Monique Dinant

Differential long-term expression and methylation of the hygromycin phosphotransferase (hph) and β-glucuronidase (GUS) genes in transgenic pearl millet (Pennisetum glaucum) callus

Stable transformation of pearl millet (Pennisetum glaucum) was achieved by microprojectile bombardment with two plasmids carrying the hygromycin phosphotransferase (hph) selectable marker and the β-glucuronidase (GUS) reporter gene. The calli were selected for their resistance to hygromycin. The presence of the hph gene in the transformants was confirmed by Southern hybridization and hygromycin phosphotransferase enzyme assay. The non-selected GUS marker was expressed in 50% of the resistant lines and was detected by DNA blot analysis in the GUS positive lines. Quantitative determination of transgene expression during long-term callus culture showed a stable expression of the hph gene contrasting with a gradual decrease of the GUS activity. This inactivation was related to a progressive methylation of the GUS gene, as demonstrated by DNA analysis with isoschizomeric enzymes responding differentially to methylation of restriction sites and by gene reactivation after treatment with 5-azacytidine.

Characterization of two genes encoding the mitochondrial alternative oxidase in Chlamydomonas reinhardtii.

Abstract Two cDNA clones (AOX1 and AOX2) and the corresponding genes encoding the alternative oxidases (AOXs) from Chlamydomonas reinhardtii were isolated and sequenced. The cDNAs, AOX1 and AOX2, contained open reading frames (ORFs) encoding putative proteins of 360 amino acids and 347 amino acids, respectively. For each of the ORFs, a potential mitochondrial-targeting sequence was found in the 5′-end regions. In comparison to AOX enzymes from plants and fungi, the predicted amino acid sequences of the ORFs showed their highest degree of identity with proteins from Aspergillus niger (38.1% and 37.2%) and Ajellomyces capsulatus (37% and 34.9%). Several residues supposed either to be Fe ligands or to be involved in the ubiquinol-binding site were fully conserved in both C. reinhardtii putative AOX proteins. In contrast, a cysteine residue conserved in the sequences of all higher plants and probably involved in the regulation of the enzyme activity was missing both from the AOX1 and AOX2 amino acid sequences and from protein sequences from various other microorganisms. The transcriptional expression of the AOX1 and AOX2 genes in wild-type cells and in mutant cells deficient in mitochondrial complex III activity was also investigated.

Regulation of the Alternative Oxidase Aox1 Gene in Chlamydomonas reinhardtii . Role of the Nitrogen Source on the Expression of a Reporter Gene under the Control of the Aox1 Promoter

In higher plants, various developmental and environmental conditions enhance expression of the alternative oxidase (AOX), whereas its induction in fungi is mainly dependent on cytochrome pathway restriction and triggering by reactive oxygen species. The AOX of the unicellular green alga Chlamydomonas reinhardtii is encoded by two different genes, the Aox1 gene being much more transcribed than Aox2. To analyze the transcriptional regulation of Aox1, we have fused its 1.4-kb promoter region to the promoterless arylsulfatase (Ars) reporter gene and measured ARS enzyme activities in transformants carrying the chimeric construct. We show that theAox1 promoter is generally unresponsive to a number of known AOX inducers, including stress agents, respiratory inhibitors, and metabolites, possibly because the AOX activity is constitutively high in the alga. In contrast, the Aox1 expression is strongly dependent on the nitrogen source, being down-regulated by ammonium and stimulated by nitrate. Inactivation of nitrate reductase leads to a further increase of expression. The stimulation by nitrate also occurs at the AOX protein and respiratory levels. A deletion analysis of the Aox1 promoter region demonstrates that a short upstream segment (−253 to +59 with respect to the transcription start site) is sufficient to ensure gene expression and regulation, but that distal elements are required for full gene expression. The observed pattern of AOX regulation points to the possible interaction between chloroplast and mitochondria in relation to a potential increase of photogenerated ATP when nitrate is used as a nitrogen source.

Mutations affecting the mitochondrial genes encoding the cytochrome oxidase subunit I and apocytochrome b of Chlamydomonas reinhardtii

Mitochondrial mutants of the green alga Chlamydomonas reinhardtii that are inactivated in the cytochrome pathway of respiration have previously been isolated. Despite the fact that the alternative oxidase pathway is still active the mutants have lost the capacity to grow heterotrophically (dark + acetate) and display reduced growth under mixotrophic conditions (light + acetate). In crosses between wild-type and mutant cells, the meiotic progeny only inherit the character transmitted by the mt− parent, which indicates that the mutations are located in the 15.8 kb linear mitochondrial genome. Two new mutants (dum-18 and dum-19) have now been isolated and characterized genetically, biochemically and at the molecular level. In addition, two previously isolated mutants (dum-11 and dum-15) were characterized in more detail. dum-11 contains two types of deleted mitochondrial DNA molecules: 15.1 kb monomers lacking the subterminal part of the genome, downstream of codon 147 of the apocytochrome b (COB) gene, and dimers resulting from head-to-head fusion of asymmetrically deleted monomers (15.1 and 9.5 kb DNA molecules, respectively). As in the wild type, the three other mutants contain only 15.8 kb mitochondrial DNA molecules. dum-15 is mutated at codon 140 of the COB gene, a serine (TCT) being changed into a tyrosine (TAC). dum-18 and dum-19 both inactivate cytochrome c oxidase, as a result of frameshift mutations (addition or deletion of 1 bp) at codons 145 and 152, respectively, of the COX1 gene encoding subunit I of cytochrome c oxidase. In a total of ten respiratory deficient mitochondrial mutants characterized thus far, only mutations located in COB or COXI have been isolated. The possibility that the inactivation of the other mitochondrial genes is lethal for the cells is discussed.

Somatic embryogenesis in pearl millet (Pennisetum glaucum): Strategies to reduce genotype limitation and to maintain long-term totipotency

Three genotypes of Pearl millet were screened in vitro for induction of embryogenic callus, somatic embryogenesis and regeneration. Shoot apices excised from in vitro germinated seedlings or immature embryos isolated from green house established plants were used as primary explants. The frequency of embryogenic callus initiation was significantly higher in shoot apices in comparison with immature zygotic embryos. Moreover, differences between genotypes were minimal when using shoot apices. Friable embryogenic calli (type II) developed on the initial nodular calli after 1 to 3 months of culture. The frequency of type II callus is related to the composition of the maintenance medium and they were more often found in ageing cultures. The transfer of embryogenic calli onto auxin-free medium was sufficient for inducing somatic embryo development in short-term culture (3 months) while a progressive loss in regeneration potential was observed with increasing time of subcultures. Maturation of embryogenic calli on medium supplemented with activated charcoal, followed by germination of somatic embryos on medium supplemented with gibberellic acid, restored regeneration in long-term cultures.

Proteomic and functional characterization of a Chlamydomonas reinhardtii mutant lacking the mitochondrial alternative oxidase 1.

In the present work, we have isolated by RNA interference and characterized at the functional and the proteomic levels a Chlamydomonas reinhardtii strain devoid of the mitochondrial alternative oxidase 1 (AOX1). The AOX1-deficient strain displays a remarkable doubling of the cell volume and biomass without alteration of the generation time or change in total respiratory rate, with a significantly higher ROS production. To identify the molecular adaptation underlying these observations, we have carried out a comparative study of both the mitochondrial and the cellular soluble proteomes. Our results indicate a strong up-regulation of the ROS scavenging systems and important quantitative modifications of proteins involved in the primary metabolism, namely an increase of enzymes involved in anabolic pathways and a concomitant general down-regulation of enzymes of the main catabolic pathways.

Transgenic Pearl Millet (Pennisetum glaucum)

Pearl millet (Pennisetum glaucum), a robust bunch grass cereal, occupies an estimated 40 million ha of the earth’s surface. Although growing in most continents of the world, the largest cultures of pearl millet are found in the arid regions of India and Pakistan. Pearl millet grows also in most African countries, but assumes its greatest importance as a cereal for human consumption in West Africa between the Sahara and the tropical forest. Pearl millet is unusually tolerant to drought and heat and has the ability to grow and produce grains on sandy, rocky soils that are too infertile, dry, acidic, or saline to cultivate maize, sorghum, or rice. Primarily used as a grain crop, pearl millet supplies 80 to 90% of the calories for millions of people in the world.

Interactions between Photosynthetic Pigments Bound to Lipid and Protein Particles. Spectroscopic Properties

Abstract The spectroscopic (visible) properties of pigment-bearing lipid and protein particles extract ed from milk show that: 1) chlorophylls a and b bound to separate particles can form aggregates provided their relative concentration is high enough. Neither pheophytin a nor β-carotene, in the same conditions, form observable aggregates. 2) Chlorophylls a and b can co-aggregate when they are bound to the same particles. Pheophytin a as well as β-carotene seem to prevent the aggregation of chlorophyll a. β-carotene has no effect on the aggregation of chlorophyll b.