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)     

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     

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     

Induction of Glucose 6-Phosphate Transport Activity in Chloroplasts of Mesembryanthemum crystallinum by the C3-CAM Transition

To study possible changes in the transport metabolites betweenchloroplasts and cytoplasm during CAM induction of Mesembryanthemumcrystallinum, we compared substrate specificity of P1¹ translocator(s)in isolated chloroplasts from the C₃ and CAM-induced plants.The [¹⁴C]glu-cose 6-phosphate (G6P) transport activity was significantonly in the chloroplasts of CAM-mode plants and not detectablein those of C₃-mode, while a similar high rate of [³²P]P_i uptakewas observed with both types of chloroplasts. Kinetic analysisof G6P uptake in the CAM chloroplasts showed a high V_max [10.6µmol (mg Chl)^–1 h^–1] and a comparatively lowK_m value (0.41 mM); the latter was similar to K_i values of P_i,3-phosphoglycerate and phospho-enolpyruvate, 0.30, 0.34 and0.47 mM, respectively. On the other hand, [32P]Pi uptake inthe CAM chloroplasts was inhibited competitively by G6P witha K_i value (8.4 mM) 20-fold higher than the K_m value for G6Puptake, while that in C₃ chloroplasts was not inhibited at all.These results suggest that a new G6P/P_i, counterexchange mechanismis induced in the chloroplast envelope of CAM-induced M. crystallinumin addition to the ordinary type of P, translocator, that cannottransport G6P, already present in the C₃-type chloroplasts. (Received March 17, 1997; Accepted May 10, 1997)     

Isolation of Chloroplasts from Poteriochromonas malhamensis with the Capacity for Translation

An improved method for the isolation of chloroplasts from Poteriochromonasmalhamensis is described. Poteriochromonas cells were brokenby passage through a nylon mesh with pores of 6µ in diameterat a flow rate of about 5 ml/15 s. After centrifugation thecrude chloroplast fraction was purified by centrifugation ina step gradient of Percoll. The isolated chloroplasts were enclosedby envelope membranes and were still surrounded in part by cytoplasmicresidues. The chloroplasts had the capacity for translation,which was both chloramphenicol-sensitive and cycloheximide-insensitive.The properties of these isolated chloroplasts from Poteriochromonasare discussed in relation to experiments on the transport intothe chloroplasts of nucleus-encoded proteins. ² Present address: Bundesgesundheitsamt, Zulassungsstelle furGentechnologie, Columbiadamm 3, D-1000 Berlin, F.R.G. (Received July 24, 1990; Accepted March 15, 1991)     

A Quantitative Study of Daily Variation in the Cellular Ultrastructure of Palisade Chlorenchyma from Sunflower Leaves

Stereology was used to describe cytological changes which occurin palisade cells of fully expanded leaves as part of theirnormal daily activity. These changes were evaluated by describingthe relationship between organelle volume and cell volume asratio values (i.e. percentage volumes, V_v; surface-to-volume,S_v). These ratios describe an average cell in terms of its volumecommitment to each organelle compartment. Cells were also describedin terms of actual volume (µm³) or surface area (µm²)of membrane present in an average cell. ANOVA-LSD and Mann-Whitneystatistics indicate significant changes occur in the ratio valuesof the vacuole, chloroplast, oil, starch and microbody compartmentsover the 24 h period. This indicates a re-allocation of cellspace to these compartments during this period. The S_v ratioof internal membranes of the chloroplast and mitochondria showedno significant change over 24 h indicating that there is a constantrelationship between volume and membrane surface area in theseorganelles. Significant changes occurred in average cell volumeover 24 h with maximum volume during the dark period. Sincechanges in cell volume affected the actual volume expressionof all of the organelle compartments there were diurnal variationin the actual size of these compartments, including the internalmembranes of chloroplasts and mitochondria which more than doubledin surface area. Helianthus annuus L, sunflower, cytology, stereology, quantitative microscopy, diurnal, morphometrics, ultrastructure, chlorenchyma, chloroplast, mitochondria, microbodies     

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     

Electron-Microscopic Study of Osmium Ferricyanide Staining in Meristematic, Extending and Differentiated Mesophyll Cells of Winter Rye Seedlings

The osmium ferricyanide (OsFeCN) cytochemical technique wasused in the electron microscopic study of organelles in cellsof functionally different leaf zones of winter rye seedlings.In meristematic cells, the endoplasmic reticulum and nuclearenvelope were the main structures reacting with OsFeCN; in cellsof the extension zone and in differentiated mesophyll cellsit was the peripheral reticulum and the intermembrane spaceof the chloroplast envelope. The change in OsFeCN staining character of organelles betweenthe various zones of leaves is considered to be due to cellexit from the meristem. The participation of the peripheralreticulum of chloroplasts in the sequestration of Ca²⁺ ionsis proposed. Secale cereale L., leaf zones, OsFeCN-staining, Ca²⁺-sequestring organelles     

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.     

Effect of benzimidazole on the senescence of wheat chloroplasts and their boat shape transformation

Benzimidazole efficiently retards the senescence of detachedwheat leaves. Electron microscopic study has revealed that nodamage was visible on the fine structure of chloroplasts inleaves which retained their green color by benzimidazole treatment,whereas chloroplasts of water floated leaves showed strikingalterations in their color, shape and fine structure as senescenceproceeded. Moreover, the chloroplast lamellar system in benzimidazoletreated leaves even seemed to be organized over those in immediatelydetached leaves. The bending tendency for lamellar array within chloroplastswas more marked in benzimidazole treated leaves than in thecontrol. Chloroplasts showed, more frequently and intensely,transformation into the "boat shape" the extreme curvature ofthe granafretwork skeleton of the chloroplasts forming the keelof the boat. ¹This work was supported by Grants to Prof. E.R. WAYGOOD fromthe Canada Department of Agriculture (EMR-14) and the NationalResearch Council of Canada (PRBT-10) ²This work was undertaken during the author's stay at the Departmentof Botany, University of Manitoba, Winnipeg, Manitoba, Canada,with Fellowship granted to the author by the National ResearchCouncil of Canada, 1963–1965 (Received July 29, 1969; )     

Interactions between the Nucleus and Cytoplasmic Organelles during the Cell Cycle of Euglena gracilis in Synchronized Cultures III. Association between the Nucleus and Chloroplasts at an Early Stage in the Cell Cycle under Photoorganotrophic Conditions (Part II)

Three-dimensional models of chloroplasts before and during theirassociation with the nucleus in Euglena gracilis Z were constructedbased on serial sections of cells taken during an early phaseof the cell cycle in synchronized cultures under photoorganotrophiccondition. Before association, the cell contained two to threechloroplasts which were composed of elongated tubular bodiesextending in different directions toward the cell periphery.These tubular arms of chloroplasts were drawn toward the nucleus,and folded, with concurrent coalescence, into a single giantbody surrounding the nucleus. A DAPI-fluorescence photomicrographof a giant chloroplast revealed that the chloroplast-nucleoidswere in the form of a continuous strand lying throughout thechloroplast body, and that some protuberances from the nucleoidstrand were in especially close proximity to the nuclear periphery. A new mode of the chloroplast-nucleus connection was observed.The nucleus had conspicuous protrusions, whose distal ends wereconnected with the chloroplast. The outer membrane of the nuclearenvelope was continuous with the outer chloroplast membrane,and at some sites, an open space was formed between the innernuclear membrane and the inner chloroplast membrane. ¹ This work was reported at the 48th Annual Meeting of the BotanicalSociety of Japan, held in Kyoto, in October, 1983. For PartI see Ehara et al. (1984).     

Control of Seed Growth in Soya Beans [Glycine max (L.) Merrill]

The seed is the primary sink for photosynthate during reproductivegrowth and an understanding of the mechanisms controlling therate of seed growth is necessary to understand completely theyield production process. The growth rate of individual seedsof seven soya bean [Glycine max (L.) Merrill] cultivars withgenetic differences in seed size varied from 10.8 to 3.9 mgseed^–1 day^–1. The growth rates were highly correlatedwith final seed size. The growth rate of cotyledons culturedin a complete nutrient medium was highly correlated with thegrowth rate of seeds developing on the plant and with finalseed size. The number of cells per seed in the cotyledons variedfrom 10.2 to 5.7 x 10⁶ across the seven cultivars. The numberof cells per seed in the cotyledons was significantly correlatedwith final seed size and the seed growth rate both on the plantand in the culture medium. The data suggest that genetic differencesin seed growth rates are controlled by the cotyledons and thenumber of cells in the cotyledons may be the mechanism of control. Glycine max L., soya bean, seed size, growth rate, cell number, sink activity     

Analysis of Rapid Light-Induced Potential Change in Cells of Chara corallina

Perfused Chara cells were used to measure the rapid light-inducedpotential change (rapid LPC) caused by activation of a K⁺ channelin the plasma membrane through photosynthesis in the presenceof various photosynthetic inhibitors. The rapid LPC was inhibitedby DCMU but recovered on addition of phenazinemethosulfate (PMS)in the presence of DCMU. Carbonylcyanide m-chlorophenylhydrazone(CCCP) stimulated the rapid LPC. DCCD partially inhibited therapid LPC with a partial inhibition of oxygen evolution. Itis concluded that both cyclic and noncyclic electron flows arecoupled with the rapid LPC. To understand the mechanism of K⁺ channel activation by photosyntheticelectron flow, the rapid LPC was measured under continuous internalperfusion. It was suggested that a diffusible substance wasnot released from chloroplasts, since vigorous continuous perfusiondid not inhibit the rapid LPC. The suggestion that the rapid LPC is caused by changes in surfacecharge density of chloroplasts was supported by the fact thatthe rapid LPC was inhibited by increasing the ionic strengthof the perfusion medium. (Received February 28, 1986; Accepted April 30, 1986)     

Interactions between the Nucleus and Cytoplasmic Organelles during the Cell Cycle of Euglena gracilis in Synchronized Cultures: II. Associations between the Nucleus and Chloroplasts at an Early Stage of the Cell Cycle under Photoautotrophic Conditions

Chloroplast-nucleus interactions were examined in cells of Euglenagracilis Z synchronized under photoautotrophic conditions. Thechloroplasts were localized near the cell periphery. At an earlystage of the cell cycle, however, some chloroplasts were transientlylocated in the inner space close to the nucleus. Electron microscopyusing serial cell sections revealed that the chloroplast formedprotrusions at several sites, which became associated with thenucleus. The outer membrane of the chloroplast envelope wasin contact, or at least continuous in part, with the outer membraneof the nuclear envelope at the sites of association, and densematerial was present in the chloroplast membrane. A chromosomewas close to each site of the association between these twoorganelles. Most of the chloroplasts including those in associationwith the nucleus were connected by fine bridges. The 4',6-diamidino-2-phenylindole-stainednucleoids in the chloroplast associated with the nucleus appearedto have a thread-like shape. There was another type of chloroplast-nucleusconnection, in which an intervening membranous body was in contactwith the outer part of the nuclear envelope on one side andwith the chloroplast envelope on the other side. ¹ This work was reported at the 48th Annual Meeting of the BotanicalSociety of Japan, Kyoto, October, 1983. (Received June 5, 1984; Accepted November 20, 1984)     

Molecular Characterisation of the 76 kDa Iron-Sulphur Protein Subunit of Potato Mitochondrial Complex I

Genes encoding subunits of complex I (EC 1.6.5.3 [EC] ) of the mitochondrialrespiratory chain vary in their locations between the mitochondrialand nuclear genomes in different organisms, whereas genes fora homologous multisub-unit complex in chloroplasts have to dateonly been found on the plastid genome. In potato (Solatium tuberosumL.), the gene coding for the mitochondrial 76 kDa iron-sulphurprotein is identified in the nuclear genome. The gene is transcribedinto polyadenylated mRNA which is most abundant in flowers,and more frequent in tubers than in leaves. The amino acid sequenceis well conserved relative to the nuclear-encoded 75 kDa and78 kDa subunits of Bos taurus and Neurospora crassa, respectively,and to the Paracoccus denitrificans homologue, most prominentlyin the region presumed to carry the iron-sulphur clusters. Polyclonalantibodies directed against the 78 kDa complex I subunit ofN. crassa recognise the 76 kDa polypeptide in potato mitochondrialcomplex I, and additionally a polypeptide of 75 kDa in solubilisedstroma thylakoids from spinach chloroplasts. The 32 amino acidresidues long presequence of the potato mitochondrial 76 kDacomplex I subunit targets the precursor polypeptide into isolatedpotato mitochondria but not into isolated chloroplasts. Theseresults suggest that chloroplast stroma thylakoids contain aprotein similar in size and antigenicity to, but geneticallydistinct from, the mitochondrial subunit. ¹ To whom correspondence should be addressed. ⁴ Present address: Max-Planck-Institut für Molekulare Genetik,Ihnestrasse 73, D-14195, Berlin, Germany. ⁵ Present address: Bioinside GmbH, Potsdamer Strasse 18A, D-14513Teltow, Germany.     

Ferredoxin Isolated from Plant Non-Photosynthetic Tissues. Purficaition and Characterization

A ferredoxin was isolated from non-photosynthetic tissues ofthe lower storage root of radish (Raphanus sativus L. var. acantiformiscultivar Miyashige) in a pure form by conventional means. Itshowed the characteristic features in its absorption spectrumof chloroplast-type ferredoxin. However, amino acid compositionand amino (N)- terminal sequence were different from those ofradish leaf ferredoxin. Root ferredoxin was able to transferelectrons from dithionite to nitrite reductase [EC 1.7.7.1 [EC] ]isolated from mung bean seedling roots and also to mediate NADP⁺photoreduction in spinach broken chloroplasts. It therefore is suggested that a set of distinctive molecularspecies of ferredoxin is present in non-photosynthetic tissuesand functions as a redox mediator in ferredox-independent enzymesystems. (Received October 18, 1985; Accepted January 16, 1986)     

Vieillissement de l'appareil photosynth?tique II. Effet synergique de la lumi?re et du vieillissement in vitro sur les activit?es photochimiques de chloroplastes isol?s d'?pinard

The consequences of chloroplast ageing in vitro were furtherinvestigated, especially on the photochemical activities ofthese organelles. Ageing of chloroplasts in dark was accompanied by decreasesin activities for photohydrolysis and cyclic and non-cyclicsyntheses of ATP, photoreduction of NADP⁺ and O₂ evolution;but there was no decrease in ferricyanide photoreduction. Therates of decrease in these activities were comparable to therate of increase in chloroplast volume. Complete inhibitionswere reached when maximum chloroplast swelling had occurred,i.e. after 5 to 6 hr of incubation at 20?C in a Tris-NaCl (pH8) medium. Ageing in the light resulted in much accelerateddecreases in activities tested; the loss of capacity for light-inducedshrinkage was also accelerated by the light during ageing. Thus,light acts synergetically towards the ageing process. Moreover,light and, to a less extent, dark ageing, resulted in an uncouplingof chloroplast photophosphorylation and associated electronflow measured by ferricyanide photoreduction. The part of theelectron flow which is induced by coupling (+ ADP, Pi, MgCl₂,pH 8) or by uncoupling (+ NH₄C1, pH 7) was found to be verysensitive to light ageing. The NADP⁺ photoreduction loss wasrestored by addition of the ascorbate-DCPIP electron donor system,suggesting that ageing interferes with the integrity of photosystemII. In many respects, these effects of ageing are comparable withthe action of detergents and fatty acids on the structure andphotochemical activities of chloroplasts, especially in thatthey attack the energy transducing mechanism in chloroplasts. (Received May 24, 1969; )