Isoforms of NADP-dependent Malic Enzyme in Tissues of the Greening Maize Leaf

Scagliarini, S., Pupillo, P. and Valenti, V. 1988. Isoformsof NADP-dependent malic enzyme in tissues of the greening maizeleaf.—J. exp. Bot. 39: 1109–1119. The compartmentation of the isoforms of NADP-dependent malicenzyme (E.C. 1.1.1.40 [EC] ) has been studied in cell-free extractsand in enzymatically-isolated protoplasts of mesophyll tissue(MT) and bundle sheath (BS) strands of greening maize leaves.The etiolated leaf of 10-d-old seedlings contains a cytosolicisozyme with a pl of 5.4 ?0.1 at low specific activity (s.a,45 ? 3 nmol min^–1 mg^–1 protein), found both in MTand BS. The green leaf on the other hand contains the dominantBS chloroplast isozyme with pl 4.6 ? 0.2 at a s.a, of 370 ?40 nmol min^–1 mg^–1 protein (3.2 ? 0.5 µmolmin^–1 mg^–1 chl) and a minor, previously undescribedisoform with pl 6.5 ? 0.1 also localized in the BS at a s.a.of 38 ? 6 nmol min^–1 mg^–1 protein. Green MT protoplastshave only traces of pl 4.6 isozyme. After illumination of dark-grown seedlings, the total leaf activityshows a rapid increase (1.5-fold within 2 h), attributed mainlyto the pl 5.4 isozyme of MT protoplasts and BS strands. Thisis followed by a large increase of enzyme activity due to thecontinued rise of pl 5.4 isozyme for about 24 h and, after aninitial lag of a few hours, to the accumulation of pl 4.6 isozyme.After 18 h illumination, pl 4.6 and 5.4 isozyme activities tendto decline in the MT whereas they are still increasing in theBS, particularly the former. This pl 4.6 species has becomethe major one by 48 h illumination. The final pattern of greenleaves is established around 96 h light, when the chloroplastisozyme has attained its maximum level, the pl 5.4 isozyme ofBS strands has been superceded by the pl 6.5 species (also supposedto be cytosolic) and MT protoplasts retain little residual activity.Some metabolic implications of the changing pattern of NADP-dependentmalic isozymes during maize leaf greening are discussed. Key words: C₄, isozymes, malic enzyme, photodifferentiation, Zea mays     

The Distribution of some NADP-Linked Dehydrogenases in Photosynthetic Tissues5

Mesophyll protoplasts of one-month-old maize leaves were separatedenzymatically from bundle sheath strands, and purified by centrifugationthrough a Percoll layer. The protoplasts and BS strands wereessentially pure as judged by microscopy, chl a/b ratios, andlevels of enzyme markers (PEP carboxylase and NADP-malic enzyme).Chioroplasts were obtained from the protoplasts and from homogenates,and purified through Percoll. The distribution of four NAD P-linked dehydrogenases in tissuesand organdies was examined. NADP-triose phosphate dehydrogenasc,used as a chloroplast marker, shows high and comparable specificactivities in both main tissues. Glucose 6-phosphate dehydrogenaseis located mainly in the mesophyll (at a specific activity of15.1 µmol h^–1 mg^–1chl in protoplasts) andis exclusively cytosolic. 6-Phosphogluconate dehydrogenase,also present in both tissue types, has a higher activity inthe BS (12.6 in purified strands versus 7.3 µmol h^–1mg^–1 chl in protoplasts). It is a cytosolic enzyme, althoughplastids may contain a low activity. Glyceraldehyde 3-phosphate:NADP reductasc is entirely in the mcsophyll cytoplasm (11.2µmol h^–1 mg^–1 chl). It is suggested that thecytoplasm of mcsophyll cells is a site of diversion of sugarphosphates for production of NADPH, at rates, however, compatiblewith the operation of the triose phosphate shuttle to bundlesheath cells for the synthesis of starch. Key words: Glucose-6-phosphate dehydrogenase, 6-phosphogluconate dchydrogenase, glyceralde-hyde-3-phosphate : NADP reductase, Zea mays     

Pyruvate Uptake by Mesophyll and Bundle Sheath Chloroplasts of a C4 Plant, Panicum miliaceum L.

Intact chloroplasts were isolated from mesophyll and bundlesheath protoplasts of a C₄ plant, Panicum miliaceum L., to measurethe uptake of [1-¹⁴C]pyruvate into their sorbitol-impermeablespaces at 4?C by the silicone oil filtering centrifugation method.When incubated in the dark, both chloroplasts showed similarslow kinetics of pyruvate uptake, and the equilibrium internalconcentrations were almost equal to the external levels. Whenincubated in the light, only mesophyll chloroplasts showed remarkableenhancement of the uptake, the internal concentration reaching10–30 times of the external level after 5 min incubation.The initial uptake rate of the mesophyll chloroplasts was enhancedabout ten fold by light and was saturated with increasing pyruvateconcentration; K_m and V_max were 0.2–0.4 mM and 20–40µmol(mg Chl)^–1 h^–1, respectively. The lightenhancement was abolished by DCMU and uncoupling reagents suchas carbonylcyanide-m-chlorophenylhydrazone and nigericin. Theseresults indicate the existence of a light-dependent pyruvatetransport system in the envelope of mesophyll chloroplasts ofP. miliaceum. The uptake activity of mesophyll chloroplastsboth in the light and the dark was inhibited by sulfhydryl reagentssuch as mersalyl and p-chloromercuriphenylsulfonate, but thebundle sheath activity was insensitive to the reagents. Thesefindings are further evidence for the differentiation of mesophylland bundle sheath chloroplasts of a C₄ plant with respect tometabolite transport. (Received July 3, 1986; Accepted October 8, 1986)     

Aldolase--An Important Enzyme in Controlling the Ribulose 1,5-bisphosphate Regeneration Rate in Photosynthesis

This study was done to explore an enzymatic mechanism for thephotosynthetic carbon reduction cycle whereby the rate of synthesisof ribulose 1,5-bisphosphate (RuBP) could be changed while thelevels of intermediates other than 3-phosphoglycerate and RuBPwere kept constant. Chloroplast aldolase was purified to homogeneityfrom spinach leaves. When the enzyme was assayed in the directionof fructose 1,6-bisphosphate synthesis in the presence of theconcentrations of the substrates reported in vivo, the activitywas severely inhibited by physiological concentrations of RuBP.The aldolase reaction proceeded with a sequential mechanism.The K_m for dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphatewere 0.45 mM and 40 µM, respectively. The activity wascompetitively inhibited by RuBP with respect to dihydroxyacetonephosphate. The K^I was 0.78 mM. The maximum activity of aldolasein spinach leaves was calculated as 1,360µmol (mg Chl)^–1h^–1 An equation to express the reaction for the synthesisof fructose 1,6-bisphosphate by aldolase was constructed topredict the metabolic rate of this reaction in vivo. The calculationclearly showed that aldolase is an important enzyme in controllingthe rate of RuBP regeneration. (Received March 25, 1991; Accepted August 12, 1991)     

Cellular Levels of Ribulose 1,5 Bisphosphate Carboxylase and Chloroplast Compartment Size in Wheat Mesophyll Cells

Pyke, K. A. and Leech, R. M. 1987. Cellular levels of ribulose1,5 bisphosphate carboxylase and chloroplast compartment sizein wheat mesophyll cells.—J. exp. Bot. 38: 1949–1956. The amount of the photosynthetic enzyme ribulose 1,5 bisphosphatecarboxylase (RUBISCO),as determined in mesophyll cells in primarywheat leaves was related to the size of the chloroplast compartmentwithin the cell for wheat species of three ploidy levels. Asimilar comparison was made for several genotypes of the hexaploidbreadwheat Triticum aestivum. Estimation of total chloroplastvolume per mesophyll cell was made assuming chloroplasts tobe oblate spheroid in shape. A significant correlation was found between the amount of RUBISCOper cell and the total chloroplast volume per cell for diploid,tetraploid and hexaploid wheat species. A significant correlationbetween cellular RUBISCO level and total chloroplast volumeper cell was also observed for a range of genotypes of the hexaploidT. aestivum but these genotypes of T. aestivutn accumulate agreater amount of RUBISCO per unit chloroplast volume than doany other wheat species. For these genotypes of T. aestivumthe stromal concentration of RUBISCO was estimated at 0·5mol m^–3 with a ribulose Msphosphate binding site concentrationof 4·0 mol m^–3. These results are discussed with respect to a gene dosage hypothesisto explain the accumulation of RUBISCO in leaf mesophyll cells. Key words: Ribulose, bisphosphate carboxylase, wheat chloroplasts, mesophyll cells     

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     

Characterization of the vacuolar-type H+-ATPase from guard cell protoplasts of Commelina

Characteristics of the vacuolar-type (V-type) H⁺-ATPase fromguard cell protoplasts of Commelina communis L. were investigatedusing a linked enzyme assay and nitrate inhibition as a diagnosticindicator of the enzyme activity. ATPase activity was completelyinhibited by about 50 mol m^–3 nitrate and activity wasoptimal near pH 8.0. The temperature optimum for activity wasabout 37 C and an Arrhenius plot indicated changes in activationenergy for the ATPase at 15C and possibly at about 30 C. Theenzyme was stimulated by Cl^– while Ca²⁺ inhibited activity(l₅₀ = 1.5 mol m^–3). The apparent K_m (MgATP) was 0.62mol m^–3. Incubation of guard cell protoplasts for up to 5 h in 50 µMabscisic acid (ABA) or 25µM fusicoccin (FC) did not affectsubsequent ATPase activity. In vitro assays with FC or ABA alsodid not affect enzyme activity. Activity was not affected bylight or potassium ferricyanide, two factors which are knownto influence stomatal activity. Beticoline was a potent inhibitorof activity (l₅₀ = 50 µM) while DCCD was less effective(l₅₀ = 90µM). On chlorophyll, protein and protoplast bases, V-type ATPaseactivity was greater in guard cell protoplasts than mesophyllcell protoplasts by 66, 13.9 and 1.9, respectively. On atonoplast surface area basis the enzyme activity was 5.6 timeshigher in guard cell protoplasts than in mesophyll cell protoplasts Thus, although the characteristics of the V-type, H ⁺-ATPaseof GCP are very similar to those found in other cell types,rates of activity and probably tonoplast enzyme density aremuch greater in guard cell protoplasts than mesophyll cell protoplastsof C. communis which corresponds with the large and rapid ionfluxes across the tonoplast associated with stomatal movements Key words: Guard cell protoplasts, stomata, V-type H ⁺-ATPase     

A Large-scale Isolation Procedure for Cereal Mesophyll Protoplasts

A method suitable for the large-scale isolation of cereal protoplastsfrom up to 50 g of leaf material is described. Surface-sterilizedleaves from cultivars of wheat, barley, maize, sorghum, andTriticale were diced and vacuum infiltrated with enzyme mixturecomposed of cellulysin (1 per cent w/v), hemicellulase (1 percent w/v), and macerozyme (0.5 per cent w/v). With this procedure,yields of between 10⁶ to 10⁷ protoplasts per gram of leavescan be reproducibly obtained after only 1.5–3 h of enzymatictreatment. These protoplasts were almost 100 per cent viable(as determined by fluorescein diacetate staining) and incorporationof ³H-uridine and ¹⁴C-leucine into an acid-insoluble fractionwas demonstrated. Almost one-third of the ribosomes of theseisolated protoplasts were present as polysomes. cereals, leaf mesophyll, protoplast isolation     

Photosynthetic Properties of Guard Cell Protoplasts from Vicia faba L.

Guard cell protoplasts were isolated enzymatically from theepidermis of Vicia faba L. and their photosynthetic activitieswere investigated. Time courses of light-induced changes inthe chlorophyll a fluorescence intensity of these protoplastsshowed essentially the same induction kinetics as found formesophyll protoplasts of Vicia. The transient change in thefluorescence intensity was affected by DCMU, an inhibitor ofphotosystem II; by phenylmercuric acetate, an inhibitor of ferredoxinand ferredoxin NADP reductase; and by methyl viologen, an acceptorof photosystem I. Low temperature (77 K) emission spectra ofthe protoplasts had peaks at 684 and 735 nm and a shoulder near695 nm. A high O₂ uptake (175 µmol mg^–1 Chl hr^–1)was observed in guard cell protoplasts kept in darkness, whichwas inhibited by 2 mM KCN or NaN₃ by about 60%. On illumination,this O₂ uptake was partially or completely suppressed, but itssuppression was removed by DCMU, which indicates that oxygenwas evolved (150 µmol mg^–1 Chl hr^–1) photosynthetically.We concluded that both photosystems I and II function in guardcell chloroplasts and that these protoplasts have high respiratoryactivity. (Received January 30, 1982; Accepted May 15, 1982)     

Inhibition of Photosynthesis by Sulfite in Mesophyll Protoplasts Isolated from Vicia faba L. in Relation to Intracellular Sulfite Accumulation

Inhibition of photosynthesis by Na₂SO₃ in mesophyll protoplastsisolated from Vicia faba leaves and uptake of sulfite by theprotoplasts were examined at various pH values of the incubationmedium containing Na₂SO₃. As the pH of the incubation mediumlowered, the rate of photosynthesis in the protoplasts decreasedand the amount of sulfite taken up by the protoplasts increased.Most of sulfite accumulated in the protoplasts was not metabolizedduring the dark incubation, as measured with an ion chromatograph.Photosynthetic O₂ evolution by the chloroplasts isolated fromVicia mesophyll protoplasts was inhibited by exogenously-appliedNa₂SO₃ over pH region examined (7.4–9.0). The sulfiteconcentration required for a half inhibition of photosynthesisby the isolated chloroplasts was similar to the intracellularsulfite level required for that by the protoplasts. These resultsindicate that the intracellular sulfite accumulated in the protoplastsin an unmetabolized state is responsible for the inhibitionof protoplast photosynthesis. (Received January 24, 1985; Accepted May 29, 1985)     

Requirements for the light-stimulated degradation of stromal proteins in isolated pea (Pisum sativum L.) chloroplasts

Chloroplasts from 17-d-old pea leaves (Pisum sativum L.) wereisolated to elucidate the requirements for the light-induceddegradation of stromal proteins. The influence of electron transportthrough the thylakoids and the influence of ATP on protein degradationwere investigated. When chloroplasts were incubated in the light(45 µmol m^–2s^–1), glutamine synthetase, thelarge subunit of ribulose-1,5-bisphosphate carboxylase and glutamatesynthase were degraded, whereas phosphoribulokinase, ferredoxin-NADP⁺reductase and the 33 kDa protein of photosystem II remainedmore stable. Major protein degradation was not observed over240 mm in darkness. The electron transport inhibitor dichlorophenyldimethylureareduced protein degradation in the light over several hours,whereas dibromothymoquinone was less effective. Inhibiting theproduction of ATP with tentoxin or by destroying the     

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     

Rapid separation of carbonic anhydrase isozymes using cellulose acetate membrane electrophoresis

A method is described for the rapid separation of carbonic anhydrase(CA) isozymes by cellulose acetate membrane electrophoresisin which CA activity is detected using the pH-indicating dye,bromcresol purple. This method can detect bovine erythrocyteCA in a 0.3 mm³ sample applied at a concentration of 100 ngcm^–3 (total of 30 pg applied) while at higher concentrationsthree isozymes were observed. It was found, using a potentiometrictechnique, that intact cells of Anabaena flos-aquae (Cyanophyceae)and Chlorella ellipsoidea had no detectable activity while C.saccharophila and Chlamydomonas reinhardtii (Chlorophyceae)had external CA activity. CA activity of the extracts suggestedthe presence of internal CA in all species. After electrophoresisit was found that C. saccharophila and C. reinhardtii had twoisozymes while A. flos-aquae and C. ellipsoidea had only a singledetectable band. Spinach had up to five detectable isozymesthat were difficult to resolve. Incubation of spinach extractwith the CA inhibitor ClO₄^– (500 mol m^–3) inhibitedCA activity by 90% using the potentiometric technique, but afterelectrophoresis had no detectable effect. This technique isuseful in identifying isozymes that are substantially differentin electrical charge and in monitoring CA isozyme activity duringenzyme purification. Key words: Carbonic anhydrase, isozymes, cyanobacteria, microalgae, spinach     

Differential Stimulation of PEP-carboxylation in Guard Cells and Mesophyll Cells by Ammonium or Fusicoccin

Dark CO₂-fixation in guard cells of Vicia faba was much moresensitive to ammonium than in mesophyll cells. Addition of ammonium(5.0 mol m^–3; pH₀ 7.6) caused up to a 7-fold increasein dark CO₂-fixation rates in guard cell protoplasts (GCP),whereas in leaf slices, mesophyll cells, and mesophyll protoplaststhe increase was only about 1.4-fold. In both cell or tissuetypes, total CO₂-fixation rates were higher in the light (2–12-foldhigher in GCP and 28-fold in mesophyll); these rates were onlyslightly changed by ammonium treatment. However, separationof ¹⁴C-labelled products after fixation of CO₂ in the lightby GCP revealed a large ammonium-induced shift in carbon flowfrom starch and sugars to typical products of C₄-metabolism(mainly malate and aspartate). In contrast, in mesophyll cellsamino acid and malate labelling was only moderately increasedby ammonium at the expense of sucrose. The data suggest thatin vivo ammonium might facilitate stomatal opening and/or delaystomatal closing through an increased production of organicacids. Key words: PEP-carboxylation, guard cell protoplasts, ammonium, fusicoccin     

Photosynthesis by developing embryos of oilseed rape (Brassica napus L.)

The aim of this study was to assess the photosynthetic potentialof developing seeds of oilseed rape (Brassica napus L.) andto compare photosynthetic properties of embryo plastids withthose of leaf chloroplasts from the same species. Measurementsof CO₂-dependent O₂ evolution show that developing seeds ofB. napus are photosynthetically active in vitro. Essentially,all of the photosynthetic activity of the developing seed isaccounted for by the embryo. The rate of photosynthesis by developingembryos increased until the onset of desiccation, after whichit declined, so that by maturity embryos were no longer photosyntheticallyactive. Photosynthetic activity was positively correlated withchlorophyll content throughout development. Comparison of thephotosynthetic characteristics of leaf and embryo chloroplastsrevealed that rates of uncoupled electron transport were 2.5-foldgreater in those from the embryo. Light-saturated rates of CO₂-dependentO₂ evolution, per unit chlorophyll, and CO₂ saturation pointswere similar for chloroplasts from both tissues. However, light-saturationpoints and chlorophyll a/b ratios were lower for embryo thanfor leaf choroplasts. Embryos and embryo chloroplasts also containedconsiderably less ribulose 1,5-bisphosphate carboxylase/oxygenaseprotein per unit total protein, than leaves. Although excisedembryos were capable of high rates of CO₂-dependent O₂ evolution(90–100 mol mg^–1 chlorophyll h^–1) under asaturating photosynthetic photon flux density (PPFD), low transmittanceof light through the silique wall (30%), together with the highPPFD required to achieve light compensation points in developingseeds (500 mol m^–2 s^–1), suggests that photosynthesisin vivo is unlikely to make a net contribution to carbon economyunder normal environmental conditions. Key words: Embryo, development, photosynthesis, chloroplast, Brassica napus L.     

Changes of Anatomical Features, Photosynthesis and Ribulose Bisphosphate Carboxylase-Oxygenase Content of Mango Leaves

Changes in anatomical and physiological features, includingchanges in amount per unit area of anthocyanin and chlorophyll,in leaves of seedling mango (Mangifera indica L. cv. Irwin)trees were determined to understand what controls the rate ofphotosynthesis (Pn) at various stages of development. The youngleaves of seedling trees contained high concentrations of anthocyanin.During enlargement of leaves, the disappearance of anthocyaninand the accumulation of chlorophyll occurred concomitantly;the anthocyanin content began to decrease markedly once theleaf area had reached a maximum. During the early period ofleaf development, the thickness of mesophyll tissue decreasedtemporarily, but when the length of the leaf reached half thatof a mature leaf, the mesophyll began to thicken again. Smallstarch grains appeared in the chloroplasts of the young leavesand chloroplast nucleoids (ct-nuclei) were distributed throughoutthe chloroplasts. When leaves matured, ct-nuclei were displacedto the periphery of chloroplasts because of the accumulationof large starch grains. Compared with young leaves, green andmature leaves contained greater concentrations of ribulose bisphosphatecarboxylase-oxygenase (RuBisCO) protein. The results of immunocytochemicalexamination of RuBisCO under the light microscope reflectedthe results of electrophoresis measurements of RuBisCO. Pn waslow during the chocolate-coloured stage of early leaf development.In green and mature leaves Pn was higher; the average Pn was7·6 mg CO₂ dm^-2 h^-1 under light at intensities above500 µmol m^-2 s^-1.Copyright 1995, 1999 Academic Press Mangifera indica L., mango leaf, chloroplast nucleoids, chloroplast ultrastructure, starch accumulation, anthocyanin, chlorophyll, DAPI staining, SDS-PAGE, immunocytochemical technique     

Role of pulvinar chloroplasts in light-driven leaf movements of the trifoliate leaf of bean (Phaseolus vulgaris L.)

Laminar pulvini of bean (Phaseolus vulgaris L.) contain numerouschloroplasts in cells of their motor tissue. The quantitativerelationships of the chloroplast pigments, chlorophyll a andb, ß-carotene, lutein, neoxanthin as well as the xanthophyllcycle carotenoids (violaxanthin, antheraxanthin and zeaxanthin)were similar to those of mesophyll chloroplasts from leafletlaminae. Exposure of pulvinules to light caused deepoxidationof violaxanthin to zeaxanthin, showing that the xanthophyllcycle is functioning. Chlorophyll fluorescence analysis of pulvinulesconfirmed that their chloroplasts are capable of both photosyntheticelectron transport and non-photochemical fluorescence quenching,showing that they build up a considerable transthylakoid protongradient in the light. Application of DCMU to excised pulvinulesand laminar discs, as well as to pulvinules of intact, attachedterminal leaflets blocked electron transport and fluorescencequenching. Application of the uncoupler CCCP to intact pulvinulesalso prevented non-photochemical fluorescence quenching. Therate of movement of the low-light-adapted terminal leaflet inresponse to exposure of its pulvinule to overhead red light(500 µmol m^–2 s^–1) was reduced when the pulvinulewas pretreated with DCMU. The pulvinar response to overheadblue light (50 µmol ^–2 s^–1), which is morepronounced than to red light, was not affected by similar pretreatmentwith DCMU. Pretreatment with CCCP caused a short lag in theresponse to red light, but did not affect its subsequent rate.The results suggest that the pulvinar response to red, but notto blue light, requires non-cyclic electron transport and theresulting generation of ATP Key words: Leaf movements, light, non-cyclic electron transport, Phaseolus, pulvinar chloroplasts     

Preservation of the Differentiated State of Mesophyll Cells In Vitro

Under a variety of light conditions, and in a modified NT mediumcontaining sucrose as a carbon source, isolated mesophyll cellsof Calystegia sepium started cell divisions after 72 h. Alternately,under light-dark alterations (12: 12 h; 1.35–1.50 W m^–2of cool white light), and culture in the NT medium lacking anorganic carbon source, and made iso-osmotic by the additionof mannitol, the cells preserved their differentiated stateas regards cell size, cell form, colour and size and distributionof chloroplasts for one week. Later on, they changed their characterand cell division was started. Calystegia sepium, mesophyll cells, differentiated state, in vitro culture     

Carbon Dioxide Fixation and Ribulose-1, 5-bisphosphate Carboxylase Activity in Intact Leaves of Sugar Beet Plants Exposed to Salinity and Water Stress

The influence of salinity in the growing media on ribulose-1,5-bisphosphate (RuBP) carboxylase and on CO₂ fixation by intactsugar beet (Beta vulgaris) leaves was investigated. RuBP carboxylase activity was mostly stimulated in young leavesafter exposure of plants for 1 week to 180 mM NaCl in the nutrientsolution. This stimulation was more effective at the higherNaHCO₂ concentrations in the reaction medium. Salinity also enhanced CO₂ fixation in intact leaves mostlyat rate-limiting light intensities. A 60 per cent stimulationin CO₂ fixation rate was obtained by salinity under 450 µEm^–2 s^–1. At quantum flux densities of 150 µEm^–2 s^–1 (400–700 nm) this stimulation was280 per cent. Under high light intensities no stimulation bysalinity was found. In contrast, water stress achieved by directleaf desiccation or by polyethylene glycol inhibited enzymeactivity up to fourfold at –1.2 MPa. Beta vulgaris, sugar beet, ribulose-1, 5-bisphosphate carboxylase, salt stress, water stress, carbon dixoide fixation, salinity     

DNA Levels in Dividing and Developing Plastids in Expanding Primary Leaves of Avena sativa

The amounts of plastid DNA in the primary leaves of 4-d-oldlight- and dark-grown seedlings of Avena sativa were measuredby microspectrofluorometry using the DNA-fluorochrome DAPI (4',6-diamidino-2-phenylindole). In the light-grown primary leaves (40–45 mm long) therewas a marked increase in DNA level per plastid from 10.2 to18.5 ? 10^–15 g between 2.0 mm and 10 mm from the leafbase, resulting from the rate of plastid DNA synthesis beinghigher than the rate of plastid division. Beyond 30 mm the plastidDNA level was reduced to 14 ? 10^–15g due to chloroplastdivision rates being higher than the rate of plastid DNA synthesis,while from 20 mm plastid DNA levels were constant at 2.2 ? 10^–12g per cell, which corresponds to 16000 plastome copies per cell. Observations of dark-grown leaves establish that, in Avena,light is not necessary for plastid division and the dark-grownleaf cells accumulate higher amounts of plastid DNA than light-grownleaf cells. Plastid nucleoids showed a change of distribution after completionof plastid DNA synthesis in light-grown leaves. A change inthe distribution of plastid nucleoids was also observed duringthe greening of etioplasts of dark-grown leaves while plastidDNA level remained constant. Such changes in plastid nucleoiddistribution appear to be independent of plastid DNA synthesisand correlate with the formation of grana stacks. Key words: Avena sativa, microspectrofluorometry, plastid DNA