Light-induced Changes in the Conformation and Configuration of the Thylakoid Membrane of Ulva and Porphyra Chloroplasts in Vivo

The ultrastructural basis of light-induced transmission and light scattering changes of thalli of Ulva and Porphyra were investigated by high resolution electron microscopy and microdensitometry. The results show that upon illumination of dark thalli (a) a reduction in thickness of thylakoid membranes (conformational change), (b) a more regular ordering, and (c) flattening of the thylakoids (configurational changes) have occurred. An explanation for the observed conformational and configurational changes was proposed in terms of correlated changes in ionic environment and osmotic properties of chloroplasts in vivo which are initiated by photosynthetic reactions.     

Seasonal and Experimentally Induced Changes in the Ultrastructure of Chloroplasts of Pinus silvestris

The ultrastructure of chloroplasts in mesophyll cells of Pinus silvesris was examined under the electron microscope. Secondary needles were regularly sampled from a tree in a natural stand for one year. Primary needles from one-year-old seedlings exposed to frost hardening and dehardening conditions in a controlled environment chamber were also studied. These seedlings were exposed to 8 or 55 W m^-2. All needles were put in fixative at the different sampling dates and stored in a refrigerator until they were prepared for electron microscopy at the end of the experimental period. During the summer the choroplasts were symmetrically shaped and heavily loaded with starch. The membrane systems were well developed and consisted of both grana and stroma thylakoids. In autumn and during early artificial frost hardening the starch content was reduced, the chloroplasts appeared amoeboid and membrane-free stroma regions were seen. Later the chloroplasts became swollen and aggregated in one part of the cell. Starch was lost and the chloroplasts aggregated earlier at 8 W m^-2 than at 55 W m^-2. During winter the stroma thylakoids were first reduced in number and later even the grana thylakoids were damaged, resulting in mostly disorganized single membranes. Also the chloroplast envelope disappeared. In spring and early summer the chloroplasts migrated to the proximity of the cell walls. The membrane systems were reorganized and starch accumulated. During the first days of artificial dehardening the photosynthetic membranes were severely damaged, especially at 55 W m^-2, but soon new membranes were formed. Starch accumulated earlier at 55 than at 8 W m^-2. The reported ultrastructural variations are discussed in relation to functional and biochemical fluctuations caused by the season or by artificial variations in the climate as demonstrated earlier.     

银杏叶片衰老过程中光合生理特性及叶绿体超微结构的变化

选取自然条件下生长的雌雄银杏植株为实验材料,测定了银杏叶片在衰老过程中部分光合生理指标及叶绿体超微结构的变化。检测结果表明：银杏叶片在衰老过程中净光合速率、叶绿素含量均呈下降趋势,SOD、CAT、APX活性均先上升后下降,MDA含量则一直呈现上升趋势。叶片衰老过程中叶绿体类囊体膜片层逐渐松散,直至膜结构逐渐解体,叶绿体内油脂颗粒增大增多,最终解体。雌雄银杏植株在各项生理指标上差异不显著。     

Relationship between Thylakoid Membrane Fluidity and the Functioning of Pea Chloroplasts : EFFECT OF CHOLESTERYL HEMISUCCINATE

Cholesteryl hemisuccinate has been incorporated into pea chloroplast thylakoids to investigate the relationship between fluidity and functioning of this membrane system. Levels of sterol which increased the apparent viscosity of the membrane, estimated by fluorescence polarization measurements using the lipophilic probe, 1,6-diphenyl-1,3,5 hexatriene, affected several photosynthetic processes. A decrease in fluidity was accompanied by an inhibition of dark limiting steps associated with electron transfer between photosystems two and one (PSII and PSI) as observed by the oxidation of the primary acceptor of PSII and by electron flow to ferricyanide. Also, treatment with cholesteryl hemisuccinate inhibited the saltinduced rise in chlorophyll fluorescence and changed the ionic conductivity of the membrane as judged by measurements of the decay of the lightinduced proton gradient. The results are discussed in terms of the effect of fluidity changes on the lateral diffusion of plastoquinone and chlorophyll protein complexes in the lipid matrix of the membrane.     

Features of Grana Organization in Pea Chloroplasts

Two fractions of membrane fragments—the pellets precipitated at 1300 and 20000 g (fractions G1.3 and G20, respectively)—were isolated from pea (Pisum sativum L.) chloroplasts after solubilization with digitonin. These fragments assigned to grana displayed the following differences: (1) in spectra of low-temperature fluorescence, the ratio of short-wave and long-wave band intensities, as well as integrated intensity of the whole spectrum, were higher for G1.3 than for G20 fraction; (2) in excitation spectra of long-wave fluorescence, the ratio of peaks at 650 and 680 nm and integrated intensity of the spectrum were higher for G1.3 than for G20 fraction; and (3) the shapes of fluorescence excitation spectra differed for G1.3 and G20. These results indicate that the two fractions examined differed in proportion of photosystem I and photosystem II complexes, as well as in organization of these complexes. The size of light-harvesting antenna was larger in PSI complexes of G1.3 fraction, owing, in particular, to a higher content of chlorophyll a/b-protein complexes in this fraction. After repeated digitonin fragmentation of G1.3 and G20 preparations, more than 80% of G1.3 fraction was decomposed into lighter fragments, whereas G20 fraction was resistant to fragmentation (it lost about 10% of its material). Analysis of the data suggests the presence of two structurally different types of thylakoids in grana. The yield of G20 fraction (about 20%) is comparable to the ratio between the number of intergranal thylakoids, connected to granum in pea chloroplasts, and the total number of thylakoids in this granum. Based on these data, we assume that G20 fraction represent the fragments of intergranal thylakoids that extend into the granum.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 499–506.Original Russian Text Copyright © 2005 by Kochubei, Shevchenko, Bondarenko.     

Activity of Thylakoid-bound Ribosomes in Pea Chloroplasts

Pea (Pisum sativum) chloroplast thylakoid membranes were prepared by washing in hypotonic buffers. These membranes contained bound ribosomes which were active in protein synthesis when supplemented with soluble components from a strain of Escherichia coli low in ribonuclease. After dissolving the membranes by Triton and purification of the ribosomes, sucrose density gradient profiles indicated the presence of polysomal material as well as monomeric ribosomes. Most of the products of protein synthesis remained associated with the thylakoid membranes even after ribosomes were removed completely by high salt concentrations in the absence of Mg²⁺. Of the newly formed products, 50% could be digested by pronase, while the remainder were protected by their association with the thylakoid membranes. The products are likely to be a mixture of intrinsic and extrinsic membrane proteins, with only the former completely protected by the membranes from attack by proteases.     

Light-induced Changes of the Electrical Potential in Chloroplasts Associated with the Activity of Photosystem I and Photosystem II

The electric potential changes induced by flashing and continuouslight were measured with microcapillary electrodes in isolatedwhole chloroplasts of Peperomia inetallica. In continuous lightthe chloroplast electrical potential rose in two phases. Theinitial rapid phase coincided in extent with the flash-inducedpotential and was insensitive to the electron transfer inhibitorDBMIB. The subsequent phase was relatively slow (20–30ms) and was inhibited by DBMIB. Electron acceptors of photosystemII (p-phenylendiamine, p-benzoquinone) added to DBMIB-treatedchloroplasts produced a suppression of the flash-induced responseand a considerable increase in the steady level of the potentialin the light. The electrical potential associated with the activityof photosystem II rose in continuous light much more slowlythan that associated with the activity of photosystem I aloneor the activities of both photosystems. Illumination of chloroplastswith successive flashes at a repetition rate 5 Hz in the presenceof oxaloacetate, a terminal acceptor of photosystem I, was accompaniedwith a gradual decline of the flash-induced potential. The specificrole of two photosystems in the light-induced H⁺ transport andthe electrogenesis across the chloroplast thylakoid membranesis discussed.     

植物光诱导延迟荧光与光合作用的内在关联性研究

光合作用是地球上最重要的化学反应,植物内源性光诱导延迟荧光是光合作用原初过程中光系统Ⅱ作用中心P680处电荷分离效率的内在探针。本文从实验上证明了延迟荧光的产生与光合作用存在本质必然的联系。实验结果表明,延迟荧光激发谱与光合作用谱存在着明显的相似,延迟荧光强度随着激发光源光强的变化表现出类似的光抑制现象,以及在室温条件下延迟荧光强度与光合速率有着很好的相关性。为从植物光诱导延迟荧光技术来研究植物的光合作用提供了重要的实验证据。     

The Effect of Root Hypoxia and Iron Deficiency on the Photosynthesis, Biochemical Composition, and Structure of Pea Chloroplasts

The combined effect of root hypoxia and iron deficiency on biochemical composition, photosynthetic indices, and structure of pea (Pisum sativum L.) chloroplasts were investigated. Both factors suppressed chlorophyll accumulation and leaf photosynthetic activity, causing chlorosis. It was shown, that iron deficiency reduced more severe the light-harvesting complexes of photosystems (PS), and root hypoxia, the reaction center complexes of the photosystem I (PSI) and photosystem II (PSII). The combined action of both factors was stronger than the effect of each factor. However, even in yellow and almost white leaves, chloroplasts contained small amounts of all pigment–protein complexes and maintained weak photosynthetic activity, although their structure was poorly developed and comprised only vesicles and small thylakoids capable to form contacts and small grana. The conclusion is that the mechanisms of root hypoxia and iron deficiency destructive action are different and these factors differently and independently influenced leaf chloroplasts.     

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.     

Changes in the Biochemical Composition, Structure, and Function of Pea Leaf Chloroplasts in Iron Deficiency and Root Anoxia

A combined effect of iron deficiency and root anoxia on the biochemical composition, function, and structure of pea leaf chloroplasts was studied. It was found that the chlorosis of apical leaves in response to iron deficiency was determined by the reduction of light-harvesting complexes I and II. Under root anoxia, complexes of the reaction centers of photosystems I and II degraded first. Weak activity was preserved even in yellow and white leaves under the effect of both factors. The ultrastructure of leaf chloroplasts gradually degraded. Initially, intergranal thylakoid sites were reduced, and the longitudinal orientation of grana was disturbed. However, yellow and white leaves still retained small thylakoids and grana. It is concluded that the degrading effects of iron deficiency and root anoxia on the complex composition and leaf chloroplast structure and function are additive because of their autonomous mechanisms.     

Isolation and Characterization of Biotin Carboxylase from Pea Chloroplasts

Pea (Pisum sativum L.) leaf acetyl-coenzyme A carboxylase (ACCase) exists as two structurally different forms: a major, chloroplastic, dissociable form and a minor, multifunctional enzyme form located in the leaf epidermis. The dissociable form is able to carboxylate free D-biotin as an alternate substrate in place of the natural substrate, biotin carboxyl carrier protein. Here we report the purification of the biotin carboxylase component of the chloroplastic pea leaf ACCase. The purified enzyme, free from carboxyltransferase activity, is composed of two firmly bound polypeptides, one of which (38 kD) is biotinylated. In contrast to bacterial biotin carboxylase, which retains full activity upon removal of the biotin carboxyl carrier component, attempts to dissociate the two subunits of the plant complex led to a complete loss of biotin carboxylase activity. Steady-state kinetic studies of the biotin carboxylase reaction reveal that addition of all substrates on the enzyme is sequential and that no product release is possible until all three substrates (MgATP, D-biotin, bicarbonate) are bound to the enzyme and all chemical processes at the active site are completed. In agreement with this mechanism, bicarbonate-dependent ATP hydrolysis by the enzyme is found to be strictly dependent on the presence of exogenous D-biotin in the reaction medium.     

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.     

Growth and Photosynthesis of Mutant Pea Seedlings

The recessive of gene, producing tendrils in place of leaves,and the recessive st gene, reducing stipule size, produce phenotypesof pea that are termed leafless (afafstst) and semi-leafless(afafStSt). Photosynthesis and growth of these two types werecompared with the conventional phenotype (AfAfStSt) during thefirst 9 days of post-emergent growth. The conventional seedlingshowed faster net photosynthesis per unit dry weight than theleafless phenotype, whilst the semi-leafless seedlings wereintermediate. Differences in dark respiration were small butleafless seedlings had significantly higher rates soon afteremergence. Where the three phenotypes used were isogenic, except for ofand st, the rates of shoot growth were in the same ranking orderas net CO₂ uptake. With three other genotypes, representingthe three phenotypes, more similar shoot growth was found betweenthe conventional and semi-leafless phenotype, possibly becauseof compensating differences in embryonic axis size. The ratesof growth of roots and the rates of dry weight loss from thecotyledons showed no consistent differences between phenotypes. The results are discussed in relation to the potential for thesemi-leafless phenotype as an alternative to the conventionalphenotype for the dried pea crop. Pea seedling, Pisum sativum, leafless pea, photosynthesis, seedling growth     

Lipid Synthesis and Ultrastructure of Isolated Barley Chloroplasts

The cell organelle contents of chloroplast preparations made from barley leaves with salt and sucrose isolation media at pH 6 and 8 were determined and compared with the acetate incorporating activity of these preparations. A chloroplast preparation obtained with 0.5 m sucrose at pH 8 gave the highest number of intact chloroplasts (with envelope and stroma), the lowest number of contaminating mitochondria, and the highest activity in light dependent acetate incorporation into lipids. In the preparations observed, the light induced lipid synthesizing capacity correlates well with the percentage of intact chloroplasts. It is suggested that the intact chloroplasts are responsible for the light induced lipid synthesis of the preparations and that the synthesizing enzymes are localized in the chloroplast stroma. Acetate is mainly incorporated into palmitic and oleic acids. The low yield of intact chloroplasts and of light induced lipid synthesis in preparations isolated at pH 6 seem to result from the action of galactolipid lipase(s).     

Limiting Factors in Photosynthesis: IV. Iron Stress-Mediated Changes in Light-Harvesting and Electron Transport Capacity and its Effects on Photosynthesis in Vivo

Using iron stress to reduce the total amount of light-harvesting and electron transport components per unit leaf area, the influence of light-harvesting and electron transport capacity on photosynthesis in sugar beet (Beta vulgaris L. cv F58-554H1) leaves was explored by monitoring net CO₂ exchange rate (P) in relation to changes in the content of Chl.

In most light/CO₂ environments, and especially those with high light (≥1000 microeinsteins photosynthetically active radiation per square meter per second) and high CO₂ (≥300 microliters CO₂ per liter air), P per area was positively correlated with changes in Chl (a + b) content (used here as an index of the total amount of light-harvesting and electron transport components). This positive correlation of P per area with Chl per area was obtained not only with Fe-deficient plants, but also over the normal range of variation in Chl contents found in healthy, Fe-sufficient plants. For example, light-saturated P per area at an ambient CO₂ concentration close to normal atmospheric levels (300 microliters CO₂ per liter air) increased by 36% with increase in Chl over the normal range, i.e. from 40 to 65 micrograms Chl per square centimeter. Iron deficiency-mediated changes in Chl content did not affect dark respiration rate or the CO₂ compensation point. The results suggest that P per area of sugar beet may be colimited by light-harvesting and electron transport capacity (per leaf area) even when CO₂ is limiting photosynthesis as occurs under field conditions.

     

The Ultrastructure of Chloroplasts in Mineral-deficient Maize Leaves

The ultrastructure of mesophyll chloroplasts in full-nutrient and mineral-deficient maize (Zea mays) leaves was examined by electron microscopy after glutaraldehyde-osmium tetroxide fixation. Nitrogen, calcium, magnesium, phosphorus, potassium, and sulfur deficiencies were induced by growing the plants in nutrient culture. Distinctive chloroplast types were observed with each deficiency. Chloroplasts from nitrogen-deficient plants were reduced in size and had prominent osmiophilic globules and large grana stacks. Magnesium deficiency was characterized by the accumulation of osmiophilic globules and the progressive disruption of the chloroplast membranes. In calcium deficiency, the chloroplast envelope was often ruptured. Chloroplasts from potassium- or phosphorus-deficient plants possessed an extensive system of stroma lamellae. Sulfur deficiency resulted in a pronounced decrease of stroma lamellae, an increase in grana stacking, and the frequent occurrence of long projections extending from the body of the chloroplast. These morphological changes were correlated with functional alterations in the chloroplasts as measured by photosystem I and II activities. In chloroplasts of the nitrogen- and sulfur-deficient plants an increase in grana stacking was associated with an increase in photosystem II activity.     

Changes in the Pattern of Enzyme Development in Gibberellin-treated Pea Internodes

A study was made on the effect of gibberellic acid on amylase,cellulase, ß-fructofuranosidase, pectinesterase, andstarch phosphorylase activities in elongating dwarf-pea internodes. Hormonal stimulation of amylase and ß-fructofuranosidaseactivities correlated closely with internode growth, the activityof starch phosphorylase less so, and gibberellic acid had noimmediate effect on cellulase and pectinesterase activities. Injection of glucose (or glucose derivatives) into pea internodesmimicked the effect of gibberellic acid on fresh- and dry-weightaccumulation, cell elongation, cell division, and cell-wallsynthesis. It is proposed that the over-all effect of gibberellic acidon enzyme development is to provide more substrate (particularlyglucose) for general cell metabolism and wall synthesis withinelongating internodes.     

Light-induced De-epoxidation in Lettuce Chloroplasts: VI. De-epoxidation in Grana and in Stoma Lamellae

Grana and stroma lamellae fractions prepared from illuminated chloroplasts (Lactuca sativa L. var. Manoa) by French press treatment contained less violaxanthin and more zeaxanthin than the corresponding fractions from dark controls. In both fractions, only part of the total violaxanthin was de-epoxidized under illumination, and the ratio of de-epoxidized and unchanged violaxanthin was similar. This not only shows that the de-epoxidation system is present in both grana and stroma thylakoids but also that violaxanthin is heterogeneous in both membranes. The presence and similarity of the de-epoxidation system in grana and stroma lamellae suggest that the function of the violaxanthin cycle is linked to photosynthetic activities which are common to both types of membranes.