protochlorophyllide
Dotaz
Zobrazit nápovědu
In most oxygenic phototrophs, including cyanobacteria, two independent enzymes catalyze the reduction of protochlorophyllide to chlorophyllide, which is the penultimate step in chlorophyll (Chl) biosynthesis. One is light-dependent NADPH:protochlorophyllide oxidoreductase (LPOR) and the second type is dark-operative protochlorophyllide oxidoreductase (DPOR). To clarify the roles of both enzymes, we assessed synthesis and accumulation of Chl-binding proteins in mutants of cyanobacterium Synechocystis PCC 6803 that either completely lack LPOR or possess low levels of the active enzyme due to its ectopic regulatable expression. The LPOR-less mutant grew photoautotrophically in moderate light and contained a maximum of 20 % of the wild-type (WT) Chl level. Both Photosystem II (PSII) and Photosystem I (PSI) were reduced to the same degree. Accumulation of PSII was mostly limited by the synthesis of antennae CP43 and especially CP47 as indicated by the accumulation of reaction center assembly complexes. The phenotype of the LPOR-less mutant was comparable to the strain lacking DPOR that also contained <25 % of the wild-type level of PSII and PSI when cultivated under light-activated heterotrophic growth conditions. However, in the latter case, we detected no reaction center assembly complexes, indicating that synthesis was almost completely inhibited for all Chl-proteins, including the D1 and D2 proteins.
- MeSH
- 2D gelová elektroforéza MeSH
- aktivace enzymů MeSH
- bakteriální proteiny genetika metabolismus MeSH
- buněčná membrána enzymologie metabolismus MeSH
- chlorofyl biosyntéza genetika MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- fenotyp MeSH
- fotosystém I (proteinový komplex) genetika metabolismus MeSH
- fotosystém II (proteinový komplex) genetika metabolismus MeSH
- fototrofní procesy MeSH
- oxidoreduktasy působící na CH-CH vazby genetika metabolismus MeSH
- protochlorofylid metabolismus MeSH
- regulace genové exprese enzymů * MeSH
- regulace genové exprese u rostlin MeSH
- světlo MeSH
- Synechocystis enzymologie genetika metabolismus účinky záření MeSH
- transformace genetická MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Unlike angiosperms, gymnosperms use two different enzymes for the reduction of protochlorophyllide to chlorophyllide: the light-dependent protochlorophyllide oxidoreductase (LPOR) and the dark-operative protochlorophyllide oxidoreductase (DPOR). In this study, we examined the specific role of both enzymes for chlorophyll synthesis in response to different light/dark and temperature conditions at different developmental stages (cotyledons and needles) of Norway spruce (Picea abies Karst.). The accumulation of chlorophyll and chlorophyll-binding proteins strongly decreased during dark growth in secondary needles at room temperature as well as in cotyledons at low temperature (7 °C) indicating suppression of DPOR activity. The levels of the three DPOR subunits ChlL, ChlN, and ChlB and the transcripts of their encoding genes were diminished in dark-grown secondary needles. The low temperature had minor effects on the transcription and translation of these genes in cotyledons, which is suggestive for post-translational control in chlorophyll biosynthesis. Taking into account the higher solubility of oxygen at low temperature and oxygen sensitivity of DPOR, we mimicked low-temperature condition by the exposure of seedlings to higher oxygen content (33%). The treatment resulted in an etiolated phenotype of dark-grown seedlings, confirming an oxygen-dependent control of DPOR activity in spruce cotyledons. Moreover, light-dependent suppression of mRNA and protein level of DPOR subunits indicates that more efficiently operating LPOR takes over the DPOR function under light conditions, especially in secondary needles.
- MeSH
- chlorofyl genetika metabolismus MeSH
- oxidoreduktasy působící na CH-CH vazby biosyntéza metabolismus MeSH
- regulace genové exprese u rostlin MeSH
- smrk enzymologie genetika metabolismus MeSH
- světlo MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- Geografické názvy
- Norsko MeSH
The negatively charged lipid phosphatidylglycerol (PG) constitutes up to 10% of total lipids in photosynthetic membranes, and its deprivation in cyanobacteria is accompanied by chlorophyll (Chl) depletion. Indeed, radioactive labeling of the PG-depleted ΔpgsA mutant of Synechocystis sp. strain PCC 6803, which is not able to synthesize PG, proved the inhibition of Chl biosynthesis caused by restriction on the formation of 5-aminolevulinic acid and protochlorophyllide. Although the mutant accumulated chlorophyllide, the last Chl precursor, we showed that it originated from dephytylation of existing Chl and not from the block in the Chl biosynthesis. The lack of de novo-produced Chl under PG depletion was accompanied by a significantly weakened biosynthesis of both monomeric and trimeric photosystem I (PSI) complexes, although the decrease in cellular content was manifested only for the trimeric form. However, our analysis of ΔpgsA mutant, which lacked trimeric PSI because of the absence of the PsaL subunit, suggested that the virtual stability of monomeric PSI is a result of disintegration of PSI trimers. Interestingly, the loss of trimeric PSI was accompanied by accumulation of monomeric PSI associated with the newly synthesized CP43 subunit of photosystem II. We conclude that the absence of PG results in the inhibition of Chl biosynthetic pathway, which impairs synthesis of PSI, despite the accumulation of chlorophyllide released from the degraded Chl proteins. Based on the knowledge about the role of PG in prokaryotes, we hypothesize that the synthesis of Chl and PSI complexes are colocated in a membrane microdomain requiring PG for integrity.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- chlorofyl biosyntéza metabolismus MeSH
- chlorofylidy metabolismus MeSH
- fosfatidylglyceroly genetika metabolismus MeSH
- fotosystém I (proteinový komplex) metabolismus MeSH
- ligasy tvořící vazby C-O metabolismus MeSH
- protochlorofylid metabolismus MeSH
- světlosběrné proteinové komplexy metabolismus MeSH
- Synechocystis genetika metabolismus MeSH
- transferasy pro jiné substituované fosfátové skupiny genetika metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Arsenic (As) pollution is a serious concern worldwide. Recent studies under environmentally relevant conditions revealed that, in the aquatic plant Ceratophyllum demersum, pigments are the first observable target of toxicity, prior to any effect on photosynthetic parameters or to oxidative stress. Lethal toxicity was initiated by a change of As species and their distribution pattern in various tissues. Here, the localization of As was investigated at the subcellular level through X-ray fluorescence using a submicron beam and a Maia detector. Further, it was possible to obtain useful tissue structural information from the ratio of the tomogram of photon flux behind the sample to the tomogram of Compton scattering. The micro-X-ray fluorescence tomograms showed that As predominantly accumulated in the nucleus of the epidermal cells in young mature leaves exposed to sublethal 1 µM As. This suggests that As may exert toxic effects in the nucleus, for example, by interfering with nucleic acid synthesis by replacing phosphorous with As. At higher cellular concentrations, As was mainly stored in the vacuole, particularly in mature leaves. An analysis of precursors of chlorophyll and degradation metabolites revealed that the observed decrease in chlorophyll concentration was associated with hindered biosynthesis, and was not due to degradation. Coproporphyrinogen III could not be detected after exposure to only 0.5 µM As. Levels of subsequent precursors, for example, protoporphyrin IX, Mg-protoporphyrin, Mg-protoporphyrin methyl ester, and divinyl protochlorophyllide, were significantly decreased at this concentration as well, indicating that the pathway was blocked upstream of tetrapyrrole synthesis.
MAIN CONCLUSION: We present an easy and effective procedure to purify plastids and mitochondria from Chromera velia. Our method enables downstream analyses of protein and metabolite content of the organelles. Chromerids are alveolate algae that are the closest known phototrophic relatives to apicomplexan parasites such as Plasmodium or Toxoplasma. While genomic and transcriptomic resources for chromerids are in place, tools and experimental conditions for proteomic studies have not been developed yet. Here we describe a rapid and efficient protocol for simultaneous isolation of plastids and mitochondria from the chromerid alga Chromera velia. This procedure involves enzymatic treatment and breakage of cells, followed by differential centrifugation. While plastids sediment in the first centrifugation step, mitochondria remain in the supernatant. Subsequently, plastids can be purified from the crude pellet by centrifugation on a discontinuous 60%/70% sucrose density gradient, while mitochondria can be obtained by centrifugation on a discontinuous 33%/80% Percoll density gradient. Isolated plastids are autofluorescent, and their multi-membrane structure was confirmed by transmission electron microscopy. Fluorescent optical microscopy was used to identify isolated mitochondria stained with MitoTrackerTM green, while their intactness and membrane potential were confirmed by staining with MitoTrackerTM orange CMTMRos. Total proteins were extracted from isolated organellar fractions, and the purity of isolated organelles was confirmed using immunoblotting. Antibodies against the beta subunit of the mitochondrial ATP synthase and the plastid protochlorophyllide oxidoreductase did not cross-react on immunoblots, suggesting that each organellar fraction is free of the residues of the other. The presented protocol represents an essential step for further proteomic, organellar, and cell biological studies of C. velia and can be employed, with minor optimizations, in other thick-walled unicellular algae.
- MeSH
- Alveolata ultrastruktura MeSH
- mikrořasy ultrastruktura MeSH
- mitochondrie ultrastruktura MeSH
- plastidy ultrastruktura MeSH
- Publikační typ
- časopisecké články MeSH
