Palisade Tissue Chloroplasts and Spongy Tissue Chloroplasts in Spinach: Biochemical and Ultrastructural Differences

Palisade tissue chloroplasts (P-Chlts) and spongy tissue chloroplasts(S-Chlts) were separately isolated from spinach leaves, andtheir photosynthetic properties were compared. The followingresults were obtained: (1) At saturating light, the activities of overall electrontransport and CO₂ fixation in P-Chlts were respectively 1.6–2.0and 2.5–3.0 times higher than those in S-Chlts on a Chlbasis. (2) The contents of PS I and PS II reaction centers (P700 and47 kDa polypeptide, respectively) were slightly higher in P-Chltsthan in S-Chlts, while the contents of plastoquinone, Cyt f,plastocyanin, ferredoxin, ferredoxin-NADP⁺ reductase, couplingfactor and ribulose-bisphosphate carboxylase were 1.6–2.2times higher in P-Chlts than in S-Chlts on a Chl basis. (3) Electron microscopic examination of chloroplast ultrastructureshowed that S-Chlts have highly stacked grana accompanied byhigher proportion of appressed thylakoids relative to non-appressedthylakoids, while P-Chlts have poorly stacked grana. The volumeratio of thylakoids to stroma was higher in S-Chlts than inP-Chlts. These results indicate that mesophyll chloroplasts adapt tothe light environment within a leaf in a similar way that thesun and shade plant chloroplasts adapt to the light environmentwithin a canopy. (Received July 19, 1984; Accepted October 13, 1984)     

A Comparative Ultrastructural Study of Cyanidium caldarium and the Unicellular Red Alga Rhodosorus marinus

The presence of nucleus, chloroplasts, mitochondria, microbodiesand a vacuole are confirmed in Cyanidium caldarium strain CCAP1355/1. Chloroplasts usually contain parallel rows of unstackedthylakoids surrounded by a peripheral thylakoid, although theconcentric arrangement has also been observed. The chloroplastenvelope consists of two closely appressed membranes which canonly be resolved after gluteraldehyde—osmium fixation.The chloroplast of Rhodosorus marinus also contains parallel,unstacked thylakoids surrounded by a peripheral thylakoid but,in addition, a prominent, stalked pyrenoid projects into thecytoplasm and is bounded by both the peripheral thylakoid andthe chloroplast envelope. This structure is covered by a capof starch grains and contains membranous vesicles in the matrix.Phycobilisomes are absent from the chloroplasts of both algae.The recognition of C. caldarium as a rhodophyte is supportedby these observations. Cyanidium caldarium, Rhodosorus marinus, chloroplast ultrastructure     

Ontogenetic Change of the PSI- and PSII-Distribution in the Thylakoid System of Euglena gracilis

The thylakoid membranes of isolated Euglena chloroplasts wereseparated into two fractions by aqueous two-phase-partitioning(mixture of dextran 500 and poly(ethylene glycol) 4000) followingpress disruption. These two fractions differ in many respectsduring most of the cell cycle of this alga in comparison withthe thylakoid characteristics of higher plants or green algae.The amount of thylakoid membranes with separation characteristicscomparable with inside-out-vesicles of higher plant chloroplastschanges depending on the cell cycle stage of Euglena gracilis.Photosystems II and I are not restricted to one fraction. Boththylakoid membrane fractions evolve oxygen photosynthetically.When chloroplast differentiation in Euglena gracilis is complete(i.e. at the end of the light-time) the composition and thephotosynthetic efficiency of the two thylakoid fractions aregenerally equal. Photosystem I-related LHCI is present in bothfractions. Photosystem II-related CP29, however, was only detectedin unfractionated thylakoid membranes. The implications forthylakoid organization in Euglena chloroplasts are discussed. Key words: Euglena gracilis, photosystem I, photosystem II, stacking, thylakoids     

Intra-Leaf and Intracellular Gradients in Chloroplast Ultrastructure of Dorsiventral Leaves Illuminated from the Adaxial or Abaxial Side during their Development

The number of thylakoids per granum, which is smaller in sun-typechloroplasts than in shade-type chloroplasts, was counted forthe chloroplasts at various positions within the single leavesof Spinacia oleracea L. and Glycine max (L.) Merrill. The thylakoidnumber increased with depth from the adaxial surface, but thistrend was not evident within respective cells. Therefore, photosyntheticproperties of chloroplasts should be similar within a cell butdifferent among cell layers. The similarity within a cell maybe due to the nuclear control of the chloroplast developmentand/or to chloroplast movement along the cell walls. Illumination of the leaves of G. max from the abaxial side duringtheir expansion resulted in the complete inversion of the intra-leafgradient in the thylakoid number, indicating that the formationof the intra-leaf gradient in chloroplast properties is influencedby the intra-leaf light environment during the later phase ofleaf development. ¹presennt address: Department of Environmental Biology, ResearchSchool of Biological Sciences, Australian National University,P.O. Box 475, Canberra, A.C.T. 2601, Australia. (Received March 13, 1986; Accepted May 30, 1986)     

Starch Accumulation and Photosynthetic Activity in Primary Leaves of Bean (Phaseolus vulgaris L.)

The effects of starch accumulation on photosynthesis and chloroplastultrastructure were studied in primary leaves of bean (Phaseolusvulgaris L. cv. Bulgarian). De-topping the shoot above the primaryleaf node, caused over an 8-day period, a considerable increasein the photosynthetic activity of the primary leaves, despitethe fact that a large quantity of starch had accumulated intheir chloroplasts. The accumulation of starch was greater inthe chloroplasts of spongy cells in comparison with that ofthe palisade cells. Initiation of starch grains was observedmainly in the peripheral part of the chloroplast, distant fromthe cell wall. As a result, most of the starch was accumulatedclose to the inner part of the cell, leaving a considerablemass of the chloroplast near the cell wall free of starch. Theaccumulation of starch was accompanied by the destruction, deformationand disorientation of grana and thylakoids. It is concludedthat the accumulation of starch is not inevitably a limitingfactor in photosynthesis and the results cast doubt on the hypothesisthat starch accumulation or dissipation is the main factor involvedin the regulation of photosynthesis. Phaseolus vulgaris L, bean, photosynthesis, starch accumulation, chloroplast ultrastructure     

Intact Chloroplasts Show Ca-Gated Switching between Localized and Delocalized Proton Gradient Energy Coupling (ATP Formation)

Intact chloroplasts were compared to isolated thylakoids as to whether storage of the organelle in high KCl medium caused the energy coupling reactions to show a delocalized or a localized proton gradient energy coupling response. With isolated thylakoids, the occurrence of one or the other energy coupling mode can be reversibly controlled by the concentration of mono- and divalent cations used for the thylakoid storage media. Calcium was shown to be the key ion and previous evidence suggested a Ca²⁺-controlled gating of H⁺ fluxes in the thylakoid membrane system (G Chiang, RA Dilley [1987] Biochemistry 26: 4911-4916). Isolated, intact chloroplasts, which retained the outer envelope membranes during the 30 min or longer storage treatments in various concentrations of KCl and CaCl₂ (with sorbitol to maintain iso-osmotic conditions), were osmotically burst in a reaction cuvette and within 3 minutes were assayed for either a localized or a delocalized proton gradient energy coupling (ATP formation) mode. The intact chloroplast system was analogous to isolated thylakoids, with regard to the effects of KCl and CaCl₂ on the energy coupling mode. For example, adding 100 millimolar KCl to the intact organelle storage medium resulted in the subsequent ATP formation assay showing delocalized proton gradient coupling just as with isolated thylakoids. Adding 5 millimolar CaCl₂ to the 100 millimolar KCl storage medium resulted in a localized proton gradient coupling mode. Suspending thylakoids in stromal material previously isolated from intact chloroplast preparations and testing the energy coupling response showed that the stromal milieu has enough Ca²⁺ to cause the localized coupling response even though there was about 80 millimolar K⁺ in the intact chloroplasts used in this study (determined by atomic absorption spectrophotometry). Extrapolating the intact chloroplast data to the whole leaf level, we suggest that proton gradient energy coupling is normally of the localized mode, but under certain conditions it could be either localized or delocalized, depending on factors that affect the putative Ca²⁺-regulated proton flux gating function.     

Membrane-bound Plastid Inclusions and Chloroplast Thylakoid Formation in Sunflower (Helianthus annuus L.)

The ultrastructure, distribution and frequency of membrane-boundplastid inclusions present in the epidermal cells of leavesof intact sunflower plants (Helianthus annuus L.) and in theepidermal and mesophyll cells of sunflower leaf discs culturedin darkness have been studied. These inclusions appear to bedilated thylakoids containing a granular material which, undernormal conditions, is probably involved in chloroplast membraneformation. It is suggested that this material accumulates, andinclusions form, in the chloroplasts of sunflower leaves intwo specific situations. Firstly, in the completely differentiatedcells of the epidermis where the chloroplasts, although at arelatively immature stage, have nevertheless reached a terminalstage of development. Secondly, in the mesophyll cells of youngleaves when chloroplast development has been arrested at animmature stage by a 5-day dark period. In the latter situationthe material can be remobilized if plastid development is restimulated.The plastids of sunflower leaf discs cultured in darkness containboth membrane-bound inclusions and prolamellar bodies, indicatingthat they are separate and distinct structures possibly containingdifferent membrane components. Helianthus annuus L., sunflower, chloroplast, ultrastructure, plastid inclusions, thylakoid formation     

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.     

Thylakoid-bound chloroplast DNA from spinach is enriched for replication forks

Chloroplast DNA is bound to the thylakoids of spinach chloroplasts. To examine a possible role for thylakoid-bound DNA in chloroplast DNA replication, vesicles formed by treating chloroplasts in 3.5 mM MgCl2 were used. Chloroplast DNA fragments are bound to the surface of these vesicles. Chloroplast DNA isolated from vesicles that had been first treated with Eco R1 contained 10% of branched fragments whereas chloroplast DNA isolated from intact chloroplasts and treated with Eco R1 contained 2% of branched fragments. This result is consistent with the growing replication fork of chloroplast DNA being associated with the chloroplast internal membrane system. Branched fragments from the chloroplast DNA digested with Eco R1 prior to the isolation from the vesicle contained fragments of unequal length. Membrane binding in chloroplasts may have a similar role in DNA replication as it does in bacteria.     

Ultrastructural Changes in Chloroplasts of Quercus rotundifolia Lam. in Response to Evernic Acid

The amount of total chlorophyll, chlorophylls a and b as wellas the ratio of a to b decreased in chloroplasts isolated fromQuercus rotundifolia leaves, kept for 17 d in a solution of35.5 µM evernic acid in 1 mM Na HCO₃, when compared withthe chloroplasts of control leaves (kept in NaHCO₃). The chloroplastsin the spongy parenchyma were smaller and the amount of starchand plastoglobuli lower. The number of grana per chloroplastsection, the number of thylakoids per grana and the height ofgrana stacks were also less in the chloroplasts of leaves treatedwith evernic acid. Quantitative ultrastructural differenceswere determined by means of electron microscopy and image analysistechniques. Quercus rotundifolia Lam., chloroplasts, ultrastructure, lichens, evernic acid     

Cryopreservation of chloroplasts and thylakoids for studies of protein import and integration

A method is presented for preservation of isolated intact chloroplasts and isolated thylakoids for use in chloroplast protein import and thylakoid protein integration studies. Chloroplasts of pea (Pisum sativum) were preserved by storage in liquid nitrogen in the presence of a cryoprotective agent. Dimethyl sulfoxide was the most effective of several cryoprotectants examined. Approximately 65 to 70% of chloroplasts stored in liquid nitrogen in the presence of dimethyl sulfoxide remained intact upon thawing and were fully functional for the import of precursor proteins. Imported proteins were correctly localized within these chloroplasts, a process that for two of the proteins tested involved transport into the thylakoids. Lysate obtained from preserved chloroplasts was functional for protein integration assays. Preserved chloroplasts retained import and localization capability for up to 6 months of storage. Thylakoids were preserved by a modification of a method previously described (Farkas DL, Malkin S [1979] Plant Physiol 64: 942-947) for preservation of photosynthetic competence. Preserved thylakoids were nearly as active for protein integration studies as freshly prepared thylakoids. The ability to store chloroplasts and subfractions for extended periods will facilitate investigations of plastid protein biogenesis.     

The Rate of Photosynthesis in Isolated Chloroplasts

Chloroplasts were isolated using aqueous and nonaqueous procedures.Aqueous chloroplasts lost approximately 50 per cent, of theirsoluble proteins during isolation. Nonaqueous chloroplasts retainedall their soluble enzymes, but lost their ability to performthe light reactions of photosynthesis. It was possible to reconstitutea chloroplast system of higher activity by adding soluble enzymesfrom nonaqueous chloroplasts to protein-deficient aqueous chloroplasts.The properties of the reconstituted chloroplast system wereas follows: 1. The CO₂ fixation rate of the reconstituted chloroplast system( 4 µM./. chlorophyll/hr.) was 3–4 times that ofthe aqueous chloroplasts ( I µM./. chlorophyll/hr.). Thefixation of aqueous chloroplasts isapparently limited in partby lack of soluble enzymes. 2. During light-fixation, the reconstituted chloroplast systemaccumulated PGA. This indicates that the reduction of PGA totriosephosphate is a rate-limiting step in this system. 3. It was possible to increase the CO₂ fixation to 12 µM.CO₂/mg. chlorophyll/ hr. by addition of ATP and TPNH to thesystem, but the reduction of PGA was still rate-limiting. 4. Further increase in the fixation rate was obtained by concentratingthe reaction mixture. Part of the striking differences of theCO₂-fixing capabilities of chloroplasts in vivo and in vitrois caused by dilution effects. Extrapolation of the dilutioneffect to the protein concentration which exists in chloroplastsyields a CO₂ fixation rate of approximately 30 µM./mg.chlorophyll/hr. 5. Inhibitors which are located in vivo outside the chloroplastsaffect the CO₂ fixation in vitro. 6. Under consideration of the examined factors which influencethe CO₂ fixation of isolated chloroplasts, it is possible toraise the fixation from approximately 1 per cent, to at least15 per cent, of the fixation in vivo.     

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)     

Effects of Crosslinking Agent-DFDNB on Energy-transducing Reactions of Chloroplast Thylakoid Membrane Proteins

The effects of crosslinking agent-DFDNB (difluoro dinitro benzene) on functions of chloroplast thylakoid membrane proteins were investigated. DFDNB inhibited activities of PSP and membrane-bound ATPase in chloroplasts. It decreased proton uptake of light-inducted chloroplast thylakoids and the relative value of fluorescence quenching of 9-aminoacridine, and inhibited the rate of fast electrogenic phase of absorption change at 515 nm in chloroplasts. In addition, the isolated CF1-ATPase was crosslinked with DFDNB. The pattern of polymers of crosslinked CF1-ATPase was observed on SDS-PAGE.     

Hill Reaction, Hydrogen Peroxide Scavenging, and Ascorbate Peroxidase Activity of Mesophyll and Bundle Sheath Chloroplasts of NADP-Malic Enzyme Type C(4) Species

Intact mesophyll and bundle sheath chloroplasts wee isolated from the NADP-malic enzyme type C₄ plants maize, sorghum (monocots), and Flaveria trinervia (dicot) using enzymic digestion and mechanical isolation techniques. Bundle sheath chloroplasts of this C₄ subgroup tend to be agranal and were previously reported to be deficient in photosystem II activity. However, following injection of intact bundle sheath chloroplasts into hypotonic medium, thylakoids had high Hill reaction activity, similar to that of mesophyll chloroplasts with the Hill oxidants dichlorophenolindophenol, p-benzoquinone, and ferricyanide (approximately 200 to 300 micromoles O₂ evolved per mg chlorophyll per hour). In comparison to that of mesophyll chloroplasts, the Hill reaction activity of bundle sheath chloroplasts of maize and sorghum was labile and lost activity during assay. Bundle sheath chloroplasts of maize also exhibited some capacity for 3-phosphoglycerate dependent O₂ evolution (29 to 58 micromoles O₂ evolved per milligram chlorophyll per hour). Both the mesophyll and bundle sheath chloroplasts were equally effective in light dependent scavenging of hydrogen peroxide. The results suggest that both chloroplast types have noncyclic electron transport and the enzymology to reduce hydrogen peroxide to water. The activities of ascorbate peroxidase from these chloroplast types was consistent with their capacity to scavenge hydrogen peroxide.     

Chloroplast biogenesis. Cell-free transfer of envelope monogalactosylglycerides to thylakoids.

An ATP- and temperature-dependent transfer of monogalactosylglycerides from the chloroplast envelope to the chloroplast thylakoids was reconstituted in a cell-free system prepared from isolated chloroplasts of garden pea (Pisum sativum) or spinach (Spinacia oleracea). Isolated envelope membranes, in which the label was present exclusively in monogalactosylglycerides, were prepared radiolabeled in vitro with [14C]galactose from UDP-[14C]galactose to label galactolipids as the donor. ATP-dependent transfer of radioactivity from donor to unlabeled acceptor thylakoids, immobilized on nitrocellulose strips, was observed. In some experiments linear transfer for longer than 30 min of incubation was facilitated by the addition of stroma proteins but in other experiments stroma was without effect or inhibitory suggesting no absolute requirements for a soluble protein carrier. Transfer was donor specific. No membrane fraction tested (plasma membrane, tonoplast, endoplasmic reticulum, nuclei, Golgi apparatus, mitochondria or thylakoids) (isolated from tissue radiolabeled in vivo with [14C]acetate) other than chloroplast envelopes demonstrated any significant ability to transfer labeled membrane lipids to immobilized thylakoids. Acceptor specificity, while not absolute, showed a 3-10-fold greater ATP-dependent transfer of labeled galactolipids from chloroplast envelopes to immobilized thylakoids than to other leaf membranes. The results provide independent confirmation of the potential for transfer of galactolipids between chloroplast envelopes and thylakoids suggested previously from ultrastructural studies and of the known location of thylakoid galactolipid biosynthetic activities in the chloroplast envelope.     

Biophysical studies on subcellular particles VII. Combined effects of alcohol and heat on photosynthetic activities in isolated spinach chloroplasts

Isolated spinach chloroplasts were warmed for 5 min at temperaturesranging between 20? and 55?C in the presence of short-chainaliphatic alcohols, and their photosynthetic activities wereassayed. Tmax, the temperature at which ferricyanide reduction is stimulatedto a maximum extent, was lowered with an increase in the concentrationof alcohol or the chain-length of n-alcohols. Values also differedamong the structural isomers of an alcohol. The Tmax shift wasobserved only when alcohol was present in a chloroplast suspensionduring warming treatment, indicating that heat and alcohol wereacting together to lower the Tmax, at which the phosphorylationactivity fell to zero. The combined effects of alcohol and heat are discussed in connectionwith the lipophilic construction of thylakoids through the partitionco-efficient of individual alcohols between water and n-octylalcohol. (Received August 5, 1972; )     

Light-Induced Release of Bound Glucose-6-phosphate Dehydrogenase to the Stroma in Pea Chloroplasts

Light affects the partitioning of glucose-6-phosphate dehydrogenase between thylakoids and stroma in the chloroplast. Illumination of intact chloroplasts changes the ratio between bound and free enzyme from approximately 1:1 to 1:2. Treatment with NADPH, inorganic phosphate, or high pH also results in release of the enzyme from isolated thylakoids.     

Electron dense substance of thylakoids in chloroplasts of tansy Tanacetum vulgare L

An intrathylakoid electron opaque substance, further referred to as loculin, is found in 80-90 % of thylakoids of tansy leaf mesophyll chloroplasts at the stage of flower bud formation and flowering. Upon conventional isolation of chloroplasts in aqueous solution, and fixation in osmium solution alone, loculin is not retained in thylakoids. Preliminary fixation of leaves in glutaraldehyde makes it possible to isolate chloroplasts without injuring the envelope and stroma (glutar chloroplasts), and loculin is retained in thylakoids under these conditions. Upon prolonged incubation of glutar chloroplasts (for 24 h), loculin leaves thylakoids in the form of drops concentrating on the chloroplast envelope. Upon crossing the thylakoid membrane and chloroplast, loculin properties remain unchanged. It is assumed that loculin is an important metabolite necessary for active growth.     

Differential effect of NaCl and polyethylene glycol on the ultrastructure of chloroplasts in rice seedlings

Ionic and osmotic effects of salinity on the ultrastructure of chloroplasts in salt-treated rice seedlings were investigated. After rice seedlings were grown in hydroponic culture for three weeks, they were treated with NaCl and polyethylene glycol (PEG) 4000 both at a water potential of -1.0 MPa for 3 days. The most notable difference in ultrastructural change between NaCl and PEG treatment was observed in the damage in chloroplast membranes. NaCl induced swelling of thylakoids and caused only a slight destruction of the chloroplast envelope. PEG caused severe destruction of the chloroplast envelope compared with NaCl, however thylakoids did not swell. Our observations suggested that in salt-treated rice plants, the ionic effects induced swelling of thylakoids and the osmotic effects caused the destruction of chloroplast envelope.