The flanking regions of PsaD drive efficient gene expression in the nucleus of the green alga Chlamydomonas reinhardtii

Fischer, Nicolas; Rochaix, Jean David

doi:10.1007/s004380100485

Cited by 219 publications

(227 citation statements)

References 23 publications

(39 reference statements)

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“…4B). Complementation of the ad-su6 VL-CO 2 lethal phenotype also was achieved by expressing CAH3 cDNA under control of the constitutive PsaD promoter and terminator (Fischer and Rochaix, 2001; data not shown). …”

Section: Complementation Of Ad-su6mentioning

confidence: 98%

Thylakoid Lumen Carbonic Anhydrase (CAH3) Mutation Suppresses Air-Dier Phenotype of LCIB Mutant in Chlamydomonas reinhardtii

2008

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An active CO 2 -concentrating mechanism is induced when Chlamydomonas reinhardtii acclimates to limiting inorganic carbon (Ci), either low-CO 2 (L-CO 2 ; air level; approximately 0.04% CO 2 ) or very low-CO 2 (VL-CO 2 ; approximately 0.01% CO 2 ) conditions. A mutant, ad1, which is defective in the limiting-CO 2 -inducible, plastid-localized LCIB, can grow in high-CO 2 or VL-CO 2 conditions but dies in L-CO 2 , indicating a deficiency in a L-CO 2 -specific Ci uptake and accumulation system. In this study, we identified two ad1 suppressors that can grow in L-CO 2 but die in VL-CO 2 . Molecular analyses revealed that both suppressors have mutations in the CAH3 gene, which encodes a thylakoid lumen localized carbonic anhydrase. Photosynthetic rates of L-CO 2 -acclimated suppressors under acclimation CO 2 concentrations were more than 2-fold higher than ad1, apparently resulting from a more than 20-fold increase in the intracellular concentration of Ci as measured by direct Ci uptake. However, photosynthetic rates of VL-CO 2 -acclimated cells under acclimation CO 2 concentrations were too low to support growth in spite of a significantly elevated intracellular Ci concentration. We conclude that LCIB functions downstream of CAH3 in the CO 2 -concentrating mechanism and probably plays a role in trapping CO 2 released by CAH3 dehydration of accumulated Ci. Apparently dehydration by the chloroplast stromal carbonic anhydrase CAH6 of the very high internal Ci caused by the defect in CAH3 provides Rubisco sufficient CO 2 to support growth in L-CO 2 -acclimated cells, but not in VL-CO 2 -acclimated cells, even in the absence of LCIB.CO 2 serves both as the substrate for photosynthesis and as an important signal to regulate plant growth and development, so variable CO 2 concentrations can impact photosynthesis, growth, and productivity of plants. Terrestrial C 4 plants have developed a CO 2 -concentrating mechanism (CCM) involving anatomical and biochemical adaptations to accumulate a higher concentration of CO 2 as substrate Rubisco and to suppress oxygenation of ribulose-1,5-bisP, a wasteful side reaction. In contrast, a different type of CCM is induced in the unicellular green microalga Chlamydomonas reinhardtii when the supply of dissolved inorganic carbon (Ci; CO 2 and HCO 3 2

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Section: Complementation Of Ad-su6mentioning

confidence: 98%

Thylakoid Lumen Carbonic Anhydrase (CAH3) Mutation Suppresses Air-Dier Phenotype of LCIB Mutant in Chlamydomonas reinhardtii

2008

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“…Ten micrograms of genomic DNA was digested for 2 h with 10 units of PstI or PvuII restriction endonucleases (New England Biolabs), the DNA fragments were separated by agarose (0.8%) gel electrophoresis, the gels were blotted overnight in 203 SSC onto nylon membranes (Bio-Rad), and the transferred DNA was cross-linked to the membrane by UV illumination. An alkaline phosphatase-labeled DNA probe was synthesized by chemically cross-linking a thermostable alkaline phosphatase to the 1.7-kb AphVIII PCR fragment (Sizova et al, 2001), which also contains the PSAD promoter and the 3# sequence from the CYTc6 gene (Fischer and Rochaix, 2001). Probe synthesis and hybridizations were performed using the Amersham AlkPhos Direct Labeling and Detection System according to the manufacturer's protocol (Amersham Biosciences).…”

Section: Southern-blot Analysesmentioning

confidence: 99%

A Mutant in theADH1Gene ofChlamydomonas reinhardtiiElicits Metabolic Restructuring during Anaerobiosis

et al. 2012

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The green alga Chlamydomonas reinhardtii has numerous genes encoding enzymes that function in fermentative pathways. Among these, the bifunctional alcohol/acetaldehyde dehydrogenase (ADH1), highly homologous to the Escherichia coli AdhE enzyme, is proposed to be a key component of fermentative metabolism. To investigate the physiological role of ADH1 in dark anoxic metabolism, a Chlamydomonas adh1 mutant was generated. We detected no ethanol synthesis in this mutant when it was placed under anoxia; the two other ADH homologs encoded on the Chlamydomonas genome do not appear to participate in ethanol production under our experimental conditions. Pyruvate formate lyase, acetate kinase, and hydrogenase protein levels were similar in wild-type cells and the adh1 mutant, while the mutant had significantly more pyruvate:ferredoxin oxidoreductase. Furthermore, a marked change in metabolite levels (in addition to ethanol) synthesized by the mutant under anoxic conditions was observed; formate levels were reduced, acetate levels were elevated, and the production of CO 2 was significantly reduced, but fermentative H 2 production was unchanged relative to wild-type cells. Of particular interest is the finding that the mutant accumulates high levels of extracellular glycerol, which requires NADH as a substrate for its synthesis. Lactate production is also increased slightly in the mutant relative to the control strain. These findings demonstrate a restructuring of fermentative metabolism in the adh1 mutant in a way that sustains the recycling (oxidation) of NADH and the survival of the mutant (similar to wild-type cell survival) during dark anoxic growth.

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“…The DNA fragments were mixed and amplified by PCR. The PCR product was digested with NdeI and EcoRI and introduced into the pGenD-ble expression vector (34). C. reinhardtii cells were transformed by electroporation and selected on TAP-agar plates containing 10 g/ml Zeocin (Invitrogen).…”

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confidence: 99%

Molecular and electrophysiological characterization of a mechanosensitive channel expressed in the chloroplasts of Chlamydomonas

Nakayama

Fujiu

Sokabe

et al. 2007

Proc. Natl. Acad. Sci. U.S.A.

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MscS is a mechanosensitive channel that is ubiquitous among bacteria. Recent progress in the genome projects has revealed that homologs of MscS are also present in eukaryotes, but whether they operate as ion channels is unknown. In this study we cloned MSC1, a homolog of MscS in Chlamydomonas, and examined its function when expressed in Escherichia coli. Full-length MSC1 was not functional when expressed in E. coli cells. However, removal of the N-terminal signal sequence (⌬N-MSC1) reversed this effect. ⌬N-MSC1 was found to open in response to membrane stretch and displayed a preference for anions over cations as permeable ions. ⌬N-MSC1 exhibited marked hysteretic behavior in response to ascending and descending stimuli. That is, channel gating occurred in response to significant stimuli but remained open until the stimulus was almost completely removed. Indirect immunofluorescence revealed that MSC1 is present as punctate spots in the cytoplasm and chloroplasts. Moreover, knockdown of MSC1 expression resulted in the abnormal localization of chlorophyll. These findings show that MSC1 is an intracellular mechanosensitive channel and is responsible for the organization of chloroplast in Chlamydomonas.MscS ͉ hysteresis ͉ heterogeneous expression ͉ knockdown ͉ green algae M echanosensation is typically involved in auditory perception, touch sensation, proprioception, and gravity perception. The mechanoreceptor potential in sensory cells is mediated by mechanosensitive channels, which are activated by stretching or deformation of the membrane. The patch-clamp technique has revealed that mechanosensitive channels are present in almost every cell type, not just sensory cells. In fact, mechanosensitive channels were first recorded with the patch-clamp method in the muscle cell (1). Mechanosensitive channels in nonsensory cells are believed to be responsible for monitoring cell deformation evoked by contact with surrounding materials and by changes in osmolarity.Bacteria harbor the mechanosensitive channels MscS and MscL, which act to protect against hypoosmotic downshock (2). The presence of MscS homologs in virtually all eubacteria and archaea suggests that MscS has an essential function in prokaryotes, but whether every MscS homolog serves as a mechanosensitive channel is uncertain. For instance, although there are at least six MscS homologs in the Escherichia coli genome, only two of them (MscS and MscK) have been detected by electrophysiological methods (3, 4). Recent genomic projects on eukaryotes have uncovered the presence of MscS homologs in some plants (Arabidopsis and Oryza), protists (Chlamydomonas), and fungi (Schizosaccharomyces). Curiously, MscS homologs have not been found in animals (5, 6). The MscS homologs of Arabidopsis (MSL2 and MSL3) have been observed as one or two foci on the plastid envelope, and their double knockout results in an abnormal size and shape of the plastids (6). This finding suggests that one of the possible functions of the MscS homologs is to control the size and shape of the intr...

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The flanking regions of PsaD drive efficient gene expression in the nucleus of the green alga Chlamydomonas reinhardtii

Cited by 219 publications

References 23 publications

Thylakoid Lumen Carbonic Anhydrase (CAH3) Mutation Suppresses Air-Dier Phenotype of LCIB Mutant in Chlamydomonas reinhardtii

Thylakoid Lumen Carbonic Anhydrase (CAH3) Mutation Suppresses Air-Dier Phenotype of LCIB Mutant in Chlamydomonas reinhardtii

A Mutant in theADH1Gene ofChlamydomonas reinhardtiiElicits Metabolic Restructuring during Anaerobiosis

Molecular and electrophysiological characterization of a mechanosensitive channel expressed in the chloroplasts of Chlamydomonas

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