Stefan Leu

Cloning and molecular characterization of a novel acyl‐CoA:diacylglycerol acyltransferase 1‐like gene (PtDGAT1) from the diatom Phaeodactylum tricornutum

We have identified and isolated a cDNA encoding a novel acyl‐CoA:diacylglycerol acyltransferase (DGAT)1‐like protein, from the diatom microalga Phaeodactylum tricornutum (PtDGAT1). The full‐length cDNA sequences of PtDGAT1 transcripts revealed that two types of mRNA, PtDGAT1short and PtDGAT1long, were transcribed from the single PtDGAT1 gene. PtDGAT1short encodes a 565 amino acid sequence that is homologous to several functionally characterized higher plant DGAT1 proteins, and 55% identical to the putative DGAT1 of the diatom Thalassiosira pseudonana, but shows little homology with other available putative and cloned algal DGAT sequences. PtDGAT1long lacks several catalytic domains, owing to a 63‐bp nucleotide insertion in the mRNA containing a stop codon. Alternative splicing consisting of intron retention appears to regulate the amount of active DGAT1 produced, providing a possible molecular mechanism for increased triacylglycerol (TAG) biosynthesis in P. tricornutum under nitrogen starvation. DGAT mediates the last committed step in TAG biosynthesis, so we investigated the changes in expression levels of the two types of mRNA following nitrogen starvation inducing TAG accumulation. The abundance of both transcripts was markedly increased under nitrogen starvation, but much less so for PtDGAT1short. PtDGAT1 activity of PtDGAT1short was confirmed in a heterologous yeast transformation system by restoring DGAT activity in a Saccharomyces cerevisiae neutral lipid‐deficient quadruple mutant strain (H1246), resulting in lipid body formation. Lipid body formation was only restored upon the expression of PtDGAT1short, and not of PtDGAT1long. The recombinant yeast appeared to display a preference for incorporating saturated C16 and C18 fatty acids into TAG.

Cell cycle-dependent transcriptional and post-transcriptional regulation of chloroplast gene expression in Chlamydomonas reinhardtii

The regulated expression of five chloroplast genes in Chlamydomonas reinhardtii during a 24 hour cell cycle (12 hours light, 12 hours dark) was analyzed. Transcription rates of the genes encoding the two reaction center proteins of Photosystem I (psaA, psaB), the subunits alpha and beta (atpA, atpB) of chloroplast ATP synthase and for chloroplast elongation factor tu (EF-tu) were measured during the cell cycle. All genes are maximally transcribed at the beginning of the light period. Transcription was induced before the onset of illumination by a light-independent mechanism. Transcript abundance of the same genes during the cell cycle was determined by quantification of Northern blots hybridized with gene-specific probes. The atpA, atpB and psaB mRNAs were most abundant in the first 6 hours of the light period and decreased to about 15% of maximum in the dark. The abundance of psaA mRNA showed less variation and was maximal around the middle of the cell cycle. The EF-tu mRNA showed a maximum early in the light period, but decreased to almost undetectable levels in the second half of the light period. Because of the similar transcriptional patterns observed, the differential steady state levels of these chloroplast transcripts appeared to be regulated at the post-transcriptional level.

Catalytic properties and sensitivity to tentoxin of Chlamydomonas reinhardtii ATP synthases changed in codon 83 of atpB by site-directed mutagenesis.

The participation of the amino acid β83 in determining the sensitivity of chloroplast ATP synthases to tentoxin was reported previously. We have changed codon 83 of the Chlamydomonas reinhardtii atpB gene by site-directed mutagenesis to further examine the role of this amino acid in the response of the ATP synthase to tentoxin and in the mechanism of ATP synthesis and hydrolysis. Amino acid β83 was changed from Glu to Asp (βE83D) and to Lys (βE83K), and the highly conserved tetrapeptide βT82-E83-G84-L85 (ΔTEGL) was deleted. Mutant strains were produced by particle gun transformation of atpB deletion mutants cw15ΔatpB and FUD50 with the mutated atpB genes. The transformants containing the βE83D and βE83K mutant genes grew well photoautotrophically. The ΔTEGL transformant did not grow photoautotrophically, and no CF1 subunits were detected by immunostaining of Western blots using CF1 specific antibodies. The rates of ATP synthesis at clamped ΔpH with thylakoids isolated from cw15 and the two mutants, βE83D and βE83K, were similar. However, only the phosphorylation activity of the mutant βE83D was inhibited by tentoxin with 50% inhibition attained at 4 μM. These results confirm that amino acid β83 is critical in determining the response of ATP synthase to tentoxin. The rates of the latent Mg-ATPase activity of the CF1s isolated from cw15, βE83D, and βE83K were similar and could be enhanced by heat, alcohols, and octylglucoside. As in the case of the membrane-bound enzyme, only CF1 from the βE83D mutant was sensitive to tentoxin. A lower alcohol concentration was required for optimal stimulation of the ATPase of the βE83K-CF1 than that of CF1 from the other two strains. Moreover, the optimal activity of the βE83K-CF1 was also lower. These results suggest that introduction of an amino acid with a positively charged side chain in position 83 in the “crown” domain affects the active conformation of the CF1-ATPase.

Downregulation of a putative plastid PDC E1α subunit impairs photosynthetic activity and triacylglycerol accumulation in nitrogen-starved photoautotrophic Chlamydomonas reinhardtii

Summary Impaired carbon precursor supply through cpPDC is detrimental for TAG biosynthesis in Chlamydomonas reinhardtii under conditions of photoautotrophy and nitrogen starvation.

SYNTHESIS OF EF-TU AND DISTRIBUTION OF ITS MRNA BETWEEN STROMA AND THYLAKOIDS DURING THE CELL CYCLE OF CHLAMYDOMONAS REINHARDII

In Chlamydomonas reinhardii the elongation factor EF-Tu is encoded in the chloroplast DNA. We identified EF-Tu in the electrophoretic product pattern of chloroplast-made proteins and showed that this protein is only synthesized in the first half of the light period in synchronized cells. The newly synthesized EF-Tu contributed little to the almost invariable content of EF-Tu in chloroplasts during the light period of the cell cycle. However, increasing cell volume and the lack of EF-Tu synthesis in the second half of the light period led to a decrease in the concentration of EF-Tu in chloroplasts. At different times in the vegetative cell cycle, the RNA was extracted from whole chloroplasts and from free and thylakoid-bound chloroplast polysomes. The content of mRNA of EF-Tu in chloroplasts and the distribution between stroma and thylakoids were determined. During the light period, the content of the mRNA for EF-Tu varied in parallel to the rate of EF-Tu synthesis. However, in the dark, some mRNA was present even in the absence of EF-Tu synthesis. Most of the mRNA was bound to thylakoids during the whole cell cycle. This suggests that synthesis of EF-Tu is associated with thylakoid membranes.

Development of a Nuclear Transformation System for Oleaginous Green Alga Lobosphaera (Parietochloris) incisa and Genetic Complementation of a Mutant Strain, Deficient in Arachidonic Acid Biosynthesis

Microalgae are considered a promising source for various high value products, such as carotenoids, ω-3 and ω-6 polyunsaturated fatty acids (PUFA). The unicellular green alga Lobosphaera (Parietochloris) incisa is an outstanding candidate for the efficient phototrophic production of arachidonic acid (AA), an essential ω-6 PUFA for infant brain development and a widely used ingredient in the baby formula industry. Although phototrophic production of such algal products has not yet been established, estimated costs are considered to be 2–5 times higher than competing heterotrophic production costs. This alga accumulates unprecedented amounts of AA within triacylglycerols and the molecular pathway of AA biosynthesis in L. incisa has been previously elucidated. Thus, progress in transformation and metabolic engineering of this high value alga could be exploited for increasing the efficient production of AA at competitive prices. We describe here the first successful transformation of L. incisa using the ble gene as a selection marker, under the control of the endogenous RBCS promoter. Furthermore, we have succeeded in the functional complementation of the L. incisa mutant strain P127, containing a mutated, inactive version of the delta-5 (Δ5) fatty acid desaturase gene. A copy of the functional Δ5 desaturase gene, linked to the ble selection marker, was transformed into the P127 mutant. The resulting transformants selected for zeocine resistant, had AA biosynthesis partially restored, indicating the functional complementation of the mutant strain with the wild-type gene. The results of this study present a platform for the successful genetic engineering of L. incisa and its long-chain PUFA metabolism.

The C. reinhardtii CF1 with the mutation βT168S has high ATPase activity

We have generated the mutation T168S in the β subunit of the chloroplast ATP synthase complex of Chlamydomonas reinhardtii by site directed mutagenesis and chloroplast transformation. CF1 and the α3β3γ complex of this mutant strain were isolated and their enzymatic activities were characterized and compared to those of the corresponding wild type complexes. Without activation the mutant CF1 exhibits MgATPase activity with at least 10 times higher rates than the wild type enzyme. The MgATPase activity could be stimulated to some extent by methanol, but less by ethanol and octylglucoside. The α3β3γ complex had an even higher MgATPase activity, which was only slightly enhanced by ethanol or methanol. The ATPase activities of the mutant complexes, like those of the wild type complexes, displayed a sharp concentration optimum for Mg2+. Free ADP inhibited neither the mutant nor the wild type ATPase significantly. Azide, which strongly inhibited the ATPase activity of the wild type enzyme, inhibited the mutant enzyme only at an about 30 times higher concentration suggesting that the mutation T168S prevents trapping of a tightly bound MgADP by a catalytic site that regulates chloroplast ATPase activity. The mutant cells grew photoautotrophically at a growth rate of about 50%. Similar to the wild type the cells survived on minimal medium in the dark. Under heterotrophic conditions with acetate as energy and carbon source the mutant cells grew much faster than the wild type cells, but the chlorophyll content per cell decreased dramatically.

Isolation of CF0CF1 from Chlamydomonas reinhardtii cw15 and the N-terminal amino acid sequences of the CF0CF1 subunits

CF0CF1 was isolated from chloroplasts of the cell wall‐deficient Chlamydomonas reinhardtii strain cw15. The subunit pattern was analyzed by SDS‐gel electrophoresis and the N‐terminal amino acid sequences of all nine subunits were determined by microsequencing. The amino acid sequences of subunits α, β, γ and ϵ match with those derived from the corresponding Chlamydomonas DNA sequences. In variance with the previously assumed N‐terminus of β; however, it was found that the first 11 amino acids are lacking. The subunits δ, I, II, III and IV were identified by comparison with known sequences of homologous polypeptides of higher plant chloroplasts and cyanobacteria, respectively.

The Effect of Planting Techniques on Arid Ecosystems in the Northern Negev

Forestation is a widely accepted way to combat desertification. This approach can have tremendous beneficial effects on soil and environment. The United Nations Food and Agriculture Organization recommended Acacia victoriae for rehabilitation of degraded arid environments. For that purpose areas in the Northern Negev were planted with Acacia victoriae in the period of 1990–1993. The planting techniques were: sparse plantings (Contour trenching and Savanna), and dense planting of woodland. We divided each of those treatments into planted and control plots. In the plots planted by contour trenching the values of annual biomass per area, nutrient and Soil Organic Carbon (SOC) contents underneath the trees canopies were the lowest, while those values in the planted savanna and woodland plots were significantly higher. Contour trenching also harmed the soil by causing erosion, and decreasing the soils water holding capacity. Therefore, Woodland and Savanna plantings should be preferred over contour trenching in arid areas.

Microalgae as a Source for VLC-PUFA Production.

Microalgae present a huge and still insufficiently tapped resource of very long-chain omega-3 and omega-6 polyunsaturated fatty acids (VLC-PUFA) for human nutrition and medicinal applications. This chapter describes the diversity of unicellular eukaryotic microalgae in respect to VLC-PUFA biosynthesis. Then, we outline the major biosynthetic pathways mediating the formation of VLC-PUFA by sequential desaturation and elongation of C18-PUFA acyl groups. We address the aspects of spatial localization of those pathways and elaborate on the role for VLC-PUFA in microalgal cells. Recent progress in microalgal genetic transformation and molecular engineering has opened the way to increased production efficiencies for VLC-PUFA. The perspectives of photobiotechnology and metabolic engineering of microalgae for altered or enhanced VLC-PUFA production are also discussed.