The CaMV-35S promoter is sensitive to shortened photoperiod in transgenic tobacco.

The CaMV-35S promoter is one the most widely used promoters in transgenic plant research because it exhibits a high level of transcriptional activity in a variety of plant tissues. Here, the CaMV-35S promoter fused to the GUS gene was used as a model for constitutive expression in transgenic Nicotiana tabacum (cv 'Xanthi') leaves. The transgenic plants were placed under a shortened photoperiod to determine if GUS expression changed; measurements were made using fluorometry and ribonuclease protection assays. When the plants were moved from a 16 : 8-h photoperiod to a 8 : 16-h photoperiod, an increase in both specific GUS activity and gus RNA levels was observed, and these levels decreased upon returning to the 16 : 8-h photoperiod. These results indicate that photoperiod plays an important role in the regulation of the CaMV-35S promoter. Studies involving a comparison of this promoter to others should be limited to constant photoperiod conditions.

Expression of a synthetic antifreeze protein in potato reduces electrolyte release at freezing temperatures.

A synthetic antifreeze protein gene was expressed in plants and reduced electrolyte leakage from the leaves at freezing temperatures. The synthetic AFP was expressed as a fusion to a signal peptide, directing it to the extracytoplasmic space where ice crystallization first occurs. The gene was introduced to Solanum tuberosum L. cv. Russet Burbank by Agrobacterium-mediated transformation. Transformants were identified by PCR screening and expression of the introduced protein was verified by immunoblot. Electrolyte-release analysis of transgenic plant leaves established a correlation between the level of transgenic protein expression and degree of tolerance to freezing. This is the first identification of a phenotype associated with antifreeze protein expression in plant tissue.

Comparison of the beta-toxins from Staphylococcus aureus and Staphylococcus intermedius.

The beta-toxins produced by Staphylococcus aureus and Staphylococcus intermedius were purified to homogeneity from culture supernatants. Although the toxin from S. aureus has been throughly studied, less is known about its unique counterpart from S. intermedius. This is the first reported purification and analysis of the S. intermedius beta-toxin. Both toxins have similar enzymatic properties, belong to the class of neutral sphingomyelinases C, and have a high specificity for sphingomyelin. They also hydrolyze lysophosphatidylcholine at a much slower rate, but have no activity toward phosphatidylcholine, phosphatidylethanolamine, or phosphatidylserine. The kinetic parameters determined for both proteins (apparent Km 1.4 mM, Vmax 100 mmol/min/microg protein) are identical. Despite these similarities, the size and amino acid composition of the two beta-toxins differ. Molecular mass values, determined by electrophoresis and gel filtration, indicate that the both enzymes are single polypeptides. The decrease in sphingomyelinase activity of S. aureus beta-toxin upon pretreatment with dithiothreitol (DTT) indicates the presence of a disulfide bond in the protein. In contrast, DTT has no effect on the enzymatic activity of S. intermedius beta-toxin. This observation is consistent with the absence of detectable cysteine residue in the protein. N-terminal amino acid sequences determined for the first 19 residues of both beta-toxins also differ, only nine of the first 19 residues are identical. Further evidence that the two proteins differ was obtained by immunological analysis which demonstrated crossreactivity but a lack of identity.

Variable expression of the herpes simplex virus thymidine kinase gene in Nicotiana tabacum affects negative selection.

The potentials and limitations of negative-selection systems based on the human herpes simplex virus thymidine kinase type-1 (HSVtk) gene, which causes sensitivity to the nucleoside analog ganciclovir, were examined in tobacco as a model system. There were great differences between individual HSVtk(+) transgenic plants in ganciclovir sensitivity. Inhibition of growth while under selection correlated with HSVtk-tianscnpt levels. Negative selection against HSVtk(+) transformants at the level of Agrobacterium-mediated transformation using a ganciclo-vir/kanamycin double-selection medium (the positive selection marker neomycin phosphotransferase-II gene was in the transformation vector) resulted in a three- to six-fold reduction in the frequency of kanamycin-resistant shoots. The efficiency of negative selection in this case was limited due to the great variation in HSVtk expression, i.e., the frequently occurring transformants with low, or no, ganciclovir sensitivity escaping negative selection. Two independently constructed HSVtk genes showed the same variability of the phenotype in Nicotiana tabacum transformants. Distinct phenotypes, ranging from no regeneration through abnormal or delayed regeneration, were observed when leaf segments were placed on shoot-inducing medium supplemented with 10(-6)-10(-3) M ganciclovir. The highest HSVtk mRNA and ganciclovir sensitivity levels were observed in plants which were transformed with the pSLJ882 chimeric construct. The pSLJ882 plant expression vector carried the coding sequence of HSVtk, whereas plasmid pCX305.1 carried an HSVtk construct retaining the untranslated 5 leader and viral 3 regions. The pCX305.1 transformants showed, at most, a delayed formation of shoots with thin stems and very narrow leaves. Ganciclovir sensitivity showed typical Mendelian segregation. A gene-dosage effect was also seen at the seedling level in the progeny of two transgenic lines.

Biochemical characterization of temperature-induced changes in lipid metabolism in a high oleic acid mutant of Brassica rapa.

We have characterized a Brassica rapa mutant (WR586) that has low levels of polyunsaturated fatty acids (D.L. Auld et al., 1992, Crop Sci. 32, 657-662). The mutant lacked oleoyl-phosphatidylcholine desaturase (ODS) activity when assayed in 6-day-old seedlings. To further characterize the mutant, the leaf fatty acid composition and major galactolipids and phospholipids were characterized in mutant (WR586) and control (cultivar "Tobin") plants grown at either 26 degrees C/26 degrees C or 10 degrees C/5 degrees C. Fatty acid profiles show significantly higher 18:1 levels in WR586 throughout 12 days of germination. The amount of saturated fatty acids decreased with a concomitant increase of 18:1. Ratios of 18:1/18:2 revealed that WR586 maintains higher mole percent of 18:1 than Tobin at all times and temperature regimes because of a lack of desaturation to 18:2. Values for monogalactosyl-diacylglycerol and digalactosyldiacylglycerol 18:1/18:2 ratios indicate a disparity in the concentration of 18:1 between WR586 and Tobin grown in either temperature during early germination. The phosphatidylcholine and phosphatidylethanolamine 18:1/18:2 ratios were higher in WR586 compared to Tobin and remained higher throughout the 12-day period. In WR586, 18:1 always accumulated to higher levels in the cooler temperature. In Tobin, 18:1 concentrations paralleled 18:2 in both temperature regimes. These results indicate that the lesion in the mutant WR586 resides at the ODS locus, 18:1 synthesis is chilling induced, and the microsomal desaturation pathway is the most prominent in early developing Brassica seedlings.

The Anti-nptII Gene (A Potential Negative Selectable Marker for Plants).

An efficient negative selection procedure is crucial to the isolation of rare homologous recombinants in gene targeting. Although gene targeting is a common practice in lower eukaryotes and is becoming routine in mammals, its application to plants has not been achieved. In this report, we have evaluated an antisense construct against the neomycin phosphotransferase gene (nptII) as a negative selectable marker. The anti-nptII gene construct was able to suppress nptII expression both transiently and in transformed tobacco (Nicotiana tabacum) calli. A construct was made which includes both a hygromycin-resistance gene and the sense plus antisense genes for neomycin phosphotransferase. Hygromy-cin-resistant calli were obtained after Agrobacterium-mediated transformation. Subsequently, hygromycin-resistant calli were tested for kanamycin sensitivity. The growth on kanamycin medium of calli harboring both the sense and antisense gene constructs was retarded, whereas that of control calli transformed with only the sense nptII gene was not inhibited. Southern blot analysis confirmed the presence of both nptII and anti-nptII genes. Northern blot analyses revealed that antisense transcripts of the nptII gene were made and that the level of sense transcripts was greatly reduced in transgenic calli. These results suggest that the anti-nptII gene could potentially be used as a negative selectable marker for gene targeting in plants.

Coenzyme A biosynthesis in plants: partial purification and characterization of pantothenate kinase from spinach.

A study of the biosynthesis of coenzyme A (CoA), a critical cofactor in the metabolism of lipids and other molecules in higher plants, was initiated. Pantothenate kinase was partially purified from spinach leaves. This enzyme was predominantly localized in the chloroplast with very little activity observed in the mitochondria or cytosol. DEAE-agarose chromatography resolved two pantothenate kinase activity peaks which differed in their requirement for reductant, stability upon boiling, and reactivity in the presence of spinach holo-acyl carrier protein (ACP) I. One active peak of this enzyme was further purified on Cibacron blue 3GA to yield a preparation containing pantothenate kinase enriched to 20% of the total protein within the fraction. Pantothenate kinase was inhibited by malonyl-CoA, but not by CoASH or acetyl-CoA, and the activity was stabilized by the phosphatase inhibitors sodium molybdate, sodium tungstate, and the phosphatase substrate glycerol 2-phosphate, but was inhibited by sodium fluoride. Further experiments demonstrated a linear increase in pantothenate kinase activity during spinach seed germination, consistent with a role for this enzyme in the developmental utilization of seed triacylglycerol.

Escherichia coli beta-hydroxydecanoyl thioester dehydrase reacts with native C10 acyl-acyl-carrier proteins of plant and bacterial origin.

beta-Hydroxydecanoyl-[acyl-carrier-protein] dehydrase catalyzes the essential step in the formation of unsaturated fatty acids in Escherichia coli. This reaction was characterized with native C10 acyl-acyl-carrier protein (ACP) structures in both an aqueous phase system and a substrate immobilization assay system. The dehydrase is equally active with E. coli ACP, recombinant ACP-I derived from spinach, or protein A:ACP-I fusion (acyl-thioesters). There were differences among the substrates in terms of the equilibrium product distribution. Both E. coli acyl-ACP and recombinant acyl-ACP-I as cosubstrates with beta-OH 10:0, trans-2 10:1, or cis-3 10:1 yielded about equal amounts (37 mol%) of the two monoenes regardless of the initial substrate. In contrast, the fusion acyl-ACP-I yielded only 17 mol% cis-3 10:1 with 49 mol% trans-2 10:1 present at equilibrium. These equilibrium values for native cis-3 10:1 are higher than those reported previously for the dehydrase using N-acetylcysteamine thioesters as substrates. The Km values for each beta-OH 10:0 ACP substrate were similar to each other and within the range of in vivo concentrations (5-10 microM). Dehydrase reactivity depends more on acyl chain length than ACP structure or origin and is therefore different from other branch point ACP-utilizing enzymes (plant and bacterial) which discriminate according to ACP structure (D. J. Guerra, J. B. Ohlrogge, and M. Frentzen, 1986, Plant Physiol. 82, 448-453).

The recombinant spinach acyl-acyl carrier protein-I expressed in Escherichia coli is the 18:1 delta 11(cis) thioester.

A synthetic spinach acyl carrier protein-I (ACP-I) gene was cloned and expressed in the Escherichia coli beta-alanine auxotroph SJ16 (P. D. Beremand et al. (1987) Arch. Biochem. Biophys. 256, 90-100). After characterization of the transformed cells and purification of the protein product it was evident that 50% of the recombinant spinach ACP-I was acylated during early log-phase growth (D. J. Guerra et al. (1988) J. Biol. Chem. 263, 4386-4391). We have purified the recombinant acyl-acyl carrier protein-I to greater than 90% homogeneity and have made a fatty acid methyl ester of the delipidated and trypsin-treated preparation. We have found that the acyl moiety attached to recombinant spinach acyl carrier protein-I is 18:1 delta 11(cis) (cis-vaccenic acid) a major unsaturated end product of Escherichia coli de novo fatty acid synthesis. This result reflects previous work (D. S. Guerra et al. (1986) Plant Physiol. 82, 448-453) which suggested the acyl carrier protein-I structure has evolved from ancestral ACP structures to accommodate the eukaryotic pathway of lipid synthesis in higher plants. The accumulation of recombinant 18:1 delta 11(cis) acyl carrier protein-I in transformed E. coli SJ16 cells attests to the poor reactivity of this substrate to acyl transferase reactions and may help explain the lack of effect on pools of fatty acids found in vivo.

Expression of an active spinach acyl carrier protein-I/protein-A gene fusion.

A synthetic gene encoding spinach acyl carrier protein I (ACP-I) was fused to a gene encoding the Fc-binding portion of staphylococcal protein A. This gene fusion, under the control of the λPR promoter, was expressed at high levels in Escherichia coli producing a 42 kDa fusion protein. This fusion protein was phosphopantethenylated in E. coli. In vitro the ACP portion of the fusion protein was able to participate in acyl ACP synthetase reactions, plant malonyl-CoA:ACP transacylase (MCT) reactions, and plant fatty acid synthetase (FAS) reactions. Inhibitory effects of high ACP concentrations on in vitro plant FAS were observed with the unfused ACP-1 but not with the fusion protein. As with unfused ACP-I, the fusion protein was a poor substrate for E. coli FAS reactions. When injected into rabbits, the fusion protein was also able to generate antiserum to spinach ACP-I.

Purification and characterization of recombinant spinach acyl carrier protein I expressed in Escherichia coli.

Expression of plant acyl carrier protein (ACP) in Escherichia coli at levels above that of constitutive E. coli ACP does not appear to substantially alter bacterial growth or fatty acid metabolism. The plant ACP expressed in E. coli contains pantetheine and approximately 50% is present in vivo as acyl-ACP. We have purified and characterized the recombinant spinach ACP-I. NH2-terminal amino acid sequencing indicated identity to authentic spinach ACP-I, and there was no evidence for terminal methionine or formylmethionine. Recombinant ACP-I was found to completely cross-react immunologically with polyclonal antibody raised to spinach ACP-I. Recombinant ACP-I was a poor substrate for E. coli fatty acid synthesis. In contrast, Brassica napus fatty acid synthetase gave similar reaction rates with both recombinant and E. coli ACP. Similarly, malonyl-coenzyme A:acyl carrier protein transacylase isolated from E. coli was only poorly able to utilize the recombinant ACP-I while the same enzyme from B. napus reacted equally well with either E. coli ACP or recombinant ACP-I. E. coli acyl-ACP synthetase showed a higher reaction rate for recombinant ACP-I than for E. coli ACP. Expression of spinach ACP-I in E. coli provides, for the first time, plant ACP in large quantities and should aid in both structural analysis of this protein and in investigations of the many ACP-dependent reactions of plant lipid metabolism.

Synthesis, cloning, and expression in Escherichia coli of a spinach acyl carrier protein-I gene.

A synthetic gene of 268 bp encoding the 82 amino acid spinach acyl carrier protein (ACP)-I was constructed based on the known amino acid sequence. Two gene fragments, one encoding the amino-terminal portion and the other the carboxy-terminal portion of the protein, were assembled from synthetic oligonucleotides and inserted into the phage M13mp19. These partial gene constructions were joined and inserted into the plasmid pTZ19R. DNA sequencing confirmed the accuracy of the constructions. The synthetic gene was then subcloned into the Escherichia coli expression vector pKK233-2, under the control of the trc promoter. Western blot analysis and radioimmunoassay indicated that E. coli cells carrying this plasmid produced up to 6 mg/liter of a protein which was immunologically cross-reactive and similar in electrophoretic mobility to authentic spinach acyl carrier protein. The bacterial cells were able to attach the phosphopantetheine prosthetic group to the synthetic plant gene product allowing it to be acylated in vitro by acyl-ACP synthetase.

Activity of acyl carrier protein isoforms in reactions of plant Fatty Acid metabolism.

Two forms of spinach acyl carrier protein (ACP-I and ACP-II) have recently been characterized and found to be expressed in a tissue-specific manner (JB Ohlrogge, TM Kuo, 1985 J Biol Chem 260: 8032). To examine possible different functions for these ACP isoforms, we have tested purified preparations of spinach leaf ACP-I and ACP-II and Escherichia coli ACP in several in vitro reactions of fatty acid metabolism. Total de novo fatty acid synthesis and malonyl-CoA:ACP transacylase do not appear to discriminate between acyl carrier protein isoforms. In contrast, the K(m) of oleoyl-ACP thioesterase for oleoyl-ACP-II is 10-fold higher than for oleoyl-ACP-I, whereas the K(m) of acyl-ACP glycerol-3-phosphate acyl transferase is 5-fold higher for oleoyl-ACP-I than for oleoyl-ACP-II. A characterization of these reactions and a possible role for ACP isoforms in regulation of fatty acid metabolism in plants are described.

Partial purification and characterization of two forms of malonyl-coenzyme A:acyl carrier protein transacylase from soybean leaf tissue.

Investigation of malonyl-CoA:acyl carrier protein transacylase from soybeans has shown that this fatty acid biosynthetic enzyme occurs in at least two isozymic forms. Both forms exist as soluble, low-molecular-mass polypeptides (approx 43 kDa) which catalyze one of the first committed steps in the synthesis of C16 and C18 fatty acids. We have partially purified the two forms of this enzyme from soybean leaf tissue 1200- and 3900-fold respectively. Isozyme 1 does not adhere to ion-exchange or blue dye affinity chromatographic supports and elutes from a polybuffer exchanger column at a pH of 7.3. Isozyme 2 requires salt to be eluted from ion-exchange and affinity matrices and elutes from a polybuffer exchanger column at a pH of 5.3. The two forms of malonyl-CoA:acyl carrier protein transacylase also differ in their sensitivity to catalytic inhibitors, heat treatment, and inhibition by acyl-CoA ester substrates. Both forms utilize malonyl-CoA as the preferred substrate, and polyacrylamide gel electrophoresis of reaction products indicated that malonyl-acyl carrier protein was the major product formed. Analysis of developing soybean seeds indicates that only one form (isozyme 1) is predominant, whereas leaf tissue possesses both isozymes.