The marginal regions of thylakoid membranes: A partial characterization by polyoxyethylene sorbitan monolaurate (Tween 20) solubilization of spinach thylakoids

The detergent Tween-20 solubilized preferentially portions of the marginal regions of Spinacea oleracea L. thylakoid membranes and, thus, opened the inside of the grana to the external media. Differential centrifugation. following Tween-20 solubilization. enabled separate fractions of grana and stromal-exposed membranes to be isolated. Analysis of Tween-20 solubilized material, after pelleting all membrane material by centrifugation at 100 000 g, revealed polypeptides associated with the coupling factor (CF₁) particles, cytochrome b₆/f and photosystem II complexes, suggesting that the marginal membranes contain these proteins. Concomitantly, the 100 000 g pellet was depleted in cytochrome b₆/f and P700, determined spectroscopically, Thus. our results reveal the margin to be a distinct membrane region, which does not contain the light-harvesting centers of photosystem II (LHC II). The implication of these results, in terms of the energetic interaction of components of granal and stromalexposed membrane regions, is discussed.     

日光温室光温因子对黄瓜叶绿体超微结构及其功能的影响

在日光温室内,研究了光温因子对黄瓜叶绿体超微结构及其功能的影响．结果表明,因季节之间光、温条件不同,日光温室黄瓜叶片显微结构和叶绿体超微结构有一定差异,1月份光照弱叶肉细胞较大,而5月份光照强叶绿体数较多．在该试验条件下,未发现叶片光合速率与叶绿体超微结构之间有直接或密切的相关性．在各生长季节其光合速率均为第4叶>初展叶>基部叶,与叶龄及各叶位的受光量有关．如果将不同叶位叶放在相同的光照下,则差异明显减少．黄瓜叶片的叶肉细胞、叶绿体和淀粉粒的大小以及叶绿体数、基粒数、基粒厚度、基粒片层数都随叶位的下降而呈增加趋势。不同品种、同品种不同生长时期的叶片显微结构和叶绿体超微结构及其功能也有一定的差异．限制日光温室冬季黄瓜光合作用的主要因素是光照弱、有效光照时数少,而在晴天温度的限制作用相对较小。阴天因光照弱而导致的室内低温则是限制黄瓜生长的关键因素．     

Effects of trypsin and cations on chloroplast membranes

A mild tryptic digestion of chloroplast membranes eliminates the effects of saturating concentrations of cations (3 to 5 millimolar MgCl₂) on chlorophyll fluorescence yield, membrane stacking, and photosystem II photochemical efficiency in spinach. At the same time, the negative surface potential of the membranes is increased (by trypsin) as revealed by studies with 9-aminoacridine. High concentrations of cations (25 to 100 millimolar MgCl₂) added after trypsin digestion are effective in restoring high fluorescence yields and membrane stacking. High concentrations of cations added after trypsin treatment do not increase the photosystem II efficiency. It is concluded that the “diffuse electrical layer” hypothesis of Barber et al. (Barber J, J Mills, A Love, 1977 FEBS Lett 74: 174-181) satisfactorily explains the effect of trypsin in eliminating the influence of saturating concentrations of cations on chlorophyll fluorescence yield and membrane stacking. However, the effect on photosystem II photochemical efficiency seems to require another mechanism.     

Effects of freezing on the structure of chloroplast membranes

Electron spin resonance (ESR) spectra of stearic acid spin labels incorporated into spinach thylakoids can be used to monitor membrane changes during freezing. Changes in the ESR parameters can be directly correlated to the extent of functional freeze damage. Freeze-induced changes in the ESR parameters strongly depend on the osmotic conditions of the incubation medium. Similar changes as on freezing can be observed by transferring thylakoids from an isotonic to a hypotonic medium, i.e., by swelling osmotically flattened thylakoids. This and computer simulations of spin label ESR spectra, which allow for variation of vesicle shape, lead to the conclusion that freeze-induced ESR spectral changes are due to swelling of the thylakoids. Indeed, van't Hoff plots of thylakoid packed volume indicate a freeze-induced increase in the apparent number of osmotically active molecules within the intrathylakoid lumen. During freezing, salt and/or sugar leak into the lumen. Simultaneously, proton channels are irreversibly opened. As the structural alterations obtained upon freezing are not accompanied by a change in bulk fluidity, these data are interpreted in terms of a local action of cryotoxic agents on critical microstructures, possibly at the rims of the thylakoid membranes.     

1H, 13C and 15N resonance assignments of the C-terminal domain of the 43 kDa subunit of the chloroplast signal recognition particle

We report the assignment of a 109 amino acid C-terminal chromo domain of the chloroplast signal recognition particle cpSRP43 subunit. cpSRP43 plays a crucial role in the targeting of light harvesting chlorophyll proteins to the thylakoids.     

Phospholipase A2 induced effects on the structural organization and physical properties of pea chloroplast membranes

Phospholipase A₂ (PLA₂)-induced effects on the membrane organization, fluidity properties and surface charge density of pea chloroplasts were investigated. It was observed that lipolytic treatment with PLA₂ altered the chloroplast structure having as a result a swelling of thylakoids and a total destruction of normal granal structure. In spite of this, the thylakoid membranes remained in close contact. At the same time, a slight decrease of surface charge density was registered, thus explaining the adhesion of swelled membranes. Fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) was measured during PLA₂ treatment. A pronounced decrease of DPH fluorescence polarization was found, indicating that phospholipase treatment resulted in considerable disordering and/or fluidization of the thylakoid membranes. The increased fluidity could be attributed to the destabilizing effect of the products of enzymatic hydrolysis of the phospholipids (free fatty acids, lysophospholipids) on the bilayer structure of thylakoids membranes.Abbreviations 9-AA 9-aminoacridine - BSA bovine serium albumin - DCMU 3-/3,4-dichlorophenyl-1,1-dimethyl/urea - DPH 1,6-diphenyl-1,3,5-hexatriene - EDTA ethylenediaminetetraacetic acid - HEPES N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid - LHC light harvesting chlorophyll a/b-protein complex of PS II - MES 2/N-morpholine/ethanesulfonic acid - PLA₂ phospholipase A₂ - PS I, PS II photosystem I and photosystem II, respectively - S lipid structural order parameter - THF tetrahydrofuran - TRICINE N-/tris/hydroxymethyl/methyl/glicine     

Effects of NaCl on photosynthetic pigments, saccharides, and chloroplast ultrastructure in leaves of tomato cultivars

In leaves of four tomato (Lycopersicon esculentum Mill.) cultivars (Red Cloud, Floradade, Peto 95, and Scorpio) the contents of chlorophyll (Chl) (a+b), Chl a, and -carotene decreased due to 100 mM NaCl treatment as compared with those of controls. The contents of soluble sugars and total saccharides were significantly increased in leaves of NaCl-treated plants, but the starch content was not significantly affected. Transmission electron microscopy indicated that in leaves of NaCl-treated plants, the chloroplasts were aggregated, the cell membranes were distorted and wrinkled, and there was no sign of grana and thylakoid structures in chloroplasts.     

Spatial organization of the cytochrome b6-f complex within chloroplast thylakoid membranes

The spatial distribution of the chloroplast thylakoid protein complex comprised of cytochromes f and b-563, and the Rieske iron-sulfur protein (Cyt b6-f) has been controversial because of conflicting results obtained by different techniques. We have combined the following biochemical and immunochemical techniques to approach this question: (1) French press disruption of thylakoids, followed by repeated two-phase aqueous polymer partitioning to separate inside-out grana from right-side-out stroma membrane fragments; (2) electrophoretic analysis followed by the 3,3',5,5'-tetramethylbenzidine stain for cytochrome hemes; (3) electroblot analysis with anti-Cyt b6-f antibodies; (4) agglutination of membrane fragments with anti-Cyt b6-f antibodies; and (5) post-embedment thin-section immunolabeling of chemically fixed or ultrarapidly frozen chloroplasts with anti-Cyt b6-f antibodies. Our results indicate that the complex is present in both of the isolated membrane fragment populations in similar amounts, with the bulk of the immunoreactive sites exposed to the thylakoidal lumen. Direct immunolabeling of thin-sectioned chloroplasts resulted in localization of the complex throughout the thylakoids, without specialized compartmentation. These results provide both the temporal and spatial resolution necessary for accurate localization of the complex. We concur with models proposing distribution of Cyt b6-f throughout all thylakoid membranes.     

Effects of spermidine on chlorophyll content,photosynthetic activity and chloroplast ultrastructure in the dark and under light

Spermidine prevented the loss of chlorophyll from detached komatsuna (Brassica campestris cv. komatsuna) leaves in the dark but caused a striking reduction in light. It also inhibited photochemical activity in leaves incubated in the dark and light. Striking changes were seen in the chloroplast ultrastructure in light; the envelope was lacking, grana were dilated and thylakoids were distributed throughout the cytoplasm.     

光质对番茄和莴苣幼苗生长及叶绿体超微结构的影响

采用发光二极管(LED)精确调制不同光谱能量分布,以荧光灯光照为对照,研究光质对番茄和莴苣幼苗生长及叶绿体超微结构的影响.结果表明：红光下番茄、莴苣幼苗的可溶性糖、淀粉和碳水化合物含量均显著高于对照,叶片叶绿体中淀粉粒膨大显著;蓝光极显著抑制了番茄下胚轴伸长,显著提高了莴苣和番茄幼苗叶片叶绿素a和类胡萝卜素含量;红蓝光下莴苣幼苗叶片的可溶性糖、淀粉、碳水化合物、蔗糖含量和C/N均达到最大值且显著高于红光处理,番茄和莴苣幼苗的主根显著伸长,幼苗叶片中叶绿体形态正常,基粒增多,基质片层清晰,淀粉粒体积明显小于红光处理.光质对植物幼苗的光形态建成、生长、碳氮代谢及叶绿体发育有显著影响;红光下光合产物积累显著但运输受阻严重,在红光中添加适量蓝光更有利于莴苣幼苗的碳水化合物积累,并可促进幼苗根系生长,有利于同化产物输出.     

Hypersalinity effects on leaf ultrastructure and physiology in the mangrove Avicennia marina

The effects of hypersalinity on leaf ultrastructure and physiology in the mangrove, Avicennia marina, were investigated by comparing leaves of adult trees growing naturally in the field under seawater and hypersalinity conditions in Richards Bay, South Africa. We tested the hypothesis that hypersalinity has a deleterious effect on membranes and cellular organelles such as chloroplasts and mitochondria, which would impact negatively physiological processes, such as ion and water relations, and photosynthetic performance. Soil ψ and soil salinity were −2.96 ± 0.07 MPa and 35 ± 2.8 psu in the seawater salinity site, compared to −5.91 ± 0.42 MPa and 58 ± 3.6 psu respectively, in the hypersaline site. In the hypersaline site, leaves were smaller and thicker, with thicker cuticles, while chloroplasts, mitochondria and nuclei exhibited swelling and disintegration, compared to those at seawater salinity. Multivesicular structures and vesicles, observed in vacuoles, chloroplasts, mitochondria, and along cell walls and plasma membranes, were more abundant in leaves from the hypersaline than the seawater site, and were probably indicative of greater plant salt uptake in the former site. Leaf concentrations of total chlorophyll and chlorophylls a and b were lower in trees from the hypersaline site by 33%, 29%, and 45% respectively, compared to those at seawater salinity. Midday minimum xylem ψ was −3.82 ± 0.33 MPa in the seawater site and −6.47 ± 0.45 MPa in the hypersaline site. In the hypersaline site, the concentration of leaf Na⁺ was 40% higher, while those of K⁺, Ca²⁺, and Mg²⁺ were lower by 45%, 44%, and 54% respectively, than those in the seawater site. CO₂ exchange and the intrinsic photochemical efficiency of PS II were significantly lower in trees from the hypersaline site by 48 and 19% respectively. The ultrastructural evidence supported the physiological data that A. marina trees in the hypersaline site are under extreme salinity stress and that this species is growing there at the upper limit of its salt tolerance.     

Phytochrome-mediated control of grana and stroma thylakoid formation in plastids of mustard cotyledons

The etioplast»chloroplast transition in the cotyledons of mustard seedlings (Sinapis alba L.) has been studied by electron microscopy. It was found that the active form of phytochrome, established by a red light pulse pretreatment, increases the initial rate and eliminates the lag of grana and stroma thylakoid formation after the onset of white light 60 h after sowing. The effect of a pretreatment with 15 s red light pulses is fully reversible by 756 nm light pulses. This reversibility is lost within 5 min. Evidence is presented which suggests that the time course of grana and stroma thylakoid formation is not correlated with the time course of the dispersal of the prolamellar body. The different functions of phytochrome and chlorophyll in controlling thylakoid formation are discussed.     

Effects of high temperature on photosynthesis, chlorophyll fluorescence, chloroplast ultrastructure, and antioxidant activities in fingered citron

Effects of heat stress on the photosynthesis system and antioxidant activities in Fingered citron (Citrus medica var. sarcodactylis Swingle) were investigated. Two-year-old Fingered citron plants were exposed to different temperature (28, 35, 40, and 45°C) for 6 h; then the photosynthetic capacity, chlorophyll fluorescence, chloroplast ultrastructure, and antioxidant activities in the leaves were evaluated. Exposure to 40 and 45°C for 6 h resulted in a significant decrease in the photosynthetic rate (P _n), carboxylation efficiency (CE), the maximal photochemical efficiency of photosystem II, and the light-saturated photosynthetic rate, which were related to the reduction of CO₂ assimilation, inactivation of photosystem II and photosynthetic electron transport. Moreover, transmission electron microscopy showed chloroplast ultrastructural alterations, including their swelling, matrix zone expanding, and lamella structure loosening. Furthermore, heat stress, especially at 45°C, caused oxidative damage resulted from ROS accumulation in Fingered citron leaves accompanied by increases in activities of superoxide dismutase, peroxidase, and catalase. However, exposure to 35°C for 6 h or 40°C for 4 h had no significant influence on the photosynthetic capacity at all. The results suggest that Fingered citron plants show no heat injury when temperature is below 40°C.     

The effect of isoproturon on root growth and ultrastructure of the photosynthetic apparatus of two wheat cultivars and a weed

The effect of isoproturon [N,N-dimethyl N'4-(1-methyl-ethyl) phenyl urea] on the germination, growth of root and ultrastructure of the photosynthetic apparatus was investigated in two wheat cultivars ( Triticum sativum L. cvs Castan and Esquilache) and a weed ( Lolium rigidum Gaud.). No inhibition of germination was apparent in any of the three plants studied. The inhibition of root growth was considerably significant, especially in cv. Esquilache. After 48 h treatment with isoproturon (340 μ M ), the chloroplasts of the two wheat cultivars were found to be similar, showing a higher number of grana with fewer thylakoids per granum (lower stacking degree), broader grana stacks than in the control and an absence of starch. However, after 60 h of treatment, swelling of the thylakoids occurred in the Esquilache cultivar.
The rye grass was the most affected by the herbicide; at low concentration (3.4 μ M ) the number and size of the grana decreased considerably and the stroma thylakoids were destroyed. At higher concentration (17 μ M ), there was a strong accumulation in certain areas of the stroma of a substance with a lipid-like appearance, probably derived from the disintegrating thylakoids. At the same concentrations some chloroplast envelopes were completely desintegrated.
The Hill reaction of the three plants exhibited a similar sensitivity towards the herbicide.     

弱光对不结球白菜光合特性与叶绿体超微结构的影响

研究了不结球白菜耐弱光品种“矮王”和不耐弱光品种“绿优”经弱光处理后,光合特性和叶绿体超微结构的变化.结果表明:遮荫处理后,“矮王”的总叶绿素和叶绿素b含量大幅度上升;2个品种的净光合速率（P_n）、光补偿点（LCP）、光饱和点（(LSP）、表观量子效率（AQY）、羧化效率（CE）均呈下降趋势;除光补偿点外,“矮王”的下降幅度均小于“绿优”.叶绿素荧光参数分析表明,在弱光条件下,“绿优”的光系统Ⅱ（PSII）受到伤害,F_v/F_m、ETR、Φ_PSⅡ和q_P的下降幅度均大于耐弱光品种“矮王”.电镜超微观察结果显示:“绿优”的叶绿体内部出现裂缝和孔洞,多数基质片层断裂,基粒片层解体,线粒体出现内含物减少、发生空洞的现象;而“矮王”的叶绿体结构良好,线粒体发育正常.表明弱光处理可以影响植物的光合机构,但对不同品种的影响不同.