Catherine B. Mason

Cloning and overexpression of two cDNAs encoding the low-CO2-inducible chloroplast envelope protein LIP-36 from Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii, a unicellular green alga, grows photoautotrophically at very low concentrations of inorganic carbon due to the presence of an inducible CO2-concentrating mechanism. During the induction of the CO2-concentrating mechanism at low-CO2 growth conditions, at least five polypeptides that are either absent or present in low amounts in cells grown on high-CO2 concentrations are induced. One of these induced polypeptides with a molecular mass of 36 kD, LIP-36, has been localized to the chloroplast envelope. The protein was purified and the partial internal amino acid sequences were obtained through lys-C digestion. Two cDNAs encoding LIP-36 have been cloned using degenerate primers based on the amino acid sequences. The two genes encoding LIP-36 are highly homologous in the coding region but are completely different in the 5[prime]-end and 3[prime]-end untranslated regions. The deduced protein sequences show strong homology to the mitochondrial carrier protein superfamily, suggesting that LIP-36 is a chloroplast carrier protein. The regulation of the expression of these two genes at high- and low-CO2 growth conditions is also different. Both genes were highly expressed under low-CO2 growth conditions, with the steady-state level of LIP-36 G1 mRNA more abundant. However, neither gene was expressed at high-CO2 growth conditions. The gene products of both clones expressed in Escherichia coli were recognized by an antibody raised against LIP-36, confirming that the two cDNAs indeed encode the C. reinhardtii chloroplast envelope carrier protein LIP-36.

The induction of the CO2-concentrating mechanism is correlated with the formation of the starch sheath around the pyrenoid of Chlamydomonas reinhardtii

The pyrenoid is a prominent proteinaceous structure found in the stroma of the chloroplast in unicellular eukaryotic algae, most multicellular algae, and some hornworts. The pyrenoid contains the enzyme ribulose-1,5-bisphosphate carboxylase/oxygenase and is sometimes surrounded by a carbohydrate sheath. We have observed in the unicellular green alga Chlamydomonas reinhardtii Dangeard that the pyrenoid starch sheath is formed rapidly in response to a decrease in the CO2 concentration in the environment. This formation of the starch sheath occurs coincidentally with the induction of the CO2-concentrating mechanism. Pyrenoid starch-sheath formation is partly inhibited by the presence of acetate in the growth medium under light and low-CO2 conditions. These growth conditions also partly inhibit the induction of the CO2-concentrating mechanism. When cells are grown with acetate in the dark, the CO2-concentrating mechanism is not induced and the pyrenoid starch sheath is not formed even though there is a large accumulation of starch in the chloroplast stroma. These observations indicate that pyrenoid starch-sheath formation correlates with induction of the CO2-concentrating mechanism under low-CO2 conditions. We suggest that this ultrastructural reorganization under lowCO2 conditions plays a role in the CO2-concentrating mechanism C. reinhardtii as well as in other eukaryotic algae.

The Low CO2-Inducible 36-Kilodalton Protein Is Localized to the Chloroplast Envelope of Chlamydomonas reinhardtii

The localization of the 36-kD polypeptide of Chlamydomonas reinhardtii induced by photoautotrophic growth on low CO2 concentrations (0.03% in air [v/v], low CO2-grown cells) has been investigated. This polypeptide was specifically localized to the chloroplast envelope membranes isolated from low CO2-grown cells and was not present in the chloroplast envelopes isolated from high (5% CO2 in air [v/v]) CO2-grown cells. The 36-kD protein does not show carbonic anhydrase activity and was not present on the plasma membranes isolated from low CO2-grown cells. This protein may, in part, account for the different inorganic carbon uptake characteristics observed in chloroplasts isolated from high and low CO2-grown cells of C. reinhardtii.

A Low-CO2-Inducible Gene Encoding an Alanine:[alpha]-Ketoglutarate Aminotransferase in Chlamydomonas reinhardtii

At low-CO2 (air) conditions, the unicellular green alga Chlamydomonas reinhardtii acquires the ability to raise its internal inorganic carbon concentration. To study this adaptation to low CO2, cDNA clones induced under low-CO2 growth conditions were selected through differential screening. One full-length clone is 2552 bp, with an open reading frame encoding 521 amino acids. The deduced amino acid sequence shows about 50% identity with alanine:[alpha]-ketoglutarate aminotransferase (Ala AT, EC 2.6.1.2) from plants and animals, and the mRNA of this clone increased 4- to 5-fold 4 h after cells were switched from high-CO2 to low-CO2 growth conditions. The expression of the enzyme and its activity also increased accordingly at low-CO2 growth conditions. To study the physiological role of Ala AT, a pyridoxal phosphate inhibitor, aminooxyacetic acid, was added at 40 [mu]M to the growth medium when cells were beginning to adapt to low CO2. This caused a 30% decrease in the maximum photosynthetic rate in air-adapting cells 8 h later. The addition of the inhibitor also caused the cells to excrete glycolate, a photorespiratory intermediate, but did not change the apparent affinity of the cell for external CO2. These physiological studies are consistent with the assumption that Ala AT is involved in the adaptation to low-CO2 conditions.

Use of the bleomycin resistance gene to generate tagged insertional mutants of Chlamydomonas reinhardtii that require elevated CO2 for optimal growth

Chlamydomonas reinhardtii Dangeard possesses a CO2 concentrating mechanism (CCM) that enables it to grow at very low CO2 concentrations. In previous studies, insertional mutagenesis was successfully used to identify genes required for growth at low CO2 in C. reinhardtii. These earlier studies used the C. reinhardtii genes, Nit1 and Arg7 to complement nit1- or arg7- strains, thereby randomly inserting a second copy of Nit1 or Arg7 into the genome. Because these genes are already present in the C. reinhardtii genome, it was often difficult to identify the location of the inserted DNA and the gene disrupted by the insertion. We have developed a transformation protocol using the BleR gene, which confers resistance to the antibiotic Zeocin. The insertion of this gene allows one to use a variety of existing polymerase chain reaction (PCR) methodologies to identify the disrupted gene. In this study the D66 strain (nit2-, cw15, mt+) was transformed by electroporation using a plasmid containing the BleR gene. Primary transformants (42 000) were obtained after growth in the dark on acetate plus Zeocin medium. Colonies were then tested for their ability to grow photosynthetically on elevated CO2 or low levels of CO2 (100 ppm). About 120 mutants were identified which grew on elevated CO2 but were unable to grow well at low CO2 concentrations. About 50% of these mutants had low affinities for inorganic carbon as assessed by K0.5(CO2), indicating a potential defect in the CCM. The location of the inserted DNA is being determined using inverse PCR (iPCR) and thermal asymmetric interlaced (TAIL) PCR. Using these methods, one can rapidly locate the inserted DNA in the genome and identify the gene that has been disrupted by the insertion.

A rapid method for chloroplast isolation from the green alga Chlamydomonas reinhardtii

This method has been developed to yield highly purified intact chloroplasts from Chlamydomonas reinhardtii. This procedure involves breaking cell-wall-deficient cells by passage through a narrow-bore syringe needle and purifying the intact chloroplasts by differential centrifugation and Percoll gradient centrifugation. This procedure can be completed in less than 3 h and is capable of generating relatively high yields of chloroplasts that should be useful for researchers studying the biochemistry and cell biology of C. reinhardtii chloroplasts.

Membrane lipid biosynthesis in Chlamydomonas reinhardtii: expression and characterization of CTP:phosphoethanolamine cytidylyltransferase

CTP:phosphoethanolamine cytidylyltransferase (ECT) is considered to be the regulatory enzyme in the CDP-ethanolamine pathway of phosphatidylethanolamine (PE) biosynthesis. The ECT cDNA of Chlamydomonas reinhardtii encodes a protein of 443 amino acid residues, which is longer than the same protein in yeast, rat or human. The translated product of cloned cDNA was expressed as a fusion protein in Escherichia coli, and was shown to have ECT activity. The deduced amino acid sequence has 41% identity with that of human or rat, and 30% with yeast. The ECT protein has a repetitive internal sequence in its N- and C-terminal halves and a signature peptide sequence, RTXGVSTT, typical of the cytidylyltransferase family. The first 70 amino acid residues do not match the N-terminal part of the cytidylyltransferases from other organisms, and we hypothesize that it is a subcellular targeting signal to mitochondria. ECT and organelle marker enzyme assays showed that the total activity of ECT correlates well with that of fumarase, a marker enzyme for mitochondria. Northern blots showed an increase in mRNA abundance during reflagellation, indicating a possibility of transcriptional regulation. A notable change in the enzyme activity in C. reinhardtii cells was observed during the cell cycle, increasing during the dark and then decreasing during the light period, while the mRNA level did not alter, providing evidence for post-translational regulation.

A mutant of Chlamydomonas reinhardtii that cannot acclimate to low CO2 conditions has an insertion in the Hdh1 gene

Photosynthetic microorganisms must acclimate to environmental conditions, such as low CO2 environments or high light intensities, which may lead to photo-oxidative stress. In an effort to understand how photosynthetic microorganisms acclimate to these conditions, Chlamydomonas reinhardtii was transformed using the BleR cassette, selected for Zeocin resistance and screened for colonies that showed poor growth at low CO2 levels. One of the insertional mutants obtained, named slc-230, was shown to have a BleR insert in the first exon of Hdh1, a novel, single copy gene. The predicted Hdh1 gene product has similarity to bacterial haloacid dehalogenase-like proteins, a protein family that includes phosphatases and epoxide hydrolases. In addition, Hdh1 is predicted to be localised to the chloroplast or mitochondria in C. reinhardtii. It was found that a genomic copy of wild type Hdh1 can complement slc-230.

Naturally occurring high oleic acid cottonseed oil: identification and functional analysis of a mutant allele of Gossypium barbadense fatty acid desaturase-2

AbstractMain conclusionSome naturally occurring cotton accessions contain commercially attractive seed oil fatty acid profiles. The likely causal factor for a high-oleate trait in pima cotton (Gossypium barbadense) accession GB-713 is described here. Vegetable oils are broadly used in the manufacture of many human and animal nutritional products, and in various industrial applications. Along with other well-known edible plant oils from soybean, corn, and canola, cottonseed oil is a valuable commodity. Cottonseed oil is a co-product derived from the processing of cottonseed fiber. In the past, it was used extensively in a variety of food applications. However, cottonseed oil has lost market share in recent years due to less than optimal ratios of the constituent fatty acids found in either traditional or partially hydrogenated oil. Increased awareness of the negative health consequences of dietary trans-fats, along with the public wariness associated with genetically modified organisms has created high demand for naturally occurring oil with high monounsaturate/polyunsaturate ratios. Here, we report the discovery of multiple exotic accessions of pima cotton that contain elevated seed oil oleate content. The genome of one such accession was sequenced, and a mutant candidate fatty acid desaturase-2 (FAD2-1D) gene was identified. The mutant protein produced significantly less linoleic acid in infiltrated Arabidopsis leaf assays, compared to a repaired version of the same enzyme. Identification of this gene provides a valuable resource. Development of markers associated with this mutant locus will be very useful in efforts to breed the high-oleate trait into agronomic fiber accessions of upland cotton.

Adaptation of Chlamydomonas Reinhardtii to Limiting CO 2 Conditions

The unicellular green alga Chlamydomonas reinhardtii can adapt to limiting CO2 conditions through the action of a CO2 concentrating mechanism (CCM). When grown on elevated CO2 (10 to 100 times ambient levels of CO2), C. reinhardtii exhibits relatively poor affinity for external inorganic carbon. However if cells grown on elevated CO2 are switched to low CO2 conditions (ambient levels of C2 the alga will adapt to the limiting CO2 environment within four hours. During this adaptation a number of new proteins are synthesized and significant morphological changes occur. We have found that over 90% of the Rubisco within the cell is localized to the pyrenoid when cells are grown on low CO2 conditions. In contrast, in cells grown on elevated CO2 a majority of the Rubisco (60%) is found in the chloroplast stroma. In addition to the redistribution of Rubisco, messages encoding three isoforms of carbonic anhydrase, a cyclophilin and three novel membrane proteins increase under low CO2 conditions. The sequence of one of these novel cDNAs named lci2, will be reported here.