A broad-specificity delta 9 desaturase gene was cloned from the cyanobacterium Anacystis nidulans. The enzyme introduces a cis-double bond at the delta 9 position of both 16 and 18 carbon saturated fatty acids linked to many kinds of membrane lipids. The gene was stably introduced into tobacco plants under transcriptional control of the cauliflower mosaic virus 35S promoter, and the enzyme was targeted into plastids by the transit peptide of the pea RuBisCO small subunit. The transgenic plants had a highly reduced level of saturated fatty acid content in most membrane lipids and exhibited a significant increase in chilling resistance.

Stromal glycerol-3-phosphate acyltransferases (GPAT) are responsible for the selective incorporation of saturated and unsaturated fatty-acyl chains into chloroplast membranes, which is an important determinant of a plant's ability to tolerate chilling temperatures. The molecular mechanisms of plant chilling tolerance were elucidated by creating chimeric GPATs between squash (Cucurbita moscata, chilling-sensitive) and spinach (Spinacea oleracea, chilling-tolerant) and the results were interpreted using structural information on squash GPAT determined by X-ray crystallography at 1.55 A Ê resolution. Enzymatic analysis of the chimeric GPATs showed that the chimeric GPATs containing the spinach region from residues 128 to 187 prefer the 18:1 unsaturated fatty acid rather than 16:0 saturated fatty acid. Structure analysis suggests that the size and character of the cavity that is formed from this region determines the speci®c recognition of acyl chains.

Eggplant seedlings (Solanum melongena) grown under red light irradiation showed a normal morphology with green, fully expanded cotyledons. When the seedlings grown under red light were irradiated with ultraviolet-containing white light, anthocyanin synthesis was induced in the hypocotyl tissues, especially when a UV light supplement was added. The accumulation of pigments was closely associated with the expression of genes involved in flavonoid synthesis. These genes include chalcone synthase (CHS) and dihydroflavonol 4-reductase (DFR). Using subtracted probes, which had been enriched for the accumulated mRNA, one white light-responsive cDNA was identified as being a P450 gene by comparison with database sequences. The maximal amino acid homology this cDNA had with other P450s was 36%. This was with CYP71 from avocado (Persea americana). Thus it represents a new P-450 family, which has been named CYP75. The mRNA of this gene was localized in the hypocotyl tissues of eggplant seedlings, which had been white light-irradiated. The transcript was accumulated by changing the light source, as in the case of other flavonoid biosynthesis genes. In delphinidin producing petunia plants, the mRNAs corresponding to the eggplant P-450 and flavonoid biosynthesis genes such as CHS and DFR were most abundant during the mid stage of flower bud development, but could not be detected in leaf tissues. These results suggest that this P-450 gene encodes a hydroxylating enzyme involved in flavonoid biosynthesis.

Carbonic anhydrase (CA) of Chlamydomonas reinhardtii is a glycoprotein of 35 kDa which is localized outside the plasma membrane. The activity of CA was increased when the C02 concentration during photoautotrophic growth was decreased to air level. After decreasing the C 0 2 concentration from 4% to 0.04%, several polypeptides including CA were induced continuously or transiently. To investigate the biosynthesis and intracellular processing of CA, the cells of wall-less mutant CW-15, which secretes CA into the culture medium, were pulse-labeled with radioactive arginine, chased, and radioactive proteins were immunoprecipitated with anti-CA serum.A 42-kDa polypeptide with isoelectric point (pl) of 7.1 -7.3 was first synthesized. Within S min the molecular mass of this polypeptide was decreased to 35 kDa and it was then secreted into the culture medium within 30 min. This indicates that the former is the precursor form and the latter the mature form of CA. The primary translation product from poly(A)-rich RNA in a cell-free reticulocyte lysate system from a rabbit was a 38-kDa polypeptide. This was cotranslationally converted into the 42-kDa precursor in vitro in the presence of dog pancreatic microsomal membranes. As the 42-kDa precursor had a high affinity to concanavalin A, it was assumed to have a high-mannose-type oligosaccharide. The mature enzyme had a pl of 6.1 -6.2 and was composed of more than two isoforms, which had a complex-type oligosaccharide with low affinity to concanavalin A. Chemical deglycosylation of the mature enzyme by trifluoromethanesulfonic acid indicated that the molecular mass of the polypeptide moiety was 32 kDa and the difference between this and the primary translation product suggests that cleavage of the polypeptide occurs during its biosynthesis.Carbonic anhydrase (CA) catalyzes the reversible hydration of C02. The cells of Chlamydomonas reinhardtii, grown photoautotrophically at air-level C 0 2 (0.04%. low-C02 cells), show a higher affinity for inorganic carbon (Ci) in photosynthesis than those grown at an elevated C 0 2 concentration (4 -5%, high-C02 cells) [I, 21. Correspondingly, the activity of CA increases in accordance with the decrease of C 0 2 concentration from 4% to air level [3,4]. It has also been established that the low-C02 cells can accumulate much more Ci within the algal cells than the high-C02 cells, indicating the operation of a Ci-influx pump in the low-C02 cells [2]. The high affinity for Ci in the low-C02 cells is explained by a higher activity of CA, which facilitates C 0 2 transport [S], and by a higher Ci-concentrating mechanism [2].In Chlamydomonas cells, CA is localized outside the plasma membrane and its activity can be assayed with the suspension of intact cells. Most of the activities are released into the suspending medium by treating the wild-type cells with a gametic wall-lysing enzyme [6] or trypsin [7]. In wallless mutant CW-15 cells, CA is secreted into the culture medium [6]. The CA from both wild-type cells and the culture

We produced transgenic chrysanthemum (Dendranthema grandiflora) plants expressing double-stranded RNA-specific ribonuclease gene (pac1) derived from Schizosaccharomyces pombe using an Agrobacteriummediated transformation method. Three transgenic lines stably expressing Pac1 protein were selected. After inoculation with chrysanthemum stunt viroid (CSVd), these lines showed a decreased frequency of infection, less accumulation of viroid, and attenuation of growth retardance compared with control plants. They also showed a significantly lower infection frequency against tomato spotted wilt virus (TSWV) than in control plants. Hybrid plants between one transgenic line and a wild chrysanthemum species, D. pacificum, were produced to test their susceptibility against TSWV infection. TSWV infected all the plants of the wild species resulting in a high mortality, whereas the hybrid plants expressing Pac1 protein showed either complete resistance or high susceptibility. These results suggest the presence of an endogenous tolerance gene against TSWV in the cultivar used for transformation. The virus infection was suppressed in transgenic plants, indicating that the commercial use of this transgene would limit the spread of viruses to wild populations of Dendranthema. This is the first study to demonstrate that a single tolerance gene in transgenic plants can attenuate both viroid and virus diseases.

Arabidopsis thaliana ⌬-12 fatty acid desaturase gene (FAD2) was overexpressed in Saccharomyces cerevisiae by using the GAL1 promoter. S. cerevisiae harboring the FAD2 gene was capable of forming hexadecadienoyl (16:2) and linoleoyl (18:2) residues in the membrane lipid when cultured in medium containing galactose. Gas-liquid chromatography analysis of total lipids indicated that the transformed S. cerevisiae accumulated these dienoic fatty acyl residues and that they accounted for approximately 50% of the total fatty acyl residues. Phospholipid analysis of this strain indicated that the oleoyl (18:1) residue binding phosphatidylcholine (PC) was mostly converted to the 18:2 residue binding PC, whereas 50% of the palmitoleoyl (16:1) residue binding PC was converted to the 16:2 residue binding PC. A marked effect on the unsaturation of 16:1 and 18:1 was observed when S. cerevisiae harboring the FAD2 gene was cultured at 8؇C. To assess the ethanol tolerance of S. cerevisiae producing polyunsaturated fatty acids, the cell viability of this strain in the presence of ethanol was examined. The results indicated that S. cerevisiae cells overexpressing the FAD2 gene had greater resistance to 15% (vol/vol) ethanol than did the control cells.

Many pathogenic plant viruses are RNA viruses, which initiate production of double-stranded RNA intermediates when they replicate in host plant cells. Introduction of double-stranded RNA-specific ribonucleases such as the Schizosaccharomyces pombe derived pac I protein and animal cell derived interferon-induced 2',5'-oligoadenylate synthetase (2-5 Aase)/ribonuclease L (RNase L) system into various plants may make plants resistant to various pathogenic viruses and viroids. We have demonstrated that pac I and 2-5 Aase/RNase L transgenic tobacco plants are resistant to various viruses including tobacco mosaic virus, cucumber mosaic virus and potato virus Y. In addition, pac I transgenic potato plants are resistant to potato spindle tuber viroid. Using Agrobacterium-mediated transformation, we have established a transformation system for chrysanthemum plants and have recently developed pac I transgenic chrysanthemum (Dendranthema grandiflora cv Reagan) resistant to chrysanthemum stunt viroid and have grown them in isolated fields for an evaluation of their effects.

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