Structure and Function of Chloroplast Membrane II. The Effects of Potassium and Magnesium Ions on the Absorption Spectrum and Photosystem II Function of Two Types of Chloroplast Me-mbranes

After exposing etiolated wheat seedlings to intermittent light (cycle of 2 min. light, 118 min. dark) for 24 hr., we obtained an incompletely developed chloroplast membrane. It was then compared with a completely developed chloroplast membrane obtaining from wheat seedlings grown under normal light-dark regime. We investgated the effect of various cations and their concentrations on the absorption spectrum and the photosystem Ⅱ function of the above two types of chloroplast membranes. A similar effect of potassium and magnesium ions on the absorption spectra of completely developed chloroplast membrane was observed. They decreased the absorption peak values at both the red and blue regions of the chloroplast membrane in the same manner. The degree of decrease in the peak value is proportional to ion concentration. But in the incompletely developed chloroplast membranes similar phenomenon was not observed. In the presence of K+ and Mg2+ of various concentrations, the absorptionn peaks at the red region overlapped almost completely, and these at the blue region only changed slightly with ion concentrations. DCIP photoreduction rate of the two types of chloroplast membranes was stimulated by the addition of K+ and Mg2+ in various concentrations. But the degree of stimulation in the two types of membranes was quite different. In the presence of l00 mM KCl or 5.0 mM MgCl2, DCIP photoreduction rate of completely developed chloroplast membranes was enhanced by 76.8% and 68.9% respectively, whereas in incompletely developed chloroplast membranes it was only increased by 56.3% and 36.4% respectively. The causes of the effects of cations on the absorption spectrum and the photosystem Ⅱ function of two types of chloroplast membranes were discussed.     

Endopolyploidy in the roots of rye,Secale cereale L.

2,4-D was applied to the roots of diploid and tetraploid corn. After the application the mitotic division in the meristem of root tips was blocked; the mitotic division in differentiated cells of cortex and central cylinder, on the other hand, was provoked. In the cortex of diploid corn (variety ?eské) predominantly tetraploid were found cells with 28 chromosomes and, to a lesser extent, octoploid and diploid ones with 56 and 14 chromosomes respectively. In the cortex of tetraploid corn (variety Bernburger Tetraroggen), most cells were octoploid with 56 chromosomes; the metaphase levels with 112 and 28 chromosomes, e.g. 16-ploid and tetraploid cells, were found less frequently. The relations between the numbers of cells with different polyploidy were similar in both the varieties. The first endoreduplication cycle was different polyploidy were similar in both the varieties. The first endoreduplication cycle was found to occur in the region where the cortex cells finish their elongation. In the central cylinder of the roots of diploid corn most cells were found to be diploid, in tetraploid corn most cells were tetraploid.     

An alternative model for photosystem II/light harvesting complex II in grana membranes based on cryo-electron microscopy studies.

The photosynthetic protein complexes in plants are located in the chloroplast thylakoid membranes. These membranes have an ultrastructure that consists of tightly stacked 'grana' regions interconnected by unstacked membrane regions. The structure of isolated grana membranes has been studied here by cryo-electron microscopy. The data reveals an unusual arrangement of the photosynthetic protein complexes, staggered over two tightly stacked planes. Chaotrope treatment of the paired grana membranes has allowed the separation and isolation of two biochemically distinct membrane fractions. These data have led us to an alternative model of the ultrastructure of the grana where segregation exists within the grana itself. This arrangement would change the existing view of plant photosynthesis, and suggests potential links between cyanobacterial and plant photosystem II light harvesting systems.     

Structure and Function of Chloroplast Membrane III. The Effects of Mg++ and K+ Ions Toward the Ultrastructure of Two Kinds of Chloroplast Thylakoid Membranes

Based upon our previous work on the relationship between structure and function of chloroplast of wheat in connection with PSⅡ reaction, we studied the effects of MgCl2 and KC1 toward two kinds of thylakoid membranes. After exposing etiolated wheat seedlings to intermittent light (cycle of 2 min. light, 118 min. dark) for 24 hr, we obtain ed an incompletely developed chleroplast membrane. Completely developed chloroplast membrane was obtained from wheat seedlings grown under normal light-dark regime. Thylakoid membranes of plants grown under intermittent light failed to form grana stacks they remained as single lamellae in the suspension containing Mg++ or K+ of high concentration although simple stackings not more than two thylakoids c.ould be found. However, thylakoids grown under normal light-dark regime showed well developed grand stacks. Isolated chloroplast samples from two kinds of seedlings were suspended in 5 mM MgCl2 and 100 mM KC1 solutions for a definite time, portions of each samples were processed for electron microscopic observations and their photosynthetic activities were measured at the same time (It will be dealt with in another article). When these two kinds of isolated plastids were suspended either in MgCl2 (5 mM) or KC1 (100), the normally developed grana thylakoids stacked closely but the incompletely developed thylakoid- membranes did not stack. The incompletely developed chloroplast thylakoid membranes,, in either Mg++ or K+ ions could not induce stacking of the scattered thylakoid membranes to form grana. Therefore, we presume that light- harvesting chlorophyll a/b protein complex is on internal factor to induce thylakoid- membranes stacking and a definite concentration of caionions is an important factor in maintaining the stacking of thylakoid membranes. These results further prove the close association between structure and function in our previous studies on the mesophyll cell of the winter wheat.     

Biogenesis of water splitting by photosystem II during de‐etiolation of barley (Hordeum vulgare L.)

Etioplasts lack thylakoid membranes and photosystem complexes. Light triggers differentiation of etioplasts into mature chloroplasts, and photosystem complexes assemble in parallel with thylakoid membrane development. Plastids isolated at various time points of de‐etiolation are ideal to study the kinetic biogenesis of photosystem complexes during chloroplast development. Here, we investigated the chronology of photosystem II (PSII) biogenesis by monitoring assembly status of chlorophyll‐binding protein complexes and development of water splitting via O₂ production in plastids (etiochloroplasts) isolated during de‐etiolation of barley (Hordeum vulgare L.). Assembly of PSII monomers, dimers and complexes binding outer light‐harvesting antenna [PSII‐light‐harvesting complex II (LHCII) supercomplexes] was identified after 1, 2 and 4 h of de‐etiolation, respectively. Water splitting was detected in parallel with assembly of PSII monomers, and its development correlated with an increase of bound Mn in the samples. After 4 h of de‐etiolation, etiochloroplasts revealed the same water‐splitting efficiency as mature chloroplasts. We conclude that the capability of PSII to split water during de‐etiolation precedes assembly of the PSII‐LHCII supercomplexes. Taken together, data show a rapid establishment of water‐splitting activity during etioplast‐to‐chloroplast transition and emphasize that assembly of the functional water‐splitting site of PSII is not the rate‐limiting step in the formation of photoactive thylakoid membranes.     

NaCl和Na2SO4胁迫下两种刺槐叶肉细胞叶绿体超微结构

二倍体刺槐(diploid Robinia pseudoacacia)是我国水土保持林的先锋树种,具有较强的适应性和抗逆性,对改善生态环境、防治水土流失、调节水文状况有重要作用。四倍体刺槐(tetraploid Robinia pseudoacacia)是二倍体刺槐的加倍品种,也称多倍体刺槐,由韩国引进,具有速生、耐盐碱、耐干旱和耐烟尘等特点。目前,关于四倍体刺槐的研究,主要集中于栽培技术和繁殖技术方面,而关于四倍体刺槐叶片超微结构与其耐盐性的关系尚缺乏报道。比较了二倍体刺槐和四倍体刺槐在NaCl和Na₂SO₄胁迫下,叶片叶绿体超微结构的变化特点,一方面可以对二者的耐盐性进行鉴定,同时也可以探讨不同盐分胁迫条件下的作用机制。利用NaCl和Na₂SO₄进行20d的盐胁迫处理,观察叶绿体超微结构的变化特点,发现:NaCl处理前,二者叶肉细胞叶绿体为梭形、形态饱满、结构完整,NaCl处理后10d时,二倍体刺槐的叶绿体出现变形、膜模糊、基粒片层松散、类囊体解体、脂质球增多等现象,NaCl处理后20d时,叶绿体肿胀、变形,基粒片层断裂,膜系统解体。Na₂SO₄处理后10d时,二倍体刺槐的叶绿体肿胀,膜模糊,基粒片层松散、类囊体解体,Na₂SO₄盐胁迫处理后20d时,膜系统全部解体,结构破坏更为严重。总体来说,四倍体刺槐在盐胁迫后叶绿体结构变化不明显,只是在Na₂SO₄处理20d时,四倍体刺槐的叶绿体出现中空、基粒片层松散、膜边缘模糊现象。在处理前,两种刺槐的叶绿体均紧贴细胞壁,分布于细胞壁边缘。在NaCl处理后10d时,二倍体刺槐的叶绿体仍呈有序排列,紧贴细胞壁,但在处理后20d时,大部分叶绿体脱离细胞壁,呈随机分布。在Na₂SO₄处理后10d时,二倍体刺槐部分叶绿体脱离细胞壁,位于细胞中央。在Na₂SO₄处理后20d时,二倍体刺槐叶绿体大部分与细胞壁脱离。四倍体刺槐在两种盐胁迫处理前后叶绿体的排列变化不明显,均分布于细胞壁边缘,紧贴细胞壁。所以在盐胁迫下,耐盐植物叶片的叶绿体表现为结构完整,基粒片层清晰,类囊体结构完整,而不耐盐植物则表现为叶绿体超微结构松散、变形,基粒片层模糊,破坏严重时基粒片层扭曲,叶绿体解体,失去完整结构。     

Ploidy of somatic embryos and the ability to regenerate plantlets in melon (Cucumis melo L.)

Summary The number of chromosomes in cells of callus, somatic embryos and regenerated plantlets during somatic embryogenesis were examined in two cultivars of melon (Cucumis melo L.). Somatic embryos were diploid (50.0%/32.1%), tetraploid (38.5%/57.5%) and octoploid (11.5%/10.4%) whereas in callus cells diploidy (41.9%/43.3%), tetraploidy (27.9%/25.8%), octoploidy (11.6%/15.5%) and a low frequency of other types of ploidy and aneuploidy were observed. Mixoploid somatic embryos were not observed. These results suggest that the somatic embryos were selectively differentiated from diploid, tetraploid and octoploid cells, and that endopolyploidization of cultured cells occurred before the start of cell division leading to somatic embryogenesis. The ratio of diploid to tetraploid (1.30/0.55) in somatic embryos was less than that in callus cells (1.50/1.68) while ratios of diploid to octoploid (4.35/3.09) and tetraploid to octoploid (3.35/5.52) in somatic embryos were greater than those in callus cells (3.61/2.80 and 2.40/1.67). Therefore, it appears that the ability of callus cell to differentiate into somatic embryos increases in the following order: octoploid < diploid < tetraploid. Regenerated plantlets were diploid (65.5%/55.1%) and tetraploid (34.5%/44.9%). No octoploid plantlets were observed. The ratio of diploid to tetraploid in regenerated plantlets (1.72/1.23) was greater than that in somatic embryos. Therefore, it appears that the ability of somatic embryos to develop into plantlets increases in the following order: octoploid < tetraploid < diploid.     

Optimization of the Leaf Mesophyll Structure in Alloploid and Diploid Wheat Species

Comparative analysis of the indices of plant growth and mesostructure of the photosynthetic apparatus was carried out with ten wheat (Triticum L.) species of various origins. Wheat alloploid forms (tetra- and hexaploids with the chromosome numbers of 28 and 42) exceeded the diploid forms (the chromosome number of 14) 2.3–2.4-fold by their absolute growth rate (AGR). As a result, the alloploid species developed a larger assimilation area; this change involved the internal reorganization of leaf phototrophic tissues and an increase in the cumulative internal assimilation area. In addition, the alloploid species manifested a higher correlation between the surface areas of cell and chloroplast membranes caused by a decrease in the cell number per the unit leaf area, a relative increase of the number of composite multifaveolate cells, a considerable expansion (in volume and surface area) of mesophyll cells, and an increase in chloroplast size and numbers. The decreased ratio between the characteristics of the cell membrane and chloroplast envelope presumes that CO₂ diffusion via cell and chloroplast membranes in the leaves was better balanced in the alloploid wheat species than in the diploid forms. All wheat species did not notably differ in their plastid–cytoplasm ratio (cell volume corresponding to one chloroplast and cell surface area per one chloroplast) and the ratio of surface area of cells to cell volume. The discriminant analysis revealed the indices of leaf growth and mesophyll structure instrumental in distinguishing between the diploid and alloploid species: leaf area, AGR, and cell size and number. The change in the latter indices optimized the structure of leaf phototrophic tissues in tetraploid and hexaploid species; as a result, the internal assimilation area was expanded and, consequently, leaf CO₂ conductance was increased.     

Photosynthetic electron transport and establishment of an associated trans-thylakoid proton electrochemical gradient during development of the wheat leaf

Abstract Photosynthetic electron transport activities and the ability to generate and maintain a trans-thylakoid proton electrochemical gradient were examined during chloroplast development in 4-day-old wheat leaves grown under a diurnal light regime. Polarographic and spectropholometric studies on leaf tissue demonstrated that poorly developed chloroplasls at the leaf base could photo-oxidize water and transfer electrons from photosystem 2 to photosystem 1. The capacity for non-cyclic whole-chain electron transport increased during chloroplast development. Thylakoids isolated from the leaf base, although capable of pumping protons into the inlrathylakoid space, could not maintain a trans-membrane proton electrochemical gradient; this ability developed at later stages of chloroplast biogenesis in the leaf. The implications of these results for the energetics of the developing leaf are discussed.     

Is There a Relationship between Infection by Rhizobia and Occurrence of Disomatic Nuclei in Nodules of Alfalfa (Medicago sativa L.)?

Cytological examination of nodules from diploid, tetraploid, and octoploid alfalfa (Medicago sativa L.) plants revealed that the proportion of nodule cells infected by rhizobia was not significantly affected by nuclear ploidy of the host plant. Flow cytometry was used to determine the influence of host plant nuclear ploidy on the nuclear ploidy of infected cells. In nodules from diploid plants, most of the nuclei were tetraploid, whereas in nodules from tetraploid plants, about half of the nodule nuclei were tetraploid and half were octoploid; in octoploid plants, most of the nodule nuclei were octoploid. The occurrence of disomatic nuclei was independent of infection of nodule cells by rhizobia, because diploid plants had mostly disomatic nodule nuclei, and octoploid plants had mostly monosomatic nodule nuclei, whereas all nodules maintained a constant proportion of infected to uninfected cells. These results do not support the earlier hypothesis that infected nodule cells contain disomatic nuclei.     

Phosphorylation of thylakoid proteins during chloroplast biogenesis in greening etiolated and light-grown wheat leaves

Phosphorylation of polypeptides in isolated thylakoids was examined during chloroplast biogenesis in greening etiolated wheat leaves and 4 day-old wheat leaves grown under a diurnal light regime. At early stages of plastid development standard thylakoid preparations were heavily contaminated with nuclear proteins, which distorted the polypeptide phosphorylation profiles. Removal of contamination from membranes by sucrose density centrifugation demonstrated that the major membrane phosphoprotein in etioplasts was at 35 kDa. During etioplast greening a number of phosphoproteins appeared, of which the 25–27 kDa apoproteins of the light-harvesting chlorophylla/b protein complex associated with photosystem II (LHCII) became the most dominant. At the early stages of thylakoid development found at the base of the 4-day-old light grown leaf the LHCII apoproteins were evident as phosphoproteins; however the major phosphoprotein was polypeptide atca. 9kDA. Phosphorylation of both the LHCII apoproteins and the 9 kDa polypeptide in these thylakoids was not light-dependent. In the older thylakoids isolated from the leaf tip the LHCII apoproteins were the major phosphoproteins and their phosphorylation had become light-regulated; however phosphorylation of the 9 kDa polypeptide remained insensitive to light.     

Electron tomography of plant thylakoid membranes

For more than half a century, electron microscopy has been a main tool for investigating the complex ultrastructure and organization of chloroplast thylakoid membranes, but, even today, the three-dimensional relationship between stroma and grana thylakoids, and the arrangement of the membrane protein complexes within them are not fully understood. Electron cryo-tomography (cryo-ET) is a powerful new technique for visualizing cellular structures, especially membranes, in three dimensions. By this technique, large membrane protein complexes, such as the photosystem II supercomplex or the chloroplast ATP synthase, can be visualized directly in the thylakoid membrane at molecular (4-5 nm) resolution. This short review compares recent advances by cryo-ET of plant thylakoid membranes with earlier results obtained by conventional electron microscopy.     

Adaptive reorganization of protein and lipid components in chloroplast membranes as associated with herbicide binding

Cultivation of Spirodela oligorrhiza ( Kurtz ) Hegelm on a sublethal dose of atrazine results in a higher linolenic to linoleic acid ratio in the thylakoid membrane lipids, less starch, more osmiophilic globules, and a reduced stroma lamellar system. Also, the grana become randomly oriented and contain more numerous and elongated lamellae. These alterations in the lipid composition and ultrastructure of the chloroplast resemble those previously observed in triazine-resistant weed biotypes and in chloroplasts developed under low light. Thylakoid membranes from atrazine-adapted plants revealed an additional high-affinity binding constant for [14C]-diuron but the number of diuron binding sites actually decreased by 20 times compared to controls. The 32,000-dalton membrane protein of the chloroplast is synthesized actively, but its breakdown appears decreased compared to control plants. The adaptive reorganization of thylakoid components may be a compensatory mechanism for maintenance of a functional interaction of the proteins and lipids of the photosystem II complex.     

Biochemical,morphological and flow cytometric evaluation of the effects of hexachlorobenzene on rat liver

This study investigated the ability of HCB (0.1% in the diet for 15 days) to cause early changes in the cellular ploidy of rat liver. Treatment caused marked hepatomegaly, increase of microsomal proteins and cytochrome P-450 content and reduction of hepatocyte microviscosity. Microscopic examination showed that the hepatocytes were enlarged, with hyaline cytoplasm and vacuoles. The size distribution of the isolated hepatocytes showed a larger percentage of bigger cells. Flow-cytometric DNA/protein analysis was performed on whole (fixed) cells and on nuclei. From the combined results of both analyses it was possible to exclude significant changes in the percentages of diploid, mononucleated tetraploid, binucleated tetraploid and octoploid hepatocytes. The DNA and protein content of each subpopulation remained unchanged. Our results suggest that HCB does not cause early diploidization of liver cells and that hepatomegaly and cytochrome P-450 induction seem not to be correlated with effects on total DNA and total protein contents.Abbreviations HCB hexachlorobenzene - PI propidium iodide - FITC fluorescein isothiocyanate - DN diploid nuclei - SN 2N-4N nuclei in S-phase - TN tetraploid nuclei - DC diploid cells - SDC 2N-4N diploid cells in S-phase - TC tetraploid cells - STC 4N-8N tetraploid cells in S-phase - OC octoploid cells - MDC mononucleated diploid cells - SMDC mononucleated diploid cells in S-phase - BOC binucleated octoploid cells - SBTC binucleated tetraploid cells in S-phase - BTC binucleated tetraploid cells - MTC mononucleated tetraploid cells.     

Study of amino acid composition and biosynthesis of protein components of the membrane system of corn plastids during biogenesis of chloroplasts]

The biosynthesis of membrane proteins in maize plastids at different stages of differentiation of the chloroplast lamellar system was studied. Prolamellar and lamellar system preparations were isolated from maize plastids, disintegrated by osmotic shock under hypotonic conditions. Changes in the amino acid composition of 14C membrane proteins were observed at all stages of chloroplast ultrastructure formation. The maximal level of the apolar amino acids was observed in the membrane fraction of chloroplasts. Washed membranes from maize proplastids and chloroplasts can be resolved into at least 14 protein bands on formic acid--urea polyacrylamide gel. It is pointed out that biogenesis process leads to the increase of lipophylic protein content in the chloroplast lamellae fraction.     

Spatial relationship of photosystem I, photosystem II, and the light- harvesting complex in chloroplast membranes

We have previously demonstrated (Armond, P. A., C. J. Arntzen, J.-M. Briantais, and C. Vernotte. 1976. Arch. Biochem. Biophys. 175:54-63; and Davis, D. J., P. A. Armond, E. L. Gross, and C. J. Arntzen. 1976. Arch. Biochem. Biophys. 175:64-70) that pea seedlings which were exposed to intermittent illumination contained incompletely developed chloroplasts. These plastids were photosynthetically competent, but did not contain grana. We now demonstrate that the incompletely developed plastids have a smaller photosynthetic unit size; this is primarily due to the absence of a major light-harvesting pigment-protein complex which is present in the mature membranes. Upon exposure of intermittent- light seedlings to continuous white light for periods up to 48 h, a ligh-harvesting chlorophyll-protein complex was inserted into the chloroplast membrane with a concomitant appearance of grana stacks and an increase in photosynthetic unit size. Plastid membranes from plants grown under intermediate light were examined by freeze-fracture electron microscopy. The membrane particles on both the outer (PF) and inner (EF) leaflets of the thylakoid membrane were found to be randomly distributed. The particle density of the PF fracture face was approx. four times that of the EF fracture face. While only small changes in particle density were observed during the greening process under continuous light, major changes in particle size were noted, particularly in the EF particles of stacked regions (EFs) of the chloroplast membrane. Both the changes in particle size and an observed aggregation of the EF particles into the newly stacked regions of the membrane were correlated with the insertion of light-harvesting pigment- protein into the membrane. Evidence is presented for identification of the EF particles as the morphological equivalent of a "complete" photosystem II complex, consisting of a phosochemically active "core" complex surrounded by discrete aggregates of the light-harvesting pigment protein. A model demonstrating the spatial relationships of photosystem I, photosystem II, and the light-harvesting complex in the chloroplast membrane is presented.     

The fluorophenylalanine sensitive and resistant tobacco cell lines,TX1 and TX 4 1. DNA contents,chromosome numbers,nuclear ultrastructures,and effects of spermidine

Summary The two tobacco cell lines TX 1 (wild type) and TX 4 (Palmer andWidholm 1975), which are respectively sensitive and resistant to pfluorophenylalanine, were studied by light and electron microscopy and scanning cytophotometry. The two cell lines differ in the following respects: TX 1 cells exhibit spherical cells, a chromatin organization like in free-grown plants ofNicotiana tabacum, and mainly diploid and haploid DNA values. TX 4 cells show large elongated (thread-like) cells, a diffuse nuclear ultrastructure, and mainly tetraploid and octoploid DNA values. Chromosome number counts indicate that dividing TX 1 cells own a karyotype close to the diploid one, while dividing TX 4 cells are around the tetraploid state. In addition, the effects of an exogenous polyamine, spermidine, on chromatin organization were investigated. While the wild type cell line, TX1, did not respond to the treatment, the mutant, fluorophenylalanine resistant cell line, TX4, exhibited reduced nuclear size and increased chromatin condensation.     

Evidence for a role of VIPP1 in the structural organization of the photosynthetic apparatus in Chlamydomonas

The vesicle-inducing protein in plastids (VIPP1) was suggested to play a role in thylakoid membrane formation via membrane vesicles. As this functional assignment is under debate, we investigated the function of VIPP1 in Chlamydomonas reinhardtii. Using immunofluorescence, we localized VIPP1 to distinct spots within the chloroplast. In VIPP1-RNA interference/artificial microRNA cells, we consistently observed aberrant, prolamellar body-like structures at the origin of multiple thylakoid membrane layers, which appear to coincide with the immunofluorescent VIPP1 spots and suggest a defect in thylakoid membrane biogenesis. Accordingly, using quantitative shotgun proteomics, we found that unstressed vipp1 mutant cells accumulate 14 to 20% less photosystems, cytochrome b(6)f complex, and ATP synthase but 30% more light-harvesting complex II than control cells, while complex assembly, thylakoid membrane ultrastructure, and bulk lipid composition appeared unaltered. Photosystems in vipp1 mutants are sensitive to high light, which coincides with a lowered midpoint potential of the Q(A)/Q(A)(-) redox couple and increased thermosensitivity of photosystem II (PSII), suggesting structural defects in PSII. Moreover, swollen thylakoids, despite reduced membrane energization, in vipp1 mutants grown on ammonium suggest defects in the supermolecular organization of thylakoid membrane complexes. Overall, our data suggest a role of VIPP1 in the biogenesis/assembly of thylakoid membrane core complexes, most likely by supplying structural lipids.