Yasushi Tasaka

Targeted mutagenesis of acyl-lipid desaturases in Synechocystis: Evidence for the important roles of polyunsaturated membrane lipids in growth, respiration and photosynthesis

Acyl‐lipid desaturases introduce double bonds (unsaturated bonds) at specifically defined positions in fatty acids that are esterified to the glycerol backbone of membrane glycerolipids. The desA, desB and desD genes of Synechocystis sp. PCC 6803 encode acyl‐lipid desaturases that introduce double bonds at the delta12, omega3 and delta6 positions of C18 fatty acids respectively. The mutation of each of these genes by insertion of an antibiotic resistance gene cartridge completely eliminated the corresponding desaturation reaction. This system allowed us to manipulate the number of unsaturated bonds in membrane glycerolipids in this organism in a step‐wise manner. Comparisons of the variously mutated cells revealed that the replacement of all polyunsaturated fatty acids by a monounsaturated fatty acid suppressed growth of the cells at low temperature and, moreover, it decreased the tolerance of the cells to photoinhibition of photosynthesis at low temperature by suppressing recovery of the photosystem II protein complex from photoinhibitory damage. However, the replacement of tri‐ and tetraunsaturated fatty acids by a diunsaturated fatty acid did not have such effects. These findings indicate that polyunsaturated fatty acids are important in protecting the photosynthetic machinery from photoinhibition at low temperatures.

Glycerol-3-phosphate acyltransferase in plants.

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the transfer of an acyl group from an acyl donor to the sn-1 position of glycerol 3-phosphate. The plant cell contains three types of GPAT, which are located in the chloroplasts, mitochondria and cytoplasm, respectively. The enzyme in chloroplasts is soluble and uses acyl-(acyl-carrier protein) as the acyl donor, whereas the enzymes in the mitochondria and the cytoplasm are bound to membranes and use acyl-CoA as the acyl donor. cDNAs for GPAT of chloroplasts have been cloned from several plants, and the gene for the enzyme has been cloned from Arabidopsis thaliana. The amino acid sequences deduced from the nucleotide sequences of cDNAs indicate that the product of translation is a precursor of about 460 amino acid residues, which consists of a leader sequence of about 70 amino acid residues and a mature protein of about 400 residues, with a molecular mass of about 42 kDa. Genetic engineering of the unsaturation of fatty acids has been achieved by manipulation of the cDNA for the GPAT found in chloroplasts and has allowed modification of the ability of tobacco to tolerate chilling temperatures.

Membrane lipid unsaturation modulates processing of the photosystem II reaction-center protein D1 at low temperatures.

The role of membrane lipid unsaturation in the restoration of photosystem II (PSII) function and in the synthesis of the D1 protein at different temperatures after photoinhibition was studied in wild-type cells and a mutant of Synechocystis sp. PCC 6803 with genetically inactivated desaturase genes. We show that posttranslational carboxyl-terminal processing of the precursor form of the D1 protein is an extremely sensitive reaction in the PSII repair cycle and is readily affected by low temperature. Furthermore, the threshold temperature at which perturbations in D1-protein processing start to emerge is specifically dependent on the extent of thylakoid membrane lipid unsaturation, as indicated by comparison of wild-type cells with the mutant defective in desaturation of 18:1 fatty acids of thylakoid membranes. When the temperature was decreased from 33[deg]C (growth temperature) to 18[deg]C, the inability of the fatty acid mutant to recover from photoinhibition was accompanied by a failure to process the newly synthesized D1 protein, which accumulated in considerable amounts as an unprocessed precursor D1 protein. Precursor D1 integrated into PSII monomer and dimer complexes even at low temperatures, but no activation of oxygen evolution occurred in these complexes in mutant cells defective in fatty acid unsaturation.

The gene and the RNA for the precursor to the plastid-located glycerol-3-phosphate acyltransferase of Arabidopsis thaliana

The gene and the RNA from Arabidopsis thaliana for the plastid-located glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) and their encoded product have been studied. The gene (designated ATS1) was isolated by screening a λDASH genomic library for cross-hybridization with a radiolabeled probe prepared from cDNA for GPAT from squash. cDNA clones representing the mRNA were isolated by screening a λZAPII cDNA library for hybridization with a radiolabeled probe prepared from a DNA fragment of ATS1. The nucleotide sequences of the gene and the cDNA were determined, and the 5′ end of the RNA was mapped by primer extension. Sequences similar to the TATA box, polyadenylation sequences and intron-splicing sequences were found at the expected locations. The pre-mRNA was 3288 nucleotides long and contained 5′ and 3′-untranslated sequences of 57 and 442 nucleotides, respectively. The coding sequence of 1377 nucleotides was interrupted by 11 introns of 1412 nucleotides in total and the 3′-untranslated sequence contained another intron of 94 nucleotides. The open-reading frame encoded a polypeptide of 459 amino acid residues, the amino acid sequence of which was highly homologous to those of precursors to plastid-located GPATs from squash and pea. The enzymatic activity of a gene product that was over-produced in Escherichia coli confirmed the indentity of the gene.

Genes for Fatty Acid Desaturases and Choline Oxidase are Responsible for Tolerance to Low-Temperature and Salinity Stresses in Cyanobacteria and Plants

Most cyanobacteria and plants contain high levels of polyunsaturated fatty acids of membrane lipids. We isolated the desA, desB and desC genes of Synechocystis sp. PCC 6803, which encode the Δ12, Δ15 (ω3) and Δ9 desaturases, respectively, of the acyl-lipid type. We disrupted the desA gene and the desD gene (for Δ6 desaturase) in Synechocystis sp. PCC 6803 by insertion of antibiotic resistance gene cartridges. These mutations greatly modified the extent of unsaturation of the fatty acids of membrane lipids. These changes in the unsaturation of membrane lipids altered the tolerance of cyanobacterial cells to low temperature. In higher plants, we isolated a cDNA for acyl-ACP: glycerol-3-phosphate acyltransferase from squash and introduced it into tobacco plants. The transformation modified the level of unsaturation of chloroplastic phosphatidylglycerol, and altered the tolerance of tobacco plants to low temperature. Glycinebetaine, a compatible solute, is synthesized in some halophilic plants. We cloned, from the soil bacterium Arthrobacter globiformis, the codA gene for choline oxidase which converts choline into glycinebetaine. introduced this gene into Synechococcus sp. PCC 7942, the resultant transformants accumulated glycinebetaine to some extent and became tolerant to salinity stresses.

The Cyanobacterial Desaturases: Aspects of Their Structure and Regulation

Desaturases introduce double bonds into fatty acids. They are important in the regulation of the degree of unsaturation of membrane glycerolipids and, thus, in the ability of certain organisms to tolerate low temperatures [1,2,3,4,5]. There are three types of desaturase, as follows. (1) Acyl-CoA desaturases introduce double bonds into fatty acids bound to coenzyme A, and these enzymes are bound to the endoplasmic reticulum in animal, yeast and fungal cells. (2) Acyl-ACP desaturases introduce double bonds into fatty acids that are bound to acyl-carrier protein and they are present in the stroma in plant chloroplasts. (3) Acyl-lipid desaturases introduce double bonds into fatty acids that have been esterified to glycerolipids, and they are bound to the membranes of plant and cyanobacterial cells. This last type of desaturase is the most efficient regulator of the extent of the unsaturation of membrane lipids in response to changes in temperature.

Fatty Acids Unsaturation of Membrane Lipids is Involved in the Tolerance to Salt Stress

Cyanobacteria exhibit considerable tolerance to salt stress and are useful for studies of acclimation and tolerance to such stress. In a previous study we isolated a desA−/desD− mutant strain of Synechocystis sp. PCC 6803 in which the desA and desD genes for the Δl2 and Δ6 desaturases, respectively, had been inactivated by targeted mutagenesis (1). The desA−/desD− cells contain monounsaturated but not polyunsaturated fatty acids, whereas wild-type cells contain polyunsaturated fatty acids such as di-, tri-, and tetraunsaturated fatty acids (2,3). In the present study, we investigated the contribution of the unsaturation of fatty acids in membrane lipids to tolerance to salt stress by comparing desA − /desD − cells to wild-type cells of Synechocystis sp. PCC 6803. We demonstrated that the unsaturation of fatty acids is associated with the ability of the photosynthetic machinery to tolerate salt stress.