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The possible involvement of chloroplast lipids in the mechanisms of NaCl tolerance was studied in leaves of two varieties of Fabaceae: Sulla carnosa and Sulla coronaria, which were subjected to 200mM NaCl over 20days. Changes in membrane lipid peroxidation, chloroplast lipids content, fatty acids (FA) composition and the ultrastructure of chloroplasts under salt stress were investigated. Chloroplast lipids were separated and quantified by high performance liquid chromatography coupled to evaporative light scattering detection (HPLC/ELSD). The results showed that salinity induced a significant decrease in digalactosyldiacylglycerol (DGDG), phosphatidylglycerol (PG) and sulfoquinovosylglycerol (SQDG) content in both S. carnosa and S. coronaria leaves, whereas monogalactosyldiacylglycerol (MGDG) content did not change significantly in S. carnosa leaves. The MGDG/DGDG ratio remained stable in S. coronaria leaves but increased in those of S. carnosa. In addition, the unsaturated-to-saturated fatty acids ratio (UFAs:SFAs) did not change under salt stress in S. coronaria leaves, while it decreased significantly in S. carnosa leaves. Moreover, salinity did not induce significant changes in MGDG and DGDG unsaturation level in S. carnosa leaves, in contrast to S. coronaria, in which salinity seems to enhance the unsaturation level in MGDG, DGDG and PG. Furthermore, the level of membrane lipid peroxidation, as expressed by malondialdehyde (MDA) levels, increased at 200mM in S. carnosa leaves, while it did not change significantly in those of S. coronaria. With respect to the ultrastructure of chloroplasts at 200mM NaCl, investigated by transmission electron microscopy (TEM), salt-stress caused the swelling of thylakoids in S. carnosa mesophyll. These ultrastructural changes were observed especially in the spongy tissue in S. coronaria. Taken together, these findings suggest that the stability of MGDG/DGDG ratio, the unchanged unsaturation level, and increasing unsaturation level in MGDG, DGDG and PG may be effective to some degree in suppressing the ultrastructural damage caused by salinity effects and may contribute to protect the chloroplast membrane integrity against salt stress., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

The surface properties of chloroplasts in leaves of some leguminous plants--Pisum sativum, Trifolium paratense, Melilotus album, and Vicia cracca--were investigated, based on the analysis of phase formation kinetics of the aqueous polymeric two-phase system dextrane-500/polyethyleneglycol-6000. The surface properties of chloroplasts have been shown to display both species- and sort-specificity. A specific influence of chloroplasts of melilot and non-chlorophyllic pea lines on phase formation kinetics has been noticed.

Chloroplasts consist of six morphologically distinct compartments. Each compartment has a specific set of polypeptides that perform distinct biochemical functions. We report here the identification of a membrane-associated protein with a novel localization. This protein was synthesized as a 37 kDa precursor and was processed to a mature protein of 30 kDa after being imported into isolated pea chloroplasts. Fractionation of chloroplasts showed that the 30 kDa mature protein was associated with both of the envelope membranes as well as with thylakoid membranes. Immunocyto-chemical localization of the 30 kDa protein revealed that the protein occurred in clusters in the vicinity of both the envelope and the thylakoid. Possible functions of this 30 kDa protein, inferred from its novel localization pattern, are discussed.

The gene product of an open reading frame of the chloroplast genome, accD, that has sequence similarity with a subunit of acetyl-CoA carboxylase from Escherichia coli was detected immunochemically in pea chloroplasts. The apparent molecular mass of the accD protein was 87 kDa on SDS-polyacrylamide gel electrophoresis. The protein was acidic and had less mobility than the calculated value, 67,116. Acetyl-CoA carboxylase activity solubilized from pea chloroplasts was inhibited by antibodies against recombinant accD protein. The antibodies precipitated a polypeptide of 35 kDa containing biotin and a polypeptide of 91 kDa together with the 87-kDa-accD protein. The accD protein formed a complex with the molecular mass of about 700 kDa, probably with the 35- and 91-kDa proteins. These results indicate that the chloroplast-encoded polypeptide, accD protein, is a component of a functional acetyl-CoA carboxylase in chloroplasts and this enzyme is a multi-subunit complex, like that from E. coli. The synthesis of accD protein was not induced by light.

In vitro assays for the import of proteins by isolated pea thylakoids have been refined and optimised with respect to (a) the method of thylakoid preparation, (b) the concentration of thylakoids in the import assay, and (c) the pH and temperature of the import assay. As a result, the 23 kDa and 16 kDa proteins of the photosynthetic oxygen-evolving complex are imported with efficiencies approaching 100%; import of the third oxygen-evolving complex protein is also observed, albeit with lower efficiencies. We have also demonstrated import of three further thylakoid proteins: plastocyanin, the CFoII subunit of the ATP synthase, and the photosystem I subunit, PSI-N, using this import assay. Import of plastocyanin, PSI-N and the 33 kDa oxygen-evolving complex protein subunit requires the presence of stromal extract whereas the other three proteins are efficiently imported in the absence of added soluble proteins. Import into isolated barley thylakoids was achieved under identical assay conditions, although with somewhat lower efficiency than into pea thylakoids.

Quantum efficient action spectra have been measured of longwave fluorescence of light chloroplast fragments at 20 and -196 degrees C. These spectra were measured as the functional dependence of fluorescence intensity on wave length of exciting light in 630--720 nm range being divided by absorption spectrum. Fluorescence intensity was measured in 8 and 4 nm spectral interval with centres at 730 and 755 nm at room and low temperatures respectively. Three maxima with centres at 650, 680 and 704 nm were revealed in the quantum efficient action spectra. It has been established that the last one resulted from self-absorption of chlorophyll aggregates with longwave fluorescence. It may be that the first maximum was due to high frequency component of multiplet, belonging to self-absorption of longwave chlorophyll aggregates. The shortwave shift of 704 nm maximum and the slight longwave one of 650 nm maximum were found in the quantum efficient action spectra of light chloroplast fragments treated with galactolipase. Good coincidence was revealed between absorption properties of longwave chlorophyll aggregates and those of Photosystem-1 centres, which were measured by other independent methods. It was concluded that longwave fluorescence of light chloroplast fragments belonged to Photosystem-1 centres.

We have developed a method to isolate intact, purified pea etioplasts. These etioplasts were capable of recognizing, transporting, and processing the precursor form of the small subunit of the ribulose-1,5-bisphosphate carboxylase, a protein which is not detectable at this developmental stage. Transport of proteins was completely dependent on ATP and could not be substituted for or stimulated by light. The transported precursor protein was processed to its proper molecular weight. The mature form of the small subunit was assembled with the large subunit of the ribulose-1,5-bisphosphate carboxylase already present at this stage to form an oligomer. Protein transport was completely abolished using the phosphatase inhibitor sodium fluoride. This is the first time protein transport has been demonstrated in isolated, purified etioplasts.

Anisotropic properties of pea chloroplasts, subchloroplast fragments (photosystem 1 particles and pigment-protein complexes) and the blue-green algae oriented in polyacrylamide gel were investigated. It was shown that linear dichroism spectra of chloroplasts are the superposition of the corresponding spectra for the main light harvesting complex (HMLC) and P 700 chlorophyll a--protein complex (CP 1). Anisotropic properties of the photosystem 1 particles and blue-green algae are mainly caused by CP 1 anisotropy. Qy-transition moments tend to perpendicular orientation to the membrane plane for the Chl. b 649, Chl. a 660 and parallel orientation--for Chl. b 654, Chl. a 682. The degree of Qy-transition moments parallel orientation is higher for the longwave forms (Chl. a 690, Chl. a 702, Chl. a 712), than for the shortwave ones and coincides with this degree for the reaction centre pigment P 700 transition moment. It is suggested that the specific orientation of the pigment-protein complexes in the chloroplast membrane is important for the regulation of the spillover between two photosystems.

Dramatic changes occur in the ultrastructure of chloroplasts within host tissues during the infection of Phaseolus by the plant pathogenic fungus Sclerotinia. Electron-opaque deposits develop initially in the peripheral region of the chloroplast stroma, in cells in advance of the hyphal front. As infection continues, the amount and intensity of deposition increases and spreads throughout the chloroplast. The deposits possess a high degree of structural integrity in the form of a crystalline square lattice with 10 nm periodicity. Enzyme digestion studies have been used to show that the deposits are proteinaceous. Similar deposits are also induced by treatment with oxalic and citric acids, but not by the potassium salts of these acids buffered at pH 7.2. It is suggested that a pH change within the chloroplasts, resulting from oxalic acid secretion by the fungus, is responsible for induction of protein deposition. The likelihood that the deposits are ribulose, 1,5-bisphosphate carboxylase-oxygenase is discussed.