Thylakoid Polypeptides Research Articles

Biogenesis of photosynthetic complexes in the chloroplast of Chlamydomonas reinhardtii requires ARSA1, a homolog of prokaryotic arsenite transporter and eukaryotic TRC40 for guided entry of tail‐anchored proteins

as1, for antenna size mutant 1, was obtained by insertion mutagenesis of the unicellular green alga Chlamydomonas reinhardtii. This strain has a low chlorophyll content, 8% with respect to the wild type, and displays a general reduction in thylakoid polypeptides. The mutant was found to carry an insertion into a homologous gene, prokaryotic arsenite transporter (ARSA), whose yeast and mammal counterparts were found to be involved in the targeting of tail-anchored (TA) proteins to cytosol-exposed membranes, essential for several cellular functions. Here we present the characterization in a photosynthetic organism of an insertion mutant in an ARSA-homolog gene. The ARSA1 protein was found to be localized in the cytosol, and yet its absence in as1 leads to a small chloroplast and a strongly decreased chlorophyll content per cell. ARSA1 appears to be required for optimal biogenesis of photosynthetic complexes because of its involvement in the accumulation of TOC34, an essential component of the outer chloroplast membrane translocon (TOC) complex, which, in turn, catalyzes the import of nucleus-encoded precursor polypeptides into the chloroplast. Remarkably, the effect of the mutation appears to be restricted to biogenesis of chlorophyll-binding polypeptides and is not compensated by the other ARSA homolog encoded by the C.reinhardtii genome, implying a non-redundant function.

Phosphorylation Controls the Localization and Activation of the Lumenal Carbonic Anhydrase in Chlamydomonas reinhardtii

BackgroundCah3 is the only carbonic anhydrase (CA) isoform located in the thylakoid lumen of Chlamydomonas reinhardtii. Previous studies demonstrated its association with the donor side of the photosystem II (PSII) where it is required for the optimal function of the water oxidizing complex. However this enzyme has also been frequently proposed to perform a critical function in inorganic carbon acquisition and CO2 fixation and all mutants lacking Cah3 exhibit very poor growth after transfer to low CO2 conditions.Results/ConclusionsIn the present work we demonstrate that after transfer to low CO2, Cah3 is phosphorylated and that phosphorylation is correlated to changes in its localization and its increase in activity. When C. reinhardtii wild-type cells were acclimated to limiting CO2 conditions, the Cah3 activity increased about 5–6 fold. Under these conditions, there were no detectable changes in the level of the Cah3 polypeptide. The increase in activity was specifically inhibited in the presence of Staurosporine, a protein kinase inhibitor, suggesting that the Cah3 protein was post-translationally regulated via phosphorylation. Immunoprecipitation and in vitro dephosphorylation experiments confirm this hypothesis. In vivo phosphorylation analysis of thylakoid polypeptides indicates that there was a 3-fold increase in the phosphorylation signal of the Cah3 polypeptide within the first two hours after transfer to low CO2 conditions. The increase in the phosphorylation signal was correlated with changes in the intracellular localization of the Cah3 protein. Under high CO2 conditions, the Cah3 protein was only associated with the donor side of PSII in the stroma thylakoids. In contrast, in cells grown at limiting CO2 the protein was partly concentrated in the thylakoids crossing the pyrenoid, which did not contain PSII and were surrounded by Rubisco molecules.SignificanceThis is the first report of a CA being post-translationally regulated and describing phosphorylation events in the thylakoid lumen.

Chloroplast ultrastructure and thylakoid polypeptide composition are affected by different salt concentrations in the halophytic plant Arthrocnemum macrostachyum

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.

Drought induced changes in physiological, biochemical and phytochemical properties of Withania somnifera Dun.

Drought (D) stress effects on growth, photosynthetic pigments, photosynthetic electron transport, thylakoid polypeptides and phytochemical variations of Withania (Solanaceae), a tropical medicinal herb were studied. Exposure of plants to D-stress led to noticeable decrease in leaf area, photosynthetic pigments, root and shoot lengths and photosynthetic activity. Well-watered plants maintained high content of total chlorophyll (Chl), root and shoot lengths, leaf area and photosynthesis. We analysed alterations at cellular level of the D-stress-associated proteins at different time intervals. A set of proteins in the range of 34 to 40 kDa showed variations in response to D-stress. Qualitative high performance thin layer chromatography (HPTLC) analysis of root extract obtained from control and D-stressed plants showed quantitative and qualitative variations. Withaferin A content increased 5% under D-stress as compared to control. Key words: Carotenoids, chlorophyll, fluorescence, leaf area, pigments photosynthesis.

Absence of the major light-harvesting antenna proteins alters the redox properties of photosystem II reaction centres in thechlorina F2mutant of barley

Although the chlorina F2 mutant of barley specifically exhibits reduced levels of the major light-harvesting polypeptides associated with photosystem II (PSII), thermoluminescence measurements of photosystem reaction centre photochemistry revealed that S2/S3QB- charge recombinations were shifted to lower temperatures, while the characteristic peak of S2QA- charge recombinations was shifted to higher temperatures compared with wild-type (WT) barley. Thus, we show that the absence of the major light-harvesting polypeptides affects the redox properties of PSII reaction centres. Radiolabeling studies in vivo and in vitro with [32P]orthophosphate or [gamma-32P]ATP, respectively, demonstrated that the D1 PSII reaction centre polypeptide is phosphorylated in both the WT and the F2 mutant. In contrast with the radiolabeling results, phosphorylation of D1 and other PSII proteins, although detected in WT barley, was ambiguous in the F2 mutant when the phosphothreonine antibody method of detection was used. Thus, caution must be exercised in the use of commercially available phosphothreonine antibodies to estimate thylakoid polypeptide phosphorylation. Furthermore, in membrano, the D1 polypeptide of the F2 mutant was less susceptible to trypsin treatment than that of WT barley. The role of the light-harvesting complex in modulating the structure and function of the D1 polypeptide of PSII reaction centers is discussed.

Consequences of C4 Differentiation for Chloroplast Membrane Proteomes in Maize Mesophyll and Bundle Sheath Cells

Chloroplasts of maize leaves differentiate into specific bundle sheath (BS) and mesophyll (M) types to accommodate C(4) photosynthesis. Chloroplasts contain thylakoid and envelope membranes that contain the photosynthetic machineries and transporters but also proteins involved in e.g. protein homeostasis. These chloroplast membranes must be specialized within each cell type to accommodate C(4) photosynthesis and regulate metabolic fluxes and activities. This quantitative study determined the differentiated state of BS and M chloroplast thylakoid and envelope membrane proteomes and their oligomeric states using innovative gel-based and mass spectrometry-based protein quantifications. This included native gels, iTRAQ, and label-free quantification using an LTQ-Orbitrap. Subunits of Photosystems I and II, the cytochrome b(6)f, and ATP synthase complexes showed average BS/M accumulation ratios of 1.6, 0.45, 1.0, and 1.33, respectively, whereas ratios for the light-harvesting complex I and II families were 1.72 and 0.68, respectively. A 1000-kDa BS-specific NAD(P)H dehydrogenase complex with associated proteins of unknown function containing more than 15 proteins was observed; we speculate that this novel complex possibly functions in inorganic carbon concentration when carboxylation rates by ribulose-bisphosphate carboxylase/oxygenase are lower than decarboxylation rates by malic enzyme. Differential accumulation of thylakoid proteases (Egy and DegP), state transition kinases (STN7,8), and Photosystem I and II assembly factors was observed, suggesting that cell-specific photosynthetic electron transport depends on post-translational regulatory mechanisms. BS/M ratios for inner envelope transporters phosphoenolpyruvate/P(i) translocator, Dit1, Dit2, and Mex1 were determined and reflect metabolic fluxes in carbon metabolism. A wide variety of hundreds of other proteins showed differential BS/M accumulation. Mass spectral information and functional annotations are available through the Plant Proteome Database. These data are integrated with previous data, resulting in a model for C(4) photosynthesis, thereby providing new rationales for metabolic engineering of C(4) pathways and targeted analysis of genetic networks that coordinate C(4) differentiation.

Comparative analysis of biochemical properties of mesophyll and bundle sheath chloroplasts from various subtypes of C4 plants grown at moderate irradiance.

The photochemical characteristics of mesophyll and bundle sheath chloroplasts isolated from the leaves of C4 species were investigated in Zea mays (NADP-ME type), Panicum miliaceum (NAD-ME type) and Panicum maximum (PEP-CK type) plants. The aim of this work was to gain information about selected photochemical properties of mesophyll and bundle sheath chloroplasts isolated from C4 plants grown in the same moderate light conditions. Enzymatic as well as mechanical methods were applied for the isolation of bundle sheath chloroplasts. In the case of Z. mays and P. maximum the enzymatic isolation resulted in the loss of some thylakoid polypeptides. It was found that the PSI and PSII activities of mesophyll and bundle sheath chloroplasts of all species studied differed significantly and the differences correlated with the composition of pigment-protein complexes, photophosphorylation efficiency and fluorescence emission characteristic of these chloroplasts. This is the first report showing differences in the photochemical activities between mesophyll chloroplasts of C4 subtypes. Our results also demonstrate that mesophyll and bundle sheath chloroplasts of C4 plants grown in identical light conditions differ significantly with respect to the activity of main thylakoid complexes, suggesting a role of factor(s) other than light in the development of photochemical activity in C4 subtypes.

Radiation inactivation analysis of thylakoid protein kinase systems in light and in darkness

Chloroplast thylakoid contains several membrane-bound protein kinases that phosphorylate thylakoid polypeptides for the regulation of photosynthesis. Thylakoid protein phosphorylation is activated when the plastoquinone pool is reduced either by light-dependent electron flow through photosystem 2 (PS2) or by adding exogenous reductants such as durohydroquinone in the dark. The major phosphorylated proteins on thylakoid are components of light-harvesting complex 2 (LHC2) and a PS2 associated 9 kDa phosphoprotein. Radiation inactivation technique was employed to determine the functional masses of various kinases for protein phosphorylation in thylakoids. Under the photosynthetically active radiation (PAR), the apparent functional masses of thylakoid protein kinase systems (TPKXs) for catalyzing phosphorylation of LHC2 27 and 25 kDa polypeptides were 540±50 and 454±35 kDa as well as it was 448±23 kDa for PS2 9 kDa protein phosphorylation. Furthermore, the functional sizes of dark-regulated TPKXs for 25 and 9 kDa proteins were 318±25 and 160±8 kDa. The 9 kDa protein phosphorylation was independent of LHC2 polypeptides phosphorylation with regard to its TPKX functional mass. Target size analysis of protein phosphorylation mentioned above indicates that thylakoid contains a group of distinct protein kinase systems. A working model is accordingly proposed to interpret the interaction between these protein kinase systems.

Effects of low temperature stress on chloroplast development in a range of Zea mays cultivars

One photosynthetic characteristic, chloroplast development, was used to evaluate tolerance to chilling in a range of Zea mays L. cultivars. Seedlings of six different cultivars of Zea mays were grown in controlled environment chambers at daytime temperatures of 14 °C, 17 °C and 25 °C and their thylakoid polypeptide profiles examined. With reduced growth temperature decreases in many polypeptides were observed, particularly those with apparent molecular masses ranging from 30–42 kDa, 43–67 kDa and 68–71 kDa. However, a number of other polypeptides with lower apparent molecular masses ranging from 14–20 kDa accumulated in some cultivars at the lower growth temperatures, especially at 14 °C. The comparatively uniform reduction in specific thylakoid polypeptides at lower growth temperatures in the six cultivars examined suggest that thylakoid polypeptide profiles may be used as a criterion for screening low temperature tolerance in maize lines.

Characterization of a pigment-deficient mutant of sunflower (Helianthus annuus L.) with abnormal chloroplast biogenesis, reduced PS II activity and low endogenous level of abscisic acid

Nuclear mutation xantha1 (xan1) arose spontaneously in sunflower and it appeared to have many pleiotropic effects. Homozygous xan1/xan1 seedlings, in field conditions, die after depletion of cotyledonary reserves and can be only recovered by selfing normal heterozygous plants (XAN1/xan1). xan1 mutant showed aberrant development of chloroplasts, deficient pigment content and reduced CO2 assimilation rate. Also, xan1 mutation negatively affected endogenous ABA levels both in non-stressed and dehydrated leaves. Photodestruction of both chlorophyll a and β-carotene in xan1 leaves directly correlated with light intensity. The activation of the xanthophyll cycle under low light (165 PPFD) and the appreciable amounts of zeaxanthin and antheraxanthin retained by xan1 mutant after a 24 h dark period were clear signals of a strong photoinhibition state. The unusual patterns of thylakoid proteins composition detected in seedlings grown under 165 PPFD, by using both denaturing and native electrophoresis analyses, were also indicative of high sensitivity of the thylakoid polypeptide complexes to light stress. Moreover, immunoblot analysis of psbA gene product (D1 protein) indicated that even at 165 PPFD the structure of photosystem (PS) II reaction center was severely damaged. Interestingly, xan1 seedlings grown in dim light (3.5 PPFD) revealed a severely altered chloroplast ultrastructure, despite the lack of photooxidative effects on leaf pigments and the lack of alterations in the electrophoretic profiles of thylakoid proteins. Electron microscopy indicated that XAN1 product is required for normal organization and distribution of thylakoids in the stroma. Chlorophyll a fluorescence analysis pointed to xan1 incompetence to fully express and retain PS II activity, even in the absence of photooxidation.

Characterization of a pigment-deficient mutant of sunflower (Helianthus annuus L.) with abnormal chloroplast biogenesis, reduced PS II activity and low endogenous level of abscisic acid

Nuclear mutation xantha1 (xan1) arose spontaneously in sunflower and it appeared to have many pleiotropic effects. Homozygous xan1/xan1 seedlings, in field conditions, die after depletion of cotyledonary reserves and can be only recovered by selfing normal heterozygous plants (XAN1/xan1). xan1 mutant showed aberrant development of chloroplasts, deficient pigment content and reduced CO2 assimilation rate. Also, xan1 mutation negatively affected endogenous ABA levels both in non-stressed and dehydrated leaves. Photodestruction of both chlorophyll a and β-carotene in xan1 leaves directly correlated with light intensity. The activation of the xanthophyll cycle under low light (165 PPFD) and the appreciable amounts of zeaxanthin and antheraxanthin retained by xan1 mutant after a 24 h dark period were clear signals of a strong photoinhibition state. The unusual patterns of thylakoid proteins composition detected in seedlings grown under 165 PPFD, by using both denaturing and native electrophoresis analyses, were also indicative of high sensitivity of the thylakoid polypeptide complexes to light stress. Moreover, immunoblot analysis of psbA gene product (D1 protein) indicated that even at 165 PPFD the structure of photosystem (PS) II reaction center was severely damaged. Interestingly, xan1 seedlings grown in dim light (3.5 PPFD) revealed a severely altered chloroplast ultrastructure, despite the lack of photooxidative effects on leaf pigments and the lack of alterations in the electrophoretic profiles of thylakoid proteins. Electron microscopy indicated that XAN1 product is required for normal organization and distribution of thylakoids in the stroma. Chlorophyll a fluorescence analysis pointed to xan1 incompetence to fully express and retain PS II activity, even in the absence of photooxidation.

Role of visible light in the recovery of photosystem II structure and function from ultraviolet-B stress in higher plants.

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.

Effects of Prolonged UV-B Enhanced Fluorescent Radiation on Some Marine Microalgae

The eukaryotic unicellular microalgae Chlorella salina, Dicrateria inornata, and Isochrysis galbana were grown under control (fluorescent 20 W m-2) and UV-B enhanced (UV-BE, 0.5 W m-2) fluorescent radiation. The growth rate showed marginal increase under UV-BE. Decrease in protein content was observed in Dicrateria cells but in Chlorella an initial increase up to 4 d and in Isochrysis an increase at days 4 and 5 was noted. The chlorophyll a content showed marked increase in Chlorella and Isochrysis but in Dicrateria a decline was found. UV-BE reduced the photosynthetic activity in all three species, but the reduction was larger in Chlorella and Dicrateria. Fluorescence excitation spectra for F682 in Chlorella cells grown for 5 d under UV-BE showed reduction in all peaks. In contrast to this, in Dicrateria and Isochrysis cells, the 530 and 590 nm excitation peaks increased with an appreciable decrease in the 466 nm peak. SDS-PAGE analysis revealed significant decrease in the contents of 47, 33, and 23 kDa polypeptides in Chlorella cells. In Dicrateria cells, significant loss in the content of 55, 38, and 18 kDa polypeptides was observed. The content of low molecular mass polypeptides (15 kDa) remained unaffected. Isochrysis cells were more stable in preserving the content of thylakoid polypeptides.

Lhca polypeptides are maintained during dark-induced senescence of leaves

Thylakoid polypeptides samples from fresh and dark-incubated (0–6 days) leaves were tested for changes in relative abundancies of Lhca polypeptides using a set of polyclonal and monoclonal antibodies in conjunction with immunoblot technique and integrating densitometry. All Lhca polypeptides were found to be more stable within 6 days of dark incubation than bulk chlorophyll itself. On chlorophyll basis the relative abundance of Lhca 1–4 increased after 6 days of dark incubation to 154, 177, 144 and 121 %, respectively, of the values found in fresh leaves.

Chronic ozone fumigation induces alterations in thylakoid functionality and composition in two poplar clones

The effects of chronic O 3 exposure (60 ppb for 5 h a day for 15 d) on photosynthetic electron transport activity and thylakoid polypeptide pattern has been studied in cuttings of two poplar clones ( Populus deltoides × maximowiczii clone Eridano and P. × euramericana clone I-214). At the end of the O 3-exposure period, there were no visible symptoms of injury on leaf surface in either clone. In comparison with the respective controls, plants of both treated clones showed a statistically significant decrease in the activity of the two photosystems (PSI and PSII), although to differing extents. In both clones, immunoblot analysis of the thylakoid proteins indicated a reduction, following O 3 treatment, in the content of all polypeptides analysed, with a more marked reduction of PSII core complex D1 protein. A generalised decrease in leaf pigment content was shown by both O 3-treated clones, and the activation of the xanthophyll cycle following ozone exposure was more evident in the Eridano clone. No alterations in the chloroplast structure were seen by electron microscope analysis in either clone. The only visible difference was a marked accumulation of starch grains in the Eridano clone. All these results suggest that chronic O 3 treatment induces a generalised decrease in the activity of electron transport chain components and in the photosystem protein and pigment content, which could be interpreted as an adaptation of the poplar plants to the adverse environmental conditions.

Water stress induced modulation in thylakoid membrane fluidity and loss of D1 protein during development of clusterbean cotyledons

Water stress causes significant changes in the accumulation of pigments, thylakoid polypeptide composition, chlorophyll-a fluorescence characteristics and photochemical efficiency of chloroplasts in the cotyledons of clusterbean (Cyamopsis tetragonoloba L.) seedlings. The cotyledons however, respond differently to the stress at their different developmental phases. During stress, a new band appears in the electrophoretic profiles of the thylakoid proteins of the young and developing cotyledons, which disappears during maturity. In contrast, the stress-induced changes in both the developing and mature cotyledons are associated with a loss in the immunologically probed D1 protein indicating a photoinhibitory damage. The stress-induced changes in the thylakoid organisation and function are correlated with relatively a low amount of carotenoids, loss in the activity of super oxide dismutase, enhancement of lipid peroxidation and subsequent increase in the chlorophyll-a fluorescence polarization. These results are suggestive of a free-radical induced alteration in membrane fluidity as reflected in an increase in polarization, leading to thylakoid rigidity as the contributory factor for the dislocation of electron transport and subsequent loss of the D1 protein in the water-stressed cotyledons of clusterbean in absence of sufficient scavenging potential of the chloroplasts.

Rice tolerance to excess Mn: Implications in the chloroplast lamellae and synthesis of a novel Mn protein

Rice ( Oryza sativa L. cv. Safari) plants were grown over a 21-d period in nutrient solutions containing Mn concentrations varying between 0.125 and 32 mg·L –1. The plant shoots and the thylakoid membranes showed similar overall Mn increases for this range of treatments, but different accumulation kinetics. It was found that the highest Mn treatment were associated to the synthesis of a new thylakoid protein with an average molecular mass of 36.5 kDa and a Mn:protein ratio of about 1. This protein exhibited superoxide dismutase activity, as well as a high content of Gln, Asp, Glu, Leu and Gly. Its EPR spectrum is characteristic of high-spin Mn(II), in a S=5/2 ground state. A comparative study of SDS polyacrylamide gel profiles of thylakoid polypeptides from the various treatments disclosed quantitative changes, as well as a new 37/36-kDa polypeptide band in the two highest Mn treatments. The photosynthetic electron transport rates coupled to PSII and PSI showed a significant increase until the 8-mg·L –1 Mn treatment. The related superoxide production of thylakoids (monitored by EPR spectroscopy) showed minimum values from the 0.5-mg·L –1 Mn treatment onwards, which, as shown by the thiobarbituric acid reaction was coupled to a non-significant variation of the acyl lipid peroxidation. It was concluded that Oryza sativa L. cv. safari has a high internal tolerance to Mn as it synthesises a new manganese protein that mimics superoxide dismutase functioning.

Differential sensitivity to ozone in two poplar clones.

The effect of acute O3 exposure (5 h at 150 ppb) on photosynthetic electron transport and the thylakoid polypeptide pattern has been studied in 2‐month‐old cuttings of two poplar clones (Populus deltoides×maximowiczii clone Eridano and P.×euramericana clone I‐214), known for their differential response to O3 in terms of visible symptoms. The more sensitive Eridano developed severe blackish bifacial necrosis concentrated in the interveinal areas of the basal and intermediate leaves, while in the less sensitive I‐214 clone, visible symptoms were slight or entirely absent. A generalized, significant decrease in leaf pigment content was shown by both of the stressed clones, although the decline in β‐carotene content was much more marked in Eridano than in I‐214. Activation of the xanthophyll cycle was not detected in either clone. In both clones, immunoblot analysis of the thylakoid proteins showed a decrease in the levels of all the PSII polypeptides tested following O3 exposure, whereas the PsaA/B protein in the PSI core was more severely affected in Eridano than in I‐214. No changes in the chloroplast structure could be seen on electron microscopic study in either clone. The only visible difference was a great accumulation of starch grains in Eridano. All of these results suggest that O3 exposure induces alterations in the photosynthetic apparatus in both poplar clones, Eridano being affected to a greater extent than I‐214.

Ultraviolet B Exposure of Whole Leaves of Barley Affects Structure and Functional Organization of Photosystem II

This study examines the effects of ecologically important levels of ultraviolet B radiation on protein D1 turnover and stability and lateral redistribution of photosystem II. It is shown that ultraviolet B light supported only limited synthesis of protein D1, one of the most important components of photosystem II, whereas it promoted significant degradation of proteins D1 and D2. Furthermore, dephosphorylation of photosystem II subunits was specifically elicited upon exposure to ultraviolet B light. Structural modifications of photosystem II and changes in its lateral distribution between granum membranes and stroma-exposed lamellae were found to be different from those observed after photoinhibition by strong visible light. In particular, more complete dismantling of photosystem II cores was observed. Altogether, the data reported here suggest that ultraviolet B radiation alone fails to activate the photosystem II repair cycle, as hypothesized for visible light. This failure may contribute to the toxic effect of ultraviolet B radiation, which is increasing as a consequence of depletion of stratospheric ozone.

Targeted disruption of the plastid RNA polymerase genes rpoA, B and C1: molecular biology, biochemistry and ultrastructure.

The plastid encoded RNA polymerase subunit genes rpoA, B and C1 of tobacco were disrupted individually by PEG-mediated plastid transformation. The resulting off-white mutant phenotype is identical for inactivation of the different genes. The mutants pass through a normal ontogenetic cycle including flower formation and production of fertile seeds. Their plastids reveal a poorly developed internal membrane system consisting of large vesicles and, occasionally, flattened membranes, reminiscent of stacked thylakoids. The rpo- material is capable of synthesising pigments and lipids, similar in composition but at lower amounts than the wild-type. Western analysis demonstrates that plastids contain nuclear-coded stroma and thylakoid polypeptides including terminally processed lumenal components of the Sec but not of the DeltapH thylakoid translocation machineries. Components using the latter route accumulate as intermediates. In striking contrast, polypeptides involved in photosynthesis encoded by plastid genes could not be detected by Western analysis, although transcription of plastid genes, including the rrn operon, by the plastid RNA polymerase of nuclear origin is found as expected. Remarkably, ultrastructural, sedimentation and Northern analyses as well as pulse experiments suggest that rpo- plastids contain functional ribosomes. The detection of the plastid-encoded ribosomal protein Rpl2 is consistent with these results. The findings demonstrate that the consequences of rpo gene disruption, and implicitly the integration of the two plastid polymerase types into the entire cellular context, are considerably more complex than presently assumed.