.). The anions oforganic acids found in high amounts (250 to 650 µM) werequinate, malate, and oxalate. Lactate, pyruvate, formate andacetate were detected in relatively low amounts with concentrationsbetween 20 and 200 µM. The total concentration of organicacids in the apoplastic sap ranged between 1.5 and 2 mM. Thetotal concentration of inorganic cations (K⁺, Mg²⁺, NH₄⁺, Ca²⁺,Na⁺) and anions (C1^–, NO₃^–, SO^2–₄ and PO^3–₄)in the apoplastic sap varied between 5 and 10 mM, and 0.35 and1.8 mM, respectively. We conclude that the concentration oforganic acid ions in the leaf apoplast depends mainly on theexchange with the leaf cells and is influenced by the electrochemicalgradient between the symplast and the apoplast in relation tothe water potential of the leaf. The determination of formateand acetate in the apoplastic compartment of leaves lend weightto the argument that the production of these acids by treesis a important emission source to the atmosphere. (Received June 9, 1998; Accepted April 8, 1999)     

Action of Thiol Proteinase on Nitrate Reductase in Leaves of Hordeum distichum L.

Nitrate reductase (NR) from the leaves of Hordeum distichumwas very susceptible to inactivation by barley leaf thiol proteinase,trypsin, and papain. A cytochrome c reductase species with amolecular weight of about 40,000 was derived from the NR complexby the proteolytic actions. The barley proteinase seemed toattack the Mo⁺-containing component of NR, just like trypsinand papain. It showed a preference for the alanine and tryptophanesters among the carbobenzoxyamino acid-nitrophenylesters tested. In vivo NR activity in the presence of leupeptin was fairlyhigher than that in its absence. Leupeptin also protected NRfrom its cleavage to small cytochrome c reductase species, suggestingthat the barley proteinase may play a role in the in vivo changein NR activity. (Received May 21, 1984; Accepted September 10, 1984)     

Response of Cultured Embryos of Phaseolus vulgaris and Hordeum vulgare to Farnesol

Excised embryos of Phaseolus vulgaris incubated in a mediumcontaining 10 mg dm^–3 farnesol showed enhanced root growthwhereas the leaves remained rudimentary At lower concentrationsof exogenous farnesol normal leaf development occurred and rootgrowth was comparable to untreated cultures. Enhanced root growthalso occurred when excised embryos of Hordeum vulgare were treatedwith farnesol but only at 10 mg dm^–3 and this treatmentdid not prevent leaf growth X-ray micro-probe analysis of leavesrevealed an increased phosphorus content in P vulgaris and adecreased sulphur content in H vulgare in comparison to untreatedplants. Hordeum vulgare L., barley, Phaseolus vulgaris, bean, embryo culture, farnesol, X-ray microprobe analysis, root growth     

Determination of apoplastic K+ in intact leaves by ratio imaging of PBFI fluorescence

The tetraammonium salt of the K⁺ binding fluorescent dye benzofuranisophthalate (PBFI) was used to investigate the influence ofpotassium nutrition (0.1–2.1 mol m^–3) on apoplasticK⁺ inVicia faba leaves by means of ratio imaging. As a referencethe infiltration-centrifugation method was used. Both methodsreflected the influence of K⁺ supply on apoplastic K⁺ concentration.The abaxial leaf side revealed significantly higher K⁺ concentrations(20-25 mol m^–3) than the adaxial side (5–8 mol m^–3).Application of CCCP led to an immediate increase in apoplasticK⁺ demonstrating the reliability of the PBFI method. Key words: Vicia faba, leaf, apoplast, K⁺, PBFI, ratio imaging, ratiometric fluorescence microscopy     

Changes in Tissue Freezing in Senecio vulgaris infected by Rust (Puccinia lagenophorae)

Freezing of healthy and rust (Puccinia lagenophorae) infectedleaves of Senecio vulgaris was compared calorimetrically bythermal analysis. In fully expanded leaves the threshold freezingtemperature was in the range –6.8 to –8.4 °Cin controls but –3.0 to –5.1 °C in leaves withsporulating rust sori. Comparable values in expanding leaveswere –5.0 to –8.9 °C and –3.9 to –6.7°C for healthy and rusted tissues, respectively. The bulktissue freezing point was between –1.0 and –4.0°C in both fully expanded and expanding healthy leaves,and was increased by infection by between +0.2 and 2.5 °C.Whereas healthy leaves supercooled by 3.1–5.8 °C,rusted leaves supercooled by only 1.8–4.9 °C Supercoolingof control leaves was reduced by dusting with aeciospores, particularlywhen leaves were wounded to simulate the rupture of the surfacecaused by sporulation, but wounding alone had no significanteffect. Supercooling of distilled water was also significantlyreduced by aeciospores, suspended at a concentration of 10⁵spores ml^–1. It is concluded that rust-induced changes in leaf freezing inS. vulgaris grown in controlled environments were due to anincrease in the number of sites for ice nucleation, caused bythe presence of the aeciospores, and increased penetration ofice into internal tissues, resulting from damage to the cuticleand epidermis. Although data for frost resistance obtained inthe growth-room are similar to previous field observations,the role of the above mechanisms under field conditions remainsunproven. Senecio vulgaris (groundsel), Puccinia lagenophorae (rust), low temperature, freezing resistance     

Changes of the Apoplastic pH and K+ Concentration in the Phaseolus Pulvinus in situ in Relation to Rhythmic Leaf Movements

The apoplastic pH and K⁺ concentration of the extensor of thePhaseolus primary-leaf pulvinus in relation to rhythmic leafmovements have been investigated with double-barrelled ion-sensitivemicro-electrodes. Simultaneous measurements of leaf movementand ion activities in a fine hole of the extensor in situ showedco-existence of ultradian and circadian leaf movements as wellas of ultradian and circadian pH changes in the Water Free Space(WFS) of the extensor apoplast in situ. During circadian leafmovement the H⁺ and K⁺ activities in the WFS of the extensorchange in an antagonistic manner. When extensor cells swell(upward movement of the lamina) the H⁺ activity increases fromapproximately pH 6.7 to 5.9 and the K⁺ concentration decreasesfrom approximately 50 to 10 mol m^–3 and vice versa whenextensor cells shrink. These changes in the ionic activitiesin the WFS must be correlated with large changes in the ioncontent of the DFS and thus support the hypothesis that thecell walls of pulvinar cells serve as reservoirs for K⁺ andH⁺. Key words: Phaseolus pulvinus, apoplastic ionic activities, rhythmic leaf movements, ion-sensitive micro-electrodes (double-barrelled)     

Stomata and Mesophyll Characteristics of Barley Leaf as Affected by Light: Stereological Analysis

The anatomical structure of the second leaf blade of barley{Hordeum vulgare L. cv. Koral) was studied in plants exposedto a photosynthetic photon flux density (PPFD) of 200 µmolm^–2 s^–1 compared with those grown under 25µmolm^–2–11. Design-based stereological methods wereused for the estimation of various leaf anatomical characteristicssuch as mesophyll volume, proportion of intercellular spaces,number of mesophyll cells, mean mesophyll cell volume, and internalleaf surface area. The structure of the mesophyll was more affectedby different levels of PPFD than were the stomatal characteristics.Increased PPFD produced thicker leaves with a larger mesophyllvolume having a higher number of less elongated mesophyll cellsand a larger internal leaf surface area. Key words: Hordeum vulgare, light effect, mesophyll, stereology, stomata     

Assimilation of gaseous ammonia and the transport of its products in barley and spinach leaves

The interactions between the assimilation and transport of nitrogenand carbon were investigated in barley and spinach leaves. Bothplants were fumigated with NH₃ (1 mg m^–3 and the contentof amino acids, sucrose and carbon intermediates of amino acidmetabolism were analysed in the leaves, apoplast and phloemsap. The following changes took place in the C- and N-metabolismof barley leaves during 5 h of fumigation with NH₃ (a) The contentsof amino acids, especially glutamine, largely increased andthe contents of sucrose, 2-oxoglutarate, phosphoenolpyruvate,and glycerate-3-phosphate declined. (b) A decrease in the phophoenolpyruvatecontent was accompanied by an increased activity of phosphoenolpyruvatecarboxylase. (c) The altered cytosolic concentrations of aminoacids and sucrose during NH₃ fumigation correlated with similarchanges in the apoplast and phloem sap. The altered percentageof each amino acid relative to the total amino acid concentrationin the cytosol, caused by NH₃ fumigation, is reflected in theapoplast and the phloem sap. The results indicate that the concentrations of amino acids in the cytosol determine their concentrationsin the phloem. Key words: Amino acids, ammonia fumigation, barley leaves, C: N partitioning, phosphoenolpyruvate carboxylase, phloem sap, spinach leaves     

Does Apoplastic Sucrose Induce the Ability for Sucrose Loading in Developing Leaves of Sugarbeet?

Dark-grown sugarbeet (Beta vulgaris L.) leaves were used toinvestigate a possible role of apoplastic sucrose in the inductionand development of the putative phloem-located sucrose carrierin relation to minor vein loading and export capacity. Unlabeledsucrose was introduced to the leaf apoplast after which veinaccumulation of [¹⁴C]sucrose was determined by autoradiogra-phy.Western blotting was used to detect the putative carrier. Anaffinity purified antibody against the sucrose binding proteinof soybean did not cross-react with the protein in a plasmalemma-enrichedfraction from sugarbeet leaves. Challenging the apoplast ofleaf discs with buffer plus sucrose for 6 h (induction) resultedin decreased [¹⁴C]sucrose uptake. When induction treatmentswere conducted with detached intact leaves in the dark, sucroseand glucose, but not buffer alone enhanced [¹⁴C]sucrose uptake.Detached leaves induced under laboratory light conditions for24 h showed enhanced [¹⁴C]sucrose uptake even in the absenceof any sugar introduced to the apoplast (buffer only). The datasuggested that in the etiolated tissue, sucrose was not a directand specific inducer of its putative carrier; instead sugarsmay have provided the energy for vein loading. Furthermore,the data suggested a role for light in the development of theputative sucrose carrier and vein accumulation of sucrose intransitional leaves of sugarbeet. The role of light may alsobe related to tissue energy level. ¹Contribution No. D-15192-1-91 from the New Jersey AgriculturalExperiment Station. This work was funded in part by the BeetSugar Development Foundation and Rutgers University ResearchCouncil and was submitted as partial fulfillment for M.S. degreeby Lynne H. Pitcher. (Received February 19, 1991; Accepted May 13, 1991)     

Determination of Nitrate Reductase Activity in Barley Leaves and Roots

The inactivation of nitrate reductase in the leaves and rootsof barley (Hordeum vulgare L. cv. Mazurka) during and afterextracting was investigated. At 0 °C in the absence of casein,25 per cent of ‘total’. i.e. maximal in vitro, nitratereductase activity was lost during the 2 min extraction process,followed by a slower loss of activity while the extract wasstored in ice. Activity was maintained by adding a minimum of1 per cent casein to the extraction medium containing 0·1M phosphate (pH 7·5), 1 mM EDTA and 1 mM dithiothreitol.Nitrate reductase was stable for several hours in these extracts,but declined in a first order manner in the absence of dithiothreitol.Casein also prevented the initial loss while making root extracts,but had less effect during storage. Using casein and thiols, nitrate reductase activity in light,(as product of maximal in vitro rates and wt g^–1) in leaveswas 98 per cent of the total activity in 31-day-old plants grownwith full nutrient in water culture and 60-day-old field-grownplants receiving no fertilizer. Field-grown plants, however,exhibited only 17 per cent of the activity of culture-grownplants. Nitrate reductase in leaves of barley plants grown in waterculture had a diurnal rhythm. During the first 3 h of the lightperiod, activity increased to 1·3 x the ‘dark’value. This was followed by a temporary decrease and then byanother increase to a maximum of 1·7 x the ‘dark’value, occurring about 8 h after illumination. Activity thendecreased during the rest of the light period and in darkness. Hordeum vulgare L., barley, nitrate reductase     

Light-induced pH and K+ changes in the apoplast of intact leaves

The K⁺-sensitive fluorescent dye benzofuran isophthalate (PBFI) and the pH-sensitive fluorescein isothiocyanate dextran (FITC-Dextran) were used to investigate the influence of light/dark transitions on apoplastic pH and K⁺ concentration in intact leaves of Vicia faba L. with fluorescence ratio imaging microscopy. Illumination by red light led to an acidification in the leaf apoplast due to light-induced H⁺ extrusion. Similar apoplastic pH responses were found on adaxial and abaxial sides of leaves after light/dark transition. Stomatal opening resulted only in a slight pH decrease (0.2 units) in the leaf apoplast. Gradients of apoplastic pH exist in the leaf apoplast, being about 0.5–1.0 units lower in the center of the xylem veins as compared with surrounding cells. The apoplastic K⁺ concentration in intact leaves declined during the light period. A steeper light-induced decrease in apoplastic K⁺, possibly caused by higher apoplastic K⁺, was found on the abaxial side of leaves concentration. Simultaneous measurements of apoplastic pH and K⁺ demonstrated that a light-induced decline in apoplastic K⁺ concentration indicative of net K⁺ uptake into leaf cells occurs independent of apoplastic pH changes. It is suggested that the driving force that is generated by H⁺ extrusion into the leaf apoplast due to H⁺-ATPase activity is sufficient for passive K⁺ influx into the leaf cells. Received: 7 March 2000 / Accepted: 12 May 2000     

Sink Activity Estimation by Sink Size and Dry Matter Increase During the Ripening Stage of Barley (Hordeum vu/gare) and Rice (Oryza sativa)

During the ripening stage of barley and rice, the sink activitywas defined as the dry matter increase per units sink size,leaf area and time, as follows: NAR = A.SinkW+NAR', where NAR is the net assimilation rate (g d.wt dm^–2d^–1);A is the sink activity [g d.wt g^–1d.wt (ear) dm^–2d^–1]; Sink W is ear wt per plant at heading (g d.wt);and NAR' is net assimilation rate excluding the assimilate ofsink organ (g d.wt dm^–2 d^–1). Plant material with 16 combinations of mutually different sink(ear) and source (leaf) size were produced at heading for eachcrop: parts of each leaf and ear were removed to produce fourgrades in barley, and also a part of each leaf was removed producingfour grades for four rice varieties showing different ear size.NAR and NAR' were determined during 26 and 21 d in barley andrice after heading, respectively. Sink activity (A), representedas the assimilation rate induced by the sink organ, was estimatedfrom the relationship between SinkW and NAR using the previousequation. The sink activity was significantly higher in ricewith a value of 0–0.028 g d.wt g^–1 d.wt (ear) dm^–2d^–1 vs. 0–0.0017 in barley, suggesting that therelative role of leaves for grain filling is considerably higherin rice than in barley. The sink activity obtained in the studymight be introduced into a model to predict the yields of barleyand rice. Hordeum vulgare L, barley, Oryza saliva L, rice, dry matter, NAR, sink, source, sink activity, model     

Regulation of Storage Protein Synthesis by Sulphur and Nitrogen Nutrition in Cultured Immature Caryopses of Barley

Techniques are described for the culture of developing barley(Hordeum vulgare L.) caryopses. Over a 7 d period in culturethe dry weights and the amounts of starch and protein increasedby at least twofold. Growth was sustained for at least 20 d.The effects of glutamine and cysteine on the amount and compositionof the hordein storage proteins were also studied. Glutaminestimulated total hordein accumulation but caused a disproportionateincrease in the amount of the S-poor ‘C’ hordeinswhen supplied at 100 mol m^–3. Addition of cysteine at1·0 mol m^–3 did not increase the amount of Srich‘B’ hordeins. The results suggest that althoughisolated caryopses are able to take up sucrose and glutamineand convert them to starch and protein there is some limitationin their ability to convert externally supplied cysteine intoproteins. Key words: Hordeum vulgare L., Caryopses, Glutamine, Cysteine, Storage proteins     

Pathway of phloem loading in the C3 tropical orchid hybrid Oncidium Goldiana

The apoplast of mature leaves of the tropical orchid OncidiumGoldiana was perfused with 0.5 mM p-chloromercuribenzenesulphonicacid (PCMBS) via the transpiration stream in order to test themode of phloem loading. The efficacy of introducing PCMBS byperfusion was shown by saffranin O dye movement in the veinsand leaf apoplast in control experiments. Photoassimilate exportas the result of phloem loading was measured by collection of¹⁴CO₂-derived photoassimilates from the basal cut-ends of intactleaves. Phloem loading and translocation of photoassimilates was inhibitedby 89% in leaves perfused with PCMBS for 1 h. The effect ofPCMBS on leaf photosynthesis was minimal. The amount of radiocarbonfixed by PCMBS-treated leaves averaged 89% of control leavesperfused with distilled water. A negative correlation betweenthe total amount of photoassimilate exuded and the calculatedconcentration of PCMBS in the leaf apoplast was also observed.The results indicate that phloem loading in Oncidium Goldianaoccurs via the apoplastic pathway. Key words: Phloem loading, apoplast, PCMBS, tropical orchid     

Interaction between barley yellow dwarf virus and rust in wheat, barley and oats, and the effects on grain yield and quality

In three separate experiments, the upper leaf surface of the fifth formed leaf of wheat cv. Highbury, the fourth and fifth leaves of barley cv. Julia and the third and fourth leaves of oat cv. Mostyn were inoculated in a spore settling tower with wheat brown rust (Puccinia recondita f. sp. tritici), barley brown rust (P. hordei) or oat crown rust (P. coronata f. sp. avenae), respectively. Fewer pustules developed on distal portions of leaves of plants infected with barley yellow dwarf virus (BYDV) than on similar portions of leaves from virus-free plants. There were no significant differences in the number of pustules on proximal leaf portions. In barley and oats, the number of pustules on distal leaf portions was negatively correlated with the amount of yellowing of the leaf areas scored. In wheat, symptoms of BYDV were mild and leaves were little affected by yellowing. The latent period of rust on wheat and oats was not affected by BYDV. In barley, BYDV reduced the latent period of rust on leaf 5, but not on leaf 4, and reduced it on proximal, but not distal, leaf portions. In other experiments, BYDV reduced the yield of wheat and oats by 44% and 66%, respectively, while BYDV-infected barley was almost sterile. The appropriate rust reduced the yield of wheat, barley and oats by 33%, 13% and 86%, respectively. When infected with both BYDV and rust, yield of wheat and oats was reduced by 63% and 91%, respectively. Neither BYDV nor rust affected the percentage crude protein content of wheat grain, nor did rust affect that of barley. In oats, BYDV and rust each significantly increased crude protein of grain, but rust infection of BYDV-infected plants tended to reduce it.     

Nitrate Effects on Growth of the First Four Main Stem Leaves of a Range of Temperate Cereals and Pasture Grasses

The effects of different applied NO₃^– concentrations onextension growth and final length and area of leaves 1–4of five cereals and six pasture grasses of temperate originwere examined. Increased applied NO₃^– in the range 0.1–0.5.0mol m^–3 caused decreased duration of growth but increasedgrowth rate and final length of leaves 2–4 of the cerealsAvena saliva, Hordeum vulgare, Secale cereale, x Triticosecaleand Triticum aestivum. For all cereals, increased NO₃^–resulted in increased area of leaves 1–4. Pasture grasseswere supplied either 0.5 or 50 mol m^–3 NO₃^–. Increasedapplied NO₃^– (0.5–5.0 mol m^–3) resulted indecreased duration of growth and increased growth rate and finalarea of leaves 1–4 of Bromus wiltdenowii, leaves 2–4ofFestuca arundinaceae and leaves 3 and 4 of Lolium muitiflorum.In addition, length of leaves 3 and 4 of B. witidenowii increasedwith increased NO₃^–. Increased NO₃^– resulted inincreased area of leaves 2–4 of Dactylis gtomerata andLolium perenne and leaves 3 and 4 of Phalaris aquaiica but hadno effect on extension growth of all three species. Avena sativa L, oat, Hordeum vulgare L, barley, Secale cereale L, rye, x Triticosecale Wittm, triticale, Triticum aestivum L, wheat, Bromus willdenowii Kunth, prairie grass, Dactylis gtomerata L, cocksfoot, Festuca arundinaceae Shreb, tall fescue, Lolium multijlorum Lam, Italian ryegrass, Lolium perenne L, perennial ryegrass, Phalaris aquatica L, nitrate, leaf extension, leaf expansion     

Proton-Chloride Symport in Barley Roots

An increase in the acidity of the incubation medium from pH6 5 to pH 4.0 increased Cl- flux into ATP-depleted Hordeum vulgareL roots more than three times This pH-dependent Cl^– fluxwas inhibited by p-chloromercuriphenyl sulphonic acid. The effectof pH on Cl- influx was eliminated when the pH gradient wasdissipated by addition of salts of permeable weak acids, andin K-loaded roots in the presence of a protonophore togetherwith valinomycin The results support the assumption that a H⁺-Cl^–symportsystem is present in barley root cells Hordeum vulgare L., barley, excised roots, ion transport, proton-chloride symport