Ozone treatment affects pigment precursor metabolism in pine seedlings.

Five-week-old seedlings of Pinus halepensis Mill. and Pinus brutia Ten. were exposed to air polluted with ozone (O3) (250 nl l-1, 12 h day-1 for 4 days) or to ambient air containing ca 10-20 nl l-1 O3, in the light (180 &mgr;mol m-2 s-1 photosynthetic photon flux density [PPFD], 12 h day-1) and then fed for 24 h in the light (100 &mgr;mol m-2 s-1 PPFD) with various radioactive precursors of chlorophyll (Chl) and carotene biosynthesis: 5-[4-14C]-aminolevulinic acid (14C-ALA), L-[14C(U)]-glutamic acid (14C-Glu), or D,L-[2-14C]-mevalonic acid (14C-MVA). Pigments were then extracted from cotyledons and fully expanded needles. Chl a and carotene were separated by thin-layer chromatography and high-performance liquid chromatography and their specific activities were determined. 14C-ALA and 14C-Glu labels were incorporated into Chl a and carotene. Exposure to O3 did not inhibit incorporation of 14C-ALA into Chl a molecules, but hydrolysis of Chl a showed that O3 inhibited phytol labelling of Chl a. Labelling of carotene was also inhibited by O3, but not when 14C-MVA was used as the label. These data suggest that O3 treatment inhibits (directly or indirectly) the biosynthesis of isoprenoids from products of ALA and Glu metabolism in the plastid, but not from MVA in the cytosol. This inhibition was more prominent when 14C-ALA was used as the label than when 14C-Glu was the labelling precursor. A significant increase in pheophorbide a, a tetrapyrrole component of Chl a labelling, and a concomitant decrease in phytol labelling was observed following incubation of O3-treated pine seedlings with 14C-ALA and 14C-Glu. Stronger inhibition of carotene biosynthesis and activation of Chl a tetrapyrrole labelling by 14C-ALA (in comparison with 14C-Glu) indicated that exposure to O3 inhibits the conversion of ALA to Glu as the first step in ALA catabolism. These results also suggested a more intensive Glu metabolism (in comparison with ALA) for carotene biosynthesis in the cytosol, as well as cooperation between two pathways of isopentenyl diphosphate biosynthesis.

Synergistic effects of a photooxidized polycyclic aromatic hydrocarbon and copper on photosynthesis and plant growth: evidence that in vivo formation of reactive oxygen species is a mechanism of copper toxicity.

Heavy metals and polycyclic aromatic hydrocarbons (PAHs) are often cocontaminants in industrialized environments, yet little is known about either the extent or mechanisms of their cotoxicity. To address this shortfall, the combined effects of an oxygenated PAH, 1,2-dihydroxyanthraquinone (1,2-dhATQ), and a heavy metal, Cu2+, on photosynthesis and growth of the duckweed (Lemna gibba) were evaluated. Using assays of chlorophyll a fluorescence and photosystem I activity, 1,2-dhATQ inhibited electron transport at the cytochrome b6/f complex. Conversely, Cu2+ alone (at low concentrations) had little effect on photosynthesis. When Cu2+ was combined with 1,2-dhATQ, an increase in transient and steady-state chlorophyll a fluorescence quenching occurred relative to 1,2-dhATQ alone. Treatment of isolated thylakoid membranes with 1,2-dhATQ inhibited whole-chain linear electron transport, measured as O2 consumption using methyl viologen as the electron acceptor. However, Cu2+ plus 1,2-dhATQ resulted in active O2 consumption with or without methyl viologen as an electron acceptor. From these data, we conclude that 1,2-dhATQ renders the plastoquinone pool to a highly reduced state by inhibiting at cytochrome b6/f. Then, Cu2+ is able to mediate the transfer of electrons from reduced plastoquinone to O2, forming reactive oxygen species. At the whole-organism level, when Cu2+ and 1,2-dhATQ were mixed at concentrations that resulted in the above-mentioned impacts on photosynthesis, synergistic inhibition of plant growth was observed. This suggests a catalytic mechanism of toxicity for redox active metals, a process that could be instrumental in explaining their impacts at low concentrations.

Oxygen uptake photosensitized by disorganized chlorophyll in model systems and thylakoids of greening barley.

Light-dependent oxygen uptake was observed and studied in thylakoids from early greening barley in comparison to oxygen uptake in chlorophyll solutions and in thylakoids from fully green leaves. Substantial oxygen uptake was observed in chlorophyll solutions supplemented with tryptophan, histidine, ascorbic acid or linoleic acid. This uptake was diminished by adding azide, beta-carotene and alpha-tocopherol, which are specific singlet-oxygen quenchers. Illuminated thylakoids from greening barley also exhibited marked oxygen uptake that, likewise, was strongly quenched by azide. In comparison, azide was found not to affect oxygen uptake that is associated with the methyl viologen-catalyzed Mehler reaction. It is reasoned that in the first two cases the oxygen uptake arises from chlorophyll-photosensitized activation of oxygen to the singlet state and its consumption by exogenous or endogenous substrates. In greening, we propose that disorganized chlorophyll photo-sensitizes the oxygen uptake.

Determination of catabolism of the photosystem II D 1 subunit by structural motifs in the polypeptide sequence.

Proteolytic mapping of the D 1 subunit of photosystem two and a degradation product which arises during its rapid catabolism shows that the latter is a result of proteolysis within the peptide motif QEEET. This motif is located in a portion of the D 1 protein thought to form a stroma-exposed connection between fourth and fifth transmembrane segments. This connection domain also contains a "PEST"-like sequence and forms part of the QB/herbicide binding niche. The QEEET motif seems to provide a major epitope in immunological studies, as judged from reaction of D 1 and its fragments with polyclonal antibodies. Antibodies against D 1 were found to react with other animal and plant proteins which contain similar sequence motifs.

Degradation of the 32 kD Herbicide Binding Protein in Far Red Light.

White light (400-700 nanometers) supports the activity of photosystem I (PSI) and photosystem II while far red light (>/=700 nanometers) supports PSI almost exclusively. In intact fronds of Spirodela oligorrhiza, turnover of the 32 kilodaltons herbicide binding protein is stimulated under both these light conditions, although not in the dark or at wavelengths >730 nanometers. As is the case in white light, the far red light induced degradation of the protein is inhibited by DCMU. The means by which far red light operates is unclear. Hypotheses considered include: PSI activated proteolysis, PSI-induced formation of semiquinone anions, and PSI-generated free radicals.

Identification of psbA and psbD gene products, D1 and D2, as reaction centre proteins of photosystem 2.

A recent report (Nanba O, Satoh K: Proc. Natl. Acad. Sci. USA 84: 109-112, 1987) described the isolation from spinach of a putative photosystem 2 reaction centre which contained cytochrome b-559 and three other electrophoretically resolvable polypeptide bands, two of which have molecular weights comparable to the D1 and D2 polypeptides. We have used in vivo labelling with radioactive methionine and probed with D1 and D2 monospecific antibodies (raised against synthetically expressed sequences of the psbA and psbD genes) for specific detection of these proteins in a similarly prepared photosystem 2 reaction centre preparation. These techniques identified a 32 000 dalton D1 band, a 30 000 dalton D2 band and a 55 000 dalton D1/D2 aggregate, the latter apparently arising from the detergent treatments employed. Digestions with a lysine-specific protease further confirmed the identification of the lysine-free D1 polypeptide and also confirmed that the D1 molecules in the 55 000 dalton band were in aggregation with other bands and not in self-aggregates. The D1 and D2 polypeptides (including the aggregate) are considerably enriched in the photosystem two reaction centre preparation compared to the other resolved fractions.

Molecular architecture of the rapidly metabolized 32-kilodalton protein of photosystem II. Indications for COOH-terminal processing of a chloroplast membrane polypeptide.

The molecular architecture of the rapidly metabolized 32-kilodalton chloroplast protein was investigated. Modified proteolytic techniques were applied to construct a cleavage map for this photosynthetic membrane component. The portion which anchors the protein to the membrane was shown to be rich in hydrophobic amino acid residues, while the surface-exposed portion was richer in polar residues. The amino to carboxy polarity of the map was established by comparing oligopeptide radiolabeling data with the polypeptide sequence decoded from the gene. This showed that the anchor domain contains the NH2 terminus, and the exposed domain the COOH terminus. The 32-kilodalton protein was previously demonstrated to derive from a 33.5-kilodalton precursor. We now show that the precursor molecule has a COOH-terminal oligopeptide extension. A model relating precursor cleavage to membrane integration is presented. COOH-terminal processing is proposed to ensure a correct sequence of events for assembly of the 32-kilodalton protein into the photosynthetic membrane.

Regulation of protein metabolism: Coupling of photosynthetic electron transport to in vivo degradation of the rapidly metabolized 32-kilodalton protein of the chloroplast membranes.

In Spirodela oligorrhiza, mature chloroplasts copiously synthesize and degrade a 32-kilodalton membrane protein. The rates of synthesis and degradation are controlled by light intensity, the protein being unstable in the light and stable in the dark. Light-driven synthesis, but not degradation, is dependent on ATP. Degradation is blocked by herbicides inhibiting photosystem II electron transport, such as diuron and atrazine. Thus, both anabolism and catabolism of the 32-kilodalton protein are photoregulated, with degradation coupled to electron transport rather than phosphorylation.

General occurrence and structural similarity of the rapidly synthesized, 32,000-dalton protein of the chloroplast membrane.

A rapidly metabolized membrane protein from Spirodela, with an apparent molecular weight of 32,000, has been implicated in allosterically regulating electron transport and mediating diuron herbicide sensitivity in the chloroplast (Mattoo, A. K., Pick, U., Hoffman-Falk, H., and Edelman, M. (1981) Proc. Natl. Acad. Sci. U. S. A. 78, 1572-1576). Rapid synthesis of a 32,000-dalton plastid membrane protein is demonstrated for several diverse angiosperms and the alga, Chlamydomonas. Comparative partial proteolytic mapping of the polypeptide showed similar patterns for all species tested. In some cases, a 33,500-dalton precursor polypeptide was identified which also displayed interspecific structural homologies. Lastly, in situ analysis of the surface-exposed 32,000-dalton membrane protein yielded a common trypsinization pattern for the organisms studied. These findings point toward a broad distribution and phylogenetic similarity of the 32,000-dalton thylakoid protein of the chloroplast at levels of precursor maturation, membrane orientation, and primary structure.