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1.
Plant Physiol Biochem ; 132: 356-362, 2018 Nov.
Article in English | MEDLINE | ID: mdl-30261469

ABSTRACT

Salicornia veneta (Pignatti et Lausi) is an extreme halophyte living in salt marsh where NaCl concentration may be as high as 1 M. Here we report on the isolation and characterization of a PSII preparation obtained by Triton X-100 solubilisation of the thylakoid membrane. By a combination of gel electrophoresis, immunoblotting and mass spectrometry, the depletion of a number of PSII proteins such as PsbQ, PsbM and PsbT was highlighted. Moreover, the requirement of Cl- and Ca2+ for optimal oxygen evolution was determined, showing that in absence of PsbQ a higher level of these ions are required. At high Cl- concentrations, oxygen evolution was inhibited in the same way in Salicornia veneta and spinach. Reconstitution of Salicornia veneta PSII preparation with partially purified spinach PsbP and PsbQ restored oxygen evolution activity at low Cl- and Ca2+ concentrations. Adaptation to high salt makes several PSII proteins dispensable.


Subject(s)
Chenopodiaceae/metabolism , Photosystem II Protein Complex/isolation & purification , Photosystem II Protein Complex/metabolism , Salt-Tolerant Plants/metabolism , Thylakoids/metabolism , Calcium/metabolism , Chlorides/metabolism , Oxygen/analysis , Plant Proteins/metabolism , Spinacia oleracea/metabolism
2.
Plant Physiol Biochem ; 57: 59-66, 2012 Aug.
Article in English | MEDLINE | ID: mdl-22683464

ABSTRACT

Tetramethylbenzidine-H(2)O(2) staining of SDS-polyacrylamide gel is a widely used method for the specific detection of proteins with heme-dependent peroxidase activity. When this method was used with thylakoids from the halophytic plant Arthrocnemum macrostachyum, besides the cytochrome f and cytochrome b6 proteins usually found in higher plants and cyanobacteria, at least four additional bands were detected. One of them, a 46-kDa protein, was shown to be an extrinsic protein, and identified by mass spectrometry and immunoblotting as a 2-cys peroxiredoxin. Peroxidase activity was insensitive to oxidizing agents such as trans-4,4-diydroxy-1,2-dithiane or hydrogen peroxide, but was inhibited by treatment of thylakoids with reducing agents such as dithiothreitol or mercaptoethanol. By immunoblotting, it was shown that loss of peroxidase activity was paralleled by disappearance of the 46-kDa band, which was converted to a 23-kDa immunoreactive form. A dimer/monomer relationship between the two proteins is suggested, with the dimeric form likely being a heme-binding protein. This possibility was further supported by anionic exchange chromatography and de novo sequencing of tryptic fragments of the protein and sequence comparison, as most of the residues previously implicated in heme binding in 2-cys peroxiredoxin from Rattus norvegicus were conserved in A. macrostachyum. The amount of this protein was modulated by environmental conditions, and increased when salt concentration in the growth medium was higher or lower than the optimal one.


Subject(s)
Amaranthaceae/metabolism , Benzidines/chemistry , Hydrogen Peroxide/chemistry , Peroxiredoxins/metabolism , Plant Proteins/metabolism , Salt-Tolerant Plants/metabolism , Thylakoids/metabolism , Peroxiredoxins/chemistry , Plant Proteins/chemistry
3.
J Plant Physiol ; 169(2): 111-6, 2012 Jan 15.
Article in English | MEDLINE | ID: mdl-22118876

ABSTRACT

The effect of different external salt concentrations, from 0 mM to 1030 mM NaCl, on photosynthetic complexes and chloroplast ultrastructure in the halophyte Arthrocnemum macrostachyum was studied. Photosystem II, but not Photosystem I or cytochrome b6/f, was affected by salt treatment. We found that the PsbQ protein was never expressed, whereas the amounts of PsbP and PsbO were influenced by salt in a complex way. Analyses of Photosystem II intrinsic proteins showed an uneven degradation of subunits with a loss of about 50% of centres in the 0 mM NaCl treated sample. Also the shape of chloroplasts, as well as the organization of thylakoid membranes were affected by NaCl concentration, with many grana containing few thylakoids at 1030 mM NaCl and thicker grana and numerous swollen thylakoids at 0 mM NaCl. The PsbQ protein was found to be depleted also in thylakoids from other halophytes.


Subject(s)
Amaranthaceae/ultrastructure , Chloroplasts/ultrastructure , Membrane Proteins/metabolism , Sodium Chloride/pharmacology , Amaranthaceae/growth & development , Amaranthaceae/metabolism , Chloroplasts/chemistry , Chloroplasts/drug effects , Chloroplasts/metabolism , Cytochromes/metabolism , Photosystem I Protein Complex/metabolism , Photosystem II Protein Complex/metabolism , Plant Growth Regulators/metabolism , Salt-Tolerant Plants/growth & development , Salt-Tolerant Plants/metabolism , Spain , Thylakoids/metabolism , Thylakoids/ultrastructure
4.
Ecotoxicol Environ Saf ; 73(8): 1988-97, 2010 Nov.
Article in English | MEDLINE | ID: mdl-20884049

ABSTRACT

The site around ACNA factory (Northern Italy) is characterized by multi-metal contamination, therefore it can be considered as a source of autochthonous plants able to tolerate or accumulate heavy metals (HMs). The hill A5, a waste dump of the chemical factory, was chosen as the study area, in order to assess the metal accumulation ability of the vegetation growing under HM stress. The plant species, biodiversity and health were related to the concentration of HMs in four areas of the hill A5, and to the metal accumulation in shoots and roots. Uptake of HMs occurred at different extent in the various plant species and differed according to the considered organ and metal. Polygonum aviculare hyperaccumulated Hg in the shoot suggesting its possible exploitation in phytoextraction. A number of species, that can be useful in phytoremediation plans, accumulated simultaneously more than two heavy metals both in the shoot and in the root.


Subject(s)
Industrial Waste , Metals, Heavy/toxicity , Polygonum/drug effects , Soil Pollutants/toxicity , Biodegradation, Environmental/drug effects , Italy , Metals, Heavy/metabolism , Plant Roots/classification , Plant Roots/drug effects , Plant Roots/growth & development , Plant Roots/metabolism , Plant Shoots/classification , Plant Shoots/drug effects , Plant Shoots/growth & development , Plant Shoots/metabolism , Polygonum/classification , Polygonum/growth & development , Polygonum/metabolism , Seasons , Soil Pollutants/chemistry , Soil Pollutants/metabolism , Species Specificity
5.
Ann Bot ; 103(3): 505-15, 2009 Feb.
Article in English | MEDLINE | ID: mdl-19033288

ABSTRACT

BACKGROUND AND AIMS: Photosystem II of oxygenic organisms is a multi-subunit protein complex made up of at least 20 subunits and requires Ca(2+) and Cl(-) as essential co-factors. While most subunits form the catalytic core responsible for water oxidation, PsbO, PsbP and PsbQ form an extrinsic domain exposed to the luminal side of the membrane. In vitro studies have shown that these subunits have a role in modulating the function of Cl(-) and Ca(2+), but their role(s) in vivo remains to be elucidated, as the relationships between ion concentrations and extrinsic polypeptides are not clear. With the aim of understanding these relationships, the photosynthetic apparatus of the extreme halophyte Salicornia veneta has been compared with that of spinach. Compared to glycophytes, halophytes have a different ionic composition, which could be expected to modulate the role of extrinsic polypeptides. METHODS: Structure and function of in vivo and in vitro PSII in S. veneta were investigated and compared to spinach. Light and electron microscopy, oxygen evolution, gel electrophoresis, immunoblotting, DNA sequencing, RT-PCR and time-resolved chlorophyll fluorescence were used. KEY RESULTS: Thylakoids of S. veneta did not contain PsbQ protein and its mRNA was absent. When compared to spinach, PsbP was partly depleted (30 %), as was its mRNA. All other thylakoid subunits were present in similar amounts in both species. PSII electron transfer was not affected. Fluorescence was strongly quenched upon irradiation of plants with high light, and relaxed only after prolonged dark incubation. Quenching of fluorescence was not linked to degradation of D1 protein. CONCLUSIONS: In S. veneta the PsbQ protein is not necessary for photosynthesis in vivo. As the amount of PsbP is sub-stoichiometric with other PSII subunits, this protein too is largely dispensable from a catalytic standpoint. One possibility is that PsbP acts as an assembly factor for PSII.


Subject(s)
Chenopodiaceae/metabolism , Oxygen/metabolism , Photosystem II Protein Complex/metabolism , Plant Proteins/metabolism , Salt-Tolerant Plants/metabolism , Amino Acid Sequence , Base Sequence , Chenopodiaceae/cytology , Chenopodiaceae/genetics , Chenopodiaceae/ultrastructure , Chlorophyll/metabolism , Conserved Sequence , Electron Transport/radiation effects , Fluorescence , Genes, Plant , Kinetics , Molecular Sequence Data , Oxidation-Reduction/radiation effects , Peptides/metabolism , Photosynthesis/radiation effects , Plant Proteins/chemistry , Plant Proteins/genetics , Plant Stems/cytology , Plant Stems/metabolism , Plant Stems/radiation effects , Protein Subunits/metabolism , Salt-Tolerant Plants/cytology , Salt-Tolerant Plants/genetics , Salt-Tolerant Plants/ultrastructure , Thylakoids/radiation effects , Thylakoids/ultrastructure
6.
J Exp Bot ; 56(414): 1239-44, 2005 Apr.
Article in English | MEDLINE | ID: mdl-15753111

ABSTRACT

As a consequence of variation in environmental factors, light being the most important one, a number of photosystem II polypeptides may be reversibly phosphorylated by thylakoid-bound kinase(s). Among them, the reaction centre D1 and D2 polypeptides, the PsbH subunit, and the inner antenna CP43. Here, the separation of two forms of CP43 by high-resolution denaturing polyacrylamide gel electrophoresis is reported. By means of immunoblotting with antibody to phosphothreonine-containing proteins and authentic CP43 and limited proteolysis, these two bands could be identified as the phosphorylated and dephosphorylated forms of CP43. Using non-denaturing isoelectrofocusing, a chromatographically derived CP43-enriched fraction could be resolved into three different native forms of CP43. Among them, one was found to be a phosphorylated form, whereas the other two were dephosphorylated forms of the protein. With respect to other methods, the procedure described here allows the isolation, for the first time, of a fully homogeneous population of this chlorophyll-protein complex, opening the way to the study of the role of phopshorylation on functional properties of this core antenna protein.


Subject(s)
Hordeum/metabolism , Photosynthetic Reaction Center Complex Proteins/metabolism , Photosystem II Protein Complex/metabolism , Phosphorylation , Phosphotransferases/metabolism , Photosynthetic Reaction Center Complex Proteins/isolation & purification , Photosystem II Protein Complex/isolation & purification , Spectrophotometry
7.
EMBO Rep ; 4(9): 900-5, 2003 Sep.
Article in English | MEDLINE | ID: mdl-12949587

ABSTRACT

The smallest extrinsic polypeptide of the water-oxidizing complex (PsbQ) was extracted and purified from spinach (Spinacia oleracea) photosystem II (PSII) membranes. It was then crystallized in the presence of Zn(2+) and its structure was determined by X-ray diffraction at 1.95-A resolution using the multi-wavelength anomalous diffraction method, with the zinc as the anomalous scatterer. The crystal structure shows that the core of the protein is a four-helix bundle, whereas the amino-terminal portion, which possibly interacts with the photosystem core, is not visible in the crystal. The distribution of positive and negative charges on the protein surface might explain the ability of PsbQ to increase the binding of Cl(-) and Ca(2+) and make them available to PSII.


Subject(s)
Arabidopsis Proteins/chemistry , Photosystem II Protein Complex/chemistry , Spinacia oleracea/chemistry , Amino Acid Motifs , Amino Acid Sequence , Arabidopsis Proteins/metabolism , Binding Sites , Crystallography, X-Ray , Molecular Sequence Data , Photosystem II Protein Complex/metabolism , Protein Structure, Tertiary , Spinacia oleracea/metabolism , Zinc/metabolism
8.
J Exp Bot ; 54(388): 1665-73, 2003 Jul.
Article in English | MEDLINE | ID: mdl-12754266

ABSTRACT

The effect of visible light on photosystem II reaction centre D1 protein in plants treated with ultraviolet-B light was studied. It was found that a 20 kDa C-terminal fragment of D1 protein generated during irradiation with ultraviolet-B light was stable when plants were incubated in the dark, but was degraded when plants were incubated in visible light. In this condition the recovery of photosynthetic activity was also observed. Even a low level of white light was sufficient to promote both further degradation of the fragment and recovery of activity. During this phase, the D1 protein is the main synthesized thylakoid polypeptide, indicating that other photosystem II proteins are recycled in the recovery process. Although both degradation of the 20 kDa fragment and resynthesis of D1 are light-dependent phenomena, they are not closely related, as degradation of the 20 kDa fragment may occur even in the absence of D1 synthesis. Comparing chemical and physical factors affecting the formation of the fragment in ultraviolet-B light and its degradation in white light, it was concluded that the formation of the fragment in ultraviolet-B light is a photochemical process, whereas the degradation of the fragment in white light is a protease-mediated process.


Subject(s)
Photosynthesis/physiology , Photosynthetic Reaction Center Complex Proteins/metabolism , Light , Lincomycin/pharmacology , Photosynthesis/radiation effects , Photosynthetic Reaction Center Complex Proteins/drug effects , Photosynthetic Reaction Center Complex Proteins/radiation effects , Photosystem II Protein Complex , Plant Leaves/physiology , Plant Leaves/radiation effects , Plant Proteins/metabolism , Plant Proteins/radiation effects , Thylakoids/physiology , Thylakoids/radiation effects , Ultraviolet Rays
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