PGRL1 Participates in Iron-induced Remodeling of the Photosynthetic Apparatus and in Energy Metabolism in Chlamydomonas reinhardtii

PGRL1 RNA and protein levels are increased in iron-deficient Chlamydomonas reinhardtii cells. In an RNAi strain, which accumulates lower PGRL1 levels in both iron-replete and -starved conditions, the photosynthetic electron transfer rate is decreased, respiratory capacity in iron-sufficient conditions is increased, and the efficiency of cyclic electron transfer under iron-deprivation is diminished. Pgrl1-kd cells exhibit iron deficiency symptoms at higher iron concentrations than wild-type cells, although the cells are not more depleted in cellular iron relative to wild-type cells as measured by mass spectrometry. Thiol-trapping experiments indicate iron-dependent and redox-induced conformational changes in PGRL1 that may provide a link between iron metabolism and the partitioning of photosynthetic electron transfer between linear and cyclic flow. We propose, therefore, that PGRL1 in C. reinhardtii may possess a dual function in the chloroplast; that is, iron sensing and modulation of electron transfer.     

Effects of Iron on Growth,Pigment Content,Photosystem II Efficiency,and Siderophores Production of <Emphasis Type="Italic">Microcystis aeruginosa</Emphasis> and <Emphasis Type="Italic">Microcystis wesenbergii</Emphasis>

Changes in growth, photosynthetic pigments, and photosystem II (PS II) photochemical efficiency as well as production of siderophores of Microcystis aeruginosa and Microcystis wesenbergii were determined in this experiment. Results showed growths of M. aeruginosa and M. wesenbergii, measured by means of optical density at 665 nm, were severely inhibited under an iron-limited condition, whereas they thrived under an iron-replete condition. The contents of chlorophyll-a, carotenoid, phycocyanin, and allophycocyanin under an iron-limited condition were lower than those under an iron-replete condition, and they all reached maximal contents on day 4 under the iron-limited condition. PS II photochemical efficiencies (maximal PS II quantum yield), saturating light levels (I _k ) and maximal electron transport rates (ETR_max) of M. aeruginosa and M. wesenbergii declined sharply under the iron-limited condition. The PS II photochemical efficiency and ETR_max of M. aeruginosa rose , whereas in the strain of M. wesenbergii, they declined gradually under the iron-replete condition. In addition, I _k of M. aeruginosa and M. wesenbergii under the iron-replete condition did not change obviously. Siderophore production of M. aeruginosa was higher than that of M. wesenbergii under the iron-limited condition. It was concluded that M. aeruginosa requires higher iron concentration for physiological and biochemical processes compared with M. wesenbergii, but its tolerance against too high a concentration of iron is weaker than M. wesenbergii.     

Effect of iron on expression of superoxide dismutase by Aeromonas salmonicida and associated resistance to superoxide anion

Abstract Superoxide dismutase activity was detected in Aeromonas salmonicida under iron-replete and iron-limited culture conditions. Under iron-replete conditions an iron superoxide dismutase, molecular mass 50,400 Da, was identified based on inhibition by hydrogen peroxide but not by millimolar concentrations of cyanide. When the available iron in the culture medium was limited by addition of the non-assimilable iron chelator 2,2-dipyridyl, a manganese superoxide dismutase, molecular mass 45,600 Da, was identified, which was resistant to inhibition by either hydrogen peroxide or cyanide. The change in enzyme production would appear to be iron dependent, as addition of FeCl₃ in excess to iron-limited broths resulted in only the iron superoxide dismutase being synthesised. Examination of the location of the superoxide dismutase enzymes revealed that the manganese superoxide dismutase expressed under iron limitation is located in the periplasm, while the iron superoxide dismutase has a cytoplasmic location. The periplasmic manganese superoxide dismutase was able to protect A. salmonicida against extracellular riboflavin-generated superoxide, with A. salmonicida grown under iron-limited conditions exhibiting a 32-fold increase in minimum bactericidal concentration of riboflavin compared to cells cultured under iron-replete conditions. Furthermore, in a time-course study of bactericidal activity of exogenously generated superoxide against A. salmonicida , bacteria grown under iron-replete conditions and expressing cytoplasmic iron superoxide dismutase were rapidly killed, whilst those grown under iron limitation expressing periplasmic manganese superoxide dismutase survived for the duration of the experiment.     

Alterations in Helicobacter pylori outer membrane and outer membrane vesicle-associated lipopolysaccharides under iron-limiting growth conditions

Outer membrane vesicles (OMVs) shed from the gastroduodenal pathogen Helicobacter pylori have measurable effects on epithelial cell responses. The aim of this study was to determine the effect of iron availability, and its basis, on the extent and nature of lipopolysaccharide (LPS) produced on H. pylori OMVs and their parental bacterial cells. Electrophoretic, immunoblotting and structural analyses revealed that LPSs of bacterial cells grown under iron-limited conditions were notably shorter than those of bacteria and OMVs obtained from iron-replete conditions. Structural analysis and serological probing showed that LPSs of iron-replete cells and OMVs expressed O-chains of Lewis(x) with a terminal Lewis(y) unit, whereas Lewis(y) expression was notably reduced on bacteria and OMVs from iron-limiting conditions. Unlike the O-chain, the core oligosaccharide and lipid A moieties of iron-replete and iron-limited bacteria and their OMVs were similar. Quantitatively, shed OMVs from iron-replete bacteria were found to be LPSenriched, whereas shed OMVs from iron-limited bacteria had a significantly reduced content of LPS. These differences were linked to bacterial ATP levels. Since iron availability affects the extent and nature of LPS expressed by H. pylori, host iron status may contribute to H. pylori pathogenesis.     

The yeast FET5 gene encodes a FET3 -related multicopper oxidase implicated in iron transport

The yeast FET3 gene encodes an integral membrane multicopper oxidase required for high-affinity iron uptake. The FET4 gene encodes an Fe(II) transporter required for low-affinity uptake. To identify other yeast genes involved in iron uptake, we isolated genes that could, when overexpressed, suppress the iron-limited growth defect of a fet3 fet4 mutant. The FET5 gene was isolated in this screen and it encodes a multicopper oxidase closely related to Fet3p. Several observations indicate that Fet5p plays a role analogous to Fet3p in iron transport. Suppression of the fet3 fet4 mutant phenotype by FET5 overexpression required the putative FTR1 transporter subunit of the high-affinity system. Fet5p is an integral membrane protein whose oxidase domain is located on the cell surface or within an intracellular compartment. Oxidase activity measured in cells with altered levels of FET5 expression suggested that Fet5p is a functional oxidase. FET5 overexpression increased the rate of iron uptake by a novel uptake system. Finally, FET5 mRNA levels are regulated by iron and are increased in cells grown in iron-limited media. These results suggest that Fet5p normally plays a role in the transport of iron.     

A salt-inducible chloroplastic monodehydroascorbate reductase from halophyte Avicennia marina confers salt stress tolerance on transgenic plants

Plant growth and productivity are adversely affected by various abiotic stress factors. In our previous study, we used Avicennia marina, a halophytic mangrove, as a model plant system for isolating genes functioning in salt stress tolerance. A large scale random EST sequencing from a salt stressed leaf tissue cDNA library of one month old A. marina plants resulted in identification of a clone showing maximum homology to Monodehydroascorbate reductase (Am-MDAR). MDAR plays a key role in regeneration of ascorbate from monodehydroascorbate for ROS scavenging. In this paper, we report the cellular localization and the ability to confer salt stress tolerance in transgenic tobacco of this salt inducible Am-MDAR. A transit peptide at the N-terminal region of Am-MDAR suggested that it encodes a chloroplastic isoform. The chloroplastic localization was confirmed by stable transformation and expression of the Am-MDAR-GFP fusion protein in tobacco. Transgenic tobacco plants overexpressing Am-MDAR survived better under conditions of salt stress compared to untransformed control plants. Assays of enzymes involved in ascorbate–glutathione cycle revealed an enhanced activity of MDAR and ascorbate peroxidase whereas the activity of dehyroascorbate reductase was reduced under salt stressed and unstressed conditions in Am-MDAR transgenic lines. The transgenic lines showed an enhanced redox state of ascorbate and reduced levels of malondialdehyde indicating its enhanced tolerance to oxidative stress. The results of our studies could be used as a starting point for genetic engineering of economically important plants tolerant to salt stress.     

Effects of Iron Limitation on the Degradation of Toluene by Pseudomonas Strains Carrying the TOL (pWWO) Plasmid

Most aerobic biodegradation pathways for hydrocarbons involve iron-containing oxygenases. In iron-limited environments, such as the rhizosphere, this may influence the rate of degradation of hydrocarbon pollutants. We investigated the effects of iron limitation on the degradation of toluene by Pseudomonas putida mt2 and the transconjugant rhizosphere bacterium P. putida WCS358(pWWO), both of which contain the pWWO (TOL) plasmid that harbors the genes for toluene degradation. The results of continuous-culture experiments showed that the activity of the upper-pathway toluene monooxygenase decreased but that the activity of benzyl alcohol dehydrogenase was not affected under iron-limited conditions. In contrast, the activities of three meta-pathway (lower-pathway) enzymes were all found to be reduced when iron concentrations were decreased. Additional experiments in which citrate was used as a growth substrate and the pathways were induced with the gratuitous inducer o-xylene showed that expression of the TOL genes increased the iron requirement in both strains. Growth yields were reduced and substrate affinities decreased under iron-limited conditions, suggesting that iron availability can be an important parameter in the oxidative breakdown of hydrocarbons.     

Impact of oxidative stress on ascorbate biosynthesis in Chlamydomonas via regulation of the VTC2 gene encoding a GDP-L-galactose phosphorylase

The L-galactose (Smirnoff-Wheeler) pathway represents the major route to L-ascorbic acid (vitamin C) biosynthesis in higher plants. Arabidopsis thaliana VTC2 and its paralogue VTC5 function as GDP-L-galactose phosphorylases converting GDP-L-galactose to L-galactose-1-P, thus catalyzing the first committed step in the biosynthesis of L-ascorbate. Here we report that the L-galactose pathway of ascorbate biosynthesis described in higher plants is conserved in green algae. The Chlamydomonas reinhardtii genome encodes all the enzymes required for vitamin C biosynthesis via the L-galactose pathway. We have characterized recombinant C. reinhardtii VTC2 as an active GDP-L-galactose phosphorylase. C. reinhardtii cells exposed to oxidative stress show increased VTC2 mRNA and L-ascorbate levels. Genes encoding enzymatic components of the ascorbate-glutathione system (e.g. ascorbate peroxidase, manganese superoxide dismutase, and dehydroascorbate reductase) are also up-regulated in response to increased oxidative stress. These results indicate that C. reinhardtii VTC2, like its plant homologs, is a highly regulated enzyme in ascorbate biosynthesis in green algae and that, together with the ascorbate recycling system, the L-galactose pathway represents the major route for providing protective levels of ascorbate in oxidatively stressed algal cells.     

Effect of ionizing radiation on the DNA damage response in <Emphasis Type="Italic">Chlamydomonas reinhardtii</Emphasis>

The DNA damage response (DDR) is induced by various DNA damaging factors and maintains genome stability in all organisms. The Chlamydomonas reinhardtii genome contains putative homologous genes involved in DDR; however, little is known about the functions and responses of these genes to DNA damage. In this study, DDR by gamma radiation was determined in C. reinhardtii. Irradiation with 80, and 200 Gy gamma radiation caused death in approximately 47 and 97 % of C. reinhardtii cells, respectively. The absolute lethality of cells was at 300 Gy. The rate of DNA breaks was also determined using comet assays after exposure to different doses of gamma radiation. Irradiation with 80 and 400 Gy resulted in 17 and 34 % of nuclear degradation in C. reinhardtii cells, respectively. To identify the major DDR pathway of C. reinhardtii induced by gamma radiation, 24 putative DDR genes were selected from the Joint Genome Institute (JGI) database. Gamma radiation significantly affected expression of 15 genes among these. Therefore, these genes displaying expressional changes by gamma radiation are involved in DDR, which indicate that C. reinhardtii may possess a fundamental conserved DDR pathway with higher plants. Furthermore, radiation responsive proteins were identified by proteomic analysis, which are involved in metabolisms of carbohydrate, energy, and photosynthesis. This is the first report to describe the responses of DDR homologous genes to gamma radiation and to identify gamma radiation-responsive proteins in C. reinhardtii. Our data should provide molecular insights into gamma radiation responses including DNA damage in green algae.