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The hormones gibberellins (GAs) have profound biological effects on several aspects of plant growth. The genes encoding enzymes of GA metabolism and proteins of GA signaling have been cloned, and their functions well characterized. Notably, progress has been made in our understanding of how GAs are synthesized and decomposed in response to various environmental cues. The homeostasis of endogenous levels of GAs is maintained at the transcriptional level of the genes by feed-back and feed-forward regulatory mechanisms through the signaling pathway. Mutations of each gene result in dwarf or succulent growth phenotypes. Some mutations cause agriculturally important phenotypes, such as ‘green revolution’ semi-dwarf in cereal crops. This indicates that the genes involved in the metabolism and signaling of GAs have the potential to improve economically important crops through the manipulation of their functions in transgenic plants.

Linoleic acid (18:2n-6) and α-linolenic acid (18:3n-3) are polyunsaturated fatty acids that are essential for mammalian nutrition, because mammals lack the desaturases required for synthesis of Δ12 (n-6) and n-3 fatty acids. Many plants can synthesize these fatty acids and, therefore, to examine the effects of a plant desaturase in mammals, we generated transgenic pigs that carried the fatty acid desaturation 2 gene for a Δ12 fatty acid desaturase from spinach. Levels of linoleic acid (18:2n-6) in adipocytes that had differentiated in vitro from cells derived from the transgenic pigs were ≈10 times higher than those from wild-type pigs. In addition, the white adipose tissue of transgenic pigs contained ≈20% more linoleic acid (18:2n-6) than that of wild-type pigs. These results demonstrate the functional expression of a plant gene for a fatty acid desaturase in mammals, opening up the possibility of modifying the fatty acid composition of products from domestic animals by transgenic technology, using plant genes for fatty acid desaturases.

The Saccharomyces cerevisiae MID1 gene encodes a stretch-activated Ca 2+ -permeable channel. In a protein database, we found a Schizosaccharomyces pombe gene whose predicted protein shows 26% identical and 62% similar to the Mid1 channel in amino acid sequence. cDNA derived from this gene, designated yam8 + , was isolated by reverse transcription-polymerase chain reaction (RT-PCR). Further analysis showed that the Yam8 protein consists of 486 amino acids and has 6 hydrophobic segments. The yam8 + cDNA, placed under the S. cerevisiae TDH3 promoter, partially complemented the mating pheromone-induced death ( mid ) phenotype of the S. cerevisiae mid1 mutant. The expression of the yam8 + cDNA in the mid1 mutant cells partially remediated the mid phenotype and resulted in a slight increase in Ca 2+ uptake activity. These findings suggest that Yam8 is a potential homologue of Mid1.

The role of unsaturated fatty acids in membrane lipids in the tolerance of the photosynthetic machinery to salt stress was studied by comparing thedesA−/desD−mutant ofSynechocystissp. PCC 6803, which contained monounsaturated fatty acids, with the wild-type strain, which contained a full complement of polyunsaturated fatty acids. In darkness, the loss of oxygen-evolving photosystem II activity in the presence of 0.5 M NaCl or 0.5 M LiCl was much more rapid indesA−/desD−cells than in wild-type cells. Oxygen-evolving activity that had been lost during incubation with 0.5 M NaCl in darkness returned when cells were transferred to conditions that allowed photosynthesis or respiration. Recovery was much greater in wild-type than indesA−/desD−cells, and it was prevented by lincomycin. Thus, the unsaturation of fatty acids is important in the tolerance of the photosynthetic machinery to salt stress. It appears also that the activity and synthesis of the Na+/H+antiporter system might be suppressed under high-salt conditions and that this effect can be reversed, in part, by the unsaturation of fatty acids in membrane lipids.

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.

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 lambda DASH 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 lambda ZAPII 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 identity of the gene.

THE chilling sensitivity of plants is closely correlated with the degree of unsaturation of fatty acids in the phosphatidylglycerol of chloroplast membranes1–5. Plants with a high proportion of cis-unsaturated fatty acids, such as spinach and Arabidopsis thaliana, are resistant to chilling, whereas species like squash with only a small proportion are not. The chloroplast enzyme glycerol-3-phosphate acyltransferase seems to be important for determining the level of phosphatidylglycerol fatty acid unsaturation6–9. Here we report that the level of fatty acid unsaturation of phosphatidylglycerol and the degree of chilling sensitivity of Nicotiana tabacum var. Samsum (tobacco) can be manipulated by transformation with complementary DNAs for glycerol-3-phosphate acyltransferases from squash and Arabidopsis. The genetic manipulation of fatty acid unsaturation is known to alter the chilling sensitivity of prokaryotes10, and we have now demonstrated that it can also do so in higher plants.

Eustoma grandiflorum Shinn requires vernalization for the induction of stem elongation and flowering. To investigate the role of gibberellins (GAs) in vernalization, the expression levels of genes encoding enzymes of GA biosynthesis, copalyl diphosphate synthetase, GA 20-oxidase and GA 3 beta-hydroxylase, were examined using two culitvars that show different responses to vernalization. The three genes were induced in a vernalization- and a cultivar-dependent manner. EgSPY, a putative negative regulator of GA signal transduction, was also induced during the vernalization period. The results suggest that the expression of the genes encoding GAs biosynthesis is regulated by vernalization. We postulate that EgSPY functions as a negative regulator of GA signal transduction during vernalization, inhibiting adventitious shoot elongation during vernalization.

In order to study the relationship between GSH and flowering, wild-type and late-flowering mutant, fca-1, of Arabidopsis thaliana were treated with L-buthionine sulfoximine (BSO), a specific inhibitor of GSH biosynthesis, under long-day conditions. BSO treatment of the fca-1 mutant starting at 17 d after imbibition promoted flowering. However, when the treatment was started at 12 d after imbibition, BSO treatment at 10(-4) M resulted in an inhibition of flowering. This inhibitory effect of BSO on flowering was abolished by GSH treatment at 10(-4) M, although GSH treatment at an increased concentration of 10(-3) M clearly delayed flowering. In contrast, BSO treatment of wild-type plants starting at 12 d after imbibition promoted flowering, whose effect was abolished by GSH application. In the fca-1 mutant, whose endogenous GSH levels were high, chilling treatment lowered the GSH levels and promoted flowering, as was the case in the BSO treatment. An A. thaliana mutant, cad2-1, which has a defect in GSH biosynthesis also exhibited late flowering. The late-flowering phenotype of this mutant tended to be strengthened by BSO and abolished by GSH treatment. These results suggest that flowering is associated with the rate of GSH biosynthesis and/or the levels of GSH in A. thaliana.

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.

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.

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.

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.

Nature 356, 710-713 (1992) THIS Letter in the 23 April issue contains errors in the figure legends, involving the definition of parts b, c and d. Figure 1 legend should begin "Chilling-induced damage to the photosyn-thetic activity of leaves of transgenic plants, a, Transformant with pBI-121 (control; Clontech).

In the present study, we analyzed the fatty acids of PG from leaves of temperate-zone evergreens, tropical evergreens and temperate-zone deciduous plants. The results are discussed in terms of estimated levels of various molecular species of the PG. It appears that the sum of the levels of saturated and trans-monounsaturated molecular species, as a proportion of the total in the PG in leaves of evergreens, is correlated with the chilling sensitivity of leaves, as previously demonstrated in herbaceous plants