Rodolphe Schantz

Is membrane potential involved in calmodulin gene expression after external stimulation in plants

In Bidens pilosa (cv. radiata), a non‐injurious stimulus induces a local and transient change in membrane potential, and an injurious stimulus induces a transmitted electrical signal described as the combination of an action potential and a slow wave. We have studied calmodulin gene expression after these stimuli. When the stimulus is non‐injurious, calmodulin mRNA accumulation is only increased in the stimulated region. In contrast, when the stimulus is injurious, mRNA accumulation takes place in both wounded and distant, unwounded tissue. We propose that the slow wave plays a role in the long‐distance transmission of a wound‐induced information in plants.

Characterization of cDNA sequences for LHCI apoproteins in Euglena gracilis: The mRNA encodes a large precursor containing several consecutive divergent polypeptides

SummaryScreening of a λgt11 cDNA expression library with antibodies directed against LHCII allowed the detection of several clones which differ markedly from the previously described cDNAs encoding LHCII apoproteins. A Northern analysis revealed a transcript size of 4.2 kb, whereas clones encoding LHCII hybridize with mRNAs in the range of 7.5 kb. Nucleotide sequencing of 2 clones showed open reading frames of 530 and 331 codons, respectively. Within these reading frames, 5 analogous motifs can be delimited corresponding to coding regions for around 180 amino acids (molecular weight, 18–19 kDa). The 5 segments share between 50% and 80% homology. Comparison with a tomato LHCI sequence indicates conserved regions at the two ends, and a central part highly divergent and containing a large deletion. By hybrid-selected translation, followed by immunoprecipitation with a monoclonal antibody directed against LHCI apoprotein, a protein of around 100 kDa is obtained. In vivo, the same antibody recognizes peptides of around 20 kDa. These results, coupled with our previous observations concerning LHCII, confirm that in Euglena at least, some chloroplast proteins encoded in the nucleus are synthesized by large mRNAs containing the information for several consecutive divergent peptides. Implications for processing and chloroplast import are discussed.

Molecular analysis of the transcripts encoding the light-harvesting chlorophyll a/b protein in Euglena gracilis: unusual size of the mRNA

SummaryA cDNA library made from Euglena polyadenylated RNA has been constructed in the expression vector λgt11. The library was screened with antibodies directed against the LHCP apoproteins. Several putative LHCP clones were isolated and analyzed. We present the nucleotide and derived amino-acid sequence of an 800 by long insert containing a poly A tail. On an aminoacid basis, the Euglena LHCP shows a 60% homology with the corresponding pea gene. Northern blot analysis revealed an unusual size of 7.5 kb. This extremely large mRNA is also recognized by a LHCP probe from Chlamydomonas. Hybrid-selected translation produces 2 proteins of 90–110 kd equivalent in size of the polypeptides immunoprecipitated from the translation products of polyadenylated RNAs.

Changes in amino acid and peptide composition of Euglena gracilis cells during chloroplast development

Abstract Amino acid composition of the free amino acid pool, the peptides, and the total protein content was analyzed in Euglena cells during chloroplast development. During this process, it appears that the total content of free amino acids in the resting cells is almost constant. Only a qualitative modification is noted, especially utilization of arginine which represents 50% of this pool. On the other hand, the peptide fraction which is composed essentially of arginine and aspartic acid, decreases markedly during cell greening. This decrease seems to be due to rapid protein turnover in the light and does not contribute entirely to chloroplast protein synthesis. The total protein content of the cells remains fairly constant during chloroplast development and no change either quantitative or qualitative is observed in the amino acid composition of this fraction. It can be concluded that the synthesis of new plastid proteins does not increase the existing protein content of the dark-grown cells. Data obtained by electron microscopy and autoradiography suggest that the newly synthesized chloroplast proteins are formed from amino acids liberated by protein hydrolysis from another cell compartment, especially mitochondria.

Photoregulation of the Synthesis of Chloroplast Membrane Proteins in Euglena gracilis

Summary Antibodies raised against the chlorophyll-binding proteins were used to establish the kinetics of protein synthesis during chloroplast development. The apoproteins, all present in trace amounts in etiolated cells, accumulate rapidly, increasing at least ten to twenty fold during the first 24 hours of cell illumination. However, the protein pattern obtained by in vitro translation of mRNAs isolated from cells at different greening stages remains fairly constant. No correlation exists between the translatable mRNA pool and chloroplast protein synthesis. To further evaluate the mRNA level of light-dependent proteins, we used chloroplast DNA regions encoding polypeptides involved in the reaction center of Photosystem I (PSI) and Photosystem II (PSII) as probes. For nuclear-encoded chloroplast proteins, we selected a cDNA clone encoding a chlorophyll a/b binding protein associated with the light-harvesting complex of PSII. Transcript levels, measured by dot-blot and Northern hybridization of total and polysomal RNAs, increase slightly (2-fold) after the first hour of cell greening and remain almost constant during the following stages of chloroplast biogenesis. Our results strongly suggest that in Euglena, many chloroplast and nuclear genes coding for chloroplast membrane proteins are mainly controlled at a post-transcriptional level. The only exception concerns the expression of the psbA gene coding for the D1 protein: a linear increase of transcript levels in response to light was found in both total and polysomal RNA fractions.

Biosynthesis of galactolipids in Euglena gracilis: I, incorporation of UDP galactose into galactosyldiglycerides

Abstract Proplastids isolated from etiolated dark-grown cells actively incorporated [ 14 C] galactose from uridine diphosphogalactose into galactolipids while neither the presence of galactolipids nor galactosyltransferase activity could be detected in isolated mitochhondria. In green cells, the formation of monogalactosyldiglyceride (MGDG) and digalactosyldiglyceride (DGDG) from uridine diphosphogalactose (UDP gal) was studied in cell-free preparations. The chloroplast envelope shows the highest specific activity and seems to be the major site of galactolipid synthesis. In contrast to higher plant, Euglena chloroplasts incorporate galactose preferentially into digalactosyldiglyceride. The apparent K m value for UDPgal was 14.5 × 10 −6 M for MGDG and 22 × 10 −6 M for DGDG. The differences found for the optimum pH and temperature ranges for the synthesis of the 2 galactolipids seem to indicate that 2 different enzymatic systems are involved in this process but both are tighly membrane bound.

Expression of Polyproteins in Euglena

Abstract In contrast to higher plants and algae, the structure and expression of the genes encoding light‐harvesting chlorophyll (LHC)‐binding proteins and the small subunit of RuBisCO (SSU) are completely different in Euglena cells. In this organism, these two important chloroplast proteins are translated as polyprotein precursors from unusual high molecular weight mRNAs. The structure of the polyproteins consists in consecutive peptides (8 for SSU and 6 to 12 for LHC proteins) separated by a decapeptide motif. Within this motif, 4 amino‐acid residues are invariant, indicating a hypothetical common recognition site for the processing protease(s). This unusual structure implies the existence of novel mechanisms of transport and processing. Light controls the gene expression at a post‐transcriptional level, either by mobilization onto the polysomes or at the translational step, depending on the greening conditions.

Biosynthesis of galactolipids in Euglena gracilis. II, changes in fatty acid composition of galactolipids during chloroplast development

Abstract Linolenic acid is the major fatty acid found in galactolipids of light-grown cells. During cell greening, on resting medium, the C 18 : 2 and C 16 : 2 content of galactolipids increased, while linolenic acid remained a minor component. Labelling experiments provided evidence that the main step in the biosynthesis of linolenic acid is the desaturation of linoleic to linolenic acid. The desaturase in Euglena seems to be regulated by various environmental conditions. The activity of the desaturase was stimulated by addition of NH 4 + to the resting medium or by saturation of the cell suspension with O 2 - or CO 2 -enriched air. The synthesis of linolenic acid in Euglena also depends on the intensity and quality of light. The temperature used for the greening experiments appeared to be the factor that most affected the activity of the desaturase; at 20°C, 33% of the fatty acids in MGDG is linolenic acid whereas at 28°C galactolipids contain only 15% linolenic acid. In contrast to higher plants, Euglena cells can form fully photosynthetic active membranes without simultaneously synthesizing linolenic acid.

Photocontrol of thylakoid protein synthesis in Euglena: differential post-transcriptional regulation depending on nutritional conditions

The expression of three chloroplastic genes, psbA (the gene for the reaction center D1 protein of PSII), psbC (the gene for the P700 apoprotein of PSI) and psaB (the gene for the intermediate antema of PSII), and a nuclear gene, cab (the gene for the chlorophyll a/bbinding protein), has been investigated during chloroplast development in Euglena gracilis Klebs. The polysomal fraction, mRNA stability and protein turnover were analysed under different conditions of cell greening. The results indicate that the main regulatory step for the nuclear and chloroplastic genes was at the translational level when greening of cells took place on a resting medium. When cell greening took place on a medium with balanced phosphate, carbon and nitrogen sources (nutritional medium), the main regulation occurred posttranscriptionally by mobilization of the transcripts onto the polysomes. These results indicate that in E. gracilis, for a given gene, regulation operates at different levels, and that although light is the principal effector in the regulation of the genes involved in chloroplast development, the effect of metabolites should also be considered. Interestingly, it appears that these various regulation levels are similar for the chloroplastic and nucleocytoplasmic compartments.