The gene family encoding the ribulose-(1,5)-bisphosphate carboxylase/oxygenase (Rubisco) small subunit of potato.

We determined the nucleotide sequences of five members of the rbcS gene family encoding the small subunit (SSU) of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) of potato. The genomic organization, structure and expression of the genes is compared to the features of the rbcS genes in tomato. Within the two species, Lycopersicon esculentum and Solanum tuberosum, both members of the Solanaceae, the rbcS genes share more interspecific sequence identity, especially in the 5'- and 3'-untranslated regions and the intron sequences, than within one species. However, the expression data of orthologous rbcS genes containing highly identical regulatory cis-acting elements were found to be different, suggesting that the simple finding of these motifs does not automatically imply similar transcriptional and/or post-transcriptional gene regulation.

A cDNA encoding 3-deoxy-D-manno-oct-2-ulosonate-8-phosphate synthase of Pisum sativum L. (pea) functionally complements a kdsA mutant of the Gram-negative bacterium Salmonella enterica.

Recombinant plasmids encoding 3-deoxy-D-manno-oct-2-ulosonate-8-phosphate (Kdo-8-P) synthase (KdsA; EC 4.1.2.16) were identified from a cDNA library of Pisum sativum L. (pea) by complementing a temperature-sensitive kdsA(ts) mutant of the Gram-negative bacterium Salmonella enterica. Sequence analysis of several inserts revealed a central open reading frame encoding a protein of 290 amino acids with a high degree of amino acid sequence similarity to bacterial KdsA. The cDNA was confirmed by amplifying a 1,812-bp DNA fragment from the chromosome of pea that encoded four exons around the 5'-end of kdsA. The recombinant enzyme was subcloned, overexpressed and characterized to synthesize Kdo-8-P from D-arabinose-5-phosphate and phosphoenolpyruvate. The pH optimum was 6.1 and the activity of the enzyme was neither stimulated by the addition of divalent cations nor inhibited by EDTA. The cDNA of kdsA could not complement Escherichia coli K-12 strain AB3257, which is defective in all three isoenzymes (AroFGH) of 3-deoxy-D-arabino-hept-2-ulosonate-7-phosphate (Dha-7-P) synthase (EC 4.1.2.15), and neither D-erythrose-4-phosphate nor D-ribose-5-phosphate could substitute for D-arabinose-5-phosphate in vitro. Thus, plant cells possess a specific enzyme for the biosynthesis of Kdo-8-P with remarkable structural and functional similarities to bacterial KdsA proteins.

Brassica napus cDNAs encoding fatty acyl-CoA synthetase.

From a cDNA library of developing siliques of rapeseed (Brassica napus L.) we have isolated five full-length clones encoding polypeptides of the AMP-binding protein family. Two cDNAs encode fatty acyl-CoA synthetase activity (EC 6.2.1.3). The deduced polypeptides share about 52% identical amino acids. After expression in Escherichia coli the predicted enzymatic activity was confirmed by in vitro assays and product analysis. The enzymatic activity for one of the clones was characterized in detail by determination of the K(m) for oleic acid ( 10.4 microm) and the pH optimum (between 7 and 8). For the three additional clones no enzymatic activities could be demonstrated after expression in E. coli, although two of them exhibit similarity to either eukaryotic or prokaryotic acyl-CoA synthetases. The sequences are compared to a number of related expressed sequence tags from Brassica and Arabidopsis. Potential subcellular locations and functions of the deduced polypeptides within plant cells are discussed.

Chilling sensitivity of Arabidopsis thaliana with genetically engineered membrane lipids.

Upon transfer of a genetically engineered Escherichia coli gene for glycerol-3-phosphate acyltransferase (plsB) to Arabidopsis thaliana (L.) Heynh., the gene is transcribed and translated into an enzymatically active polypeptide. This leads to an alteration in fatty acid composition of membrane lipids. From these alterations it is evident that the enzyme is located mainly inside the plastids. The amount of saturated fatty acids in plastidial membrane lipids increased. In particular, the fraction of high-temperature melting species of phosphatidylglycerol is elevated. These molecules are thought to play a crucial role in determining chilling sensitivity of plants. An increase in sensitivity could be observed in the transgenic plants during recultivation after chilling treatment. Implications for the hypothesis of phosphatidylglycerol-determined chilling sensitivity are discussed.

The fadD gene of Escherichia coli K12 is located close to rnd at 39.6 min of the chromosomal map and is a new member of the AMP-binding protein family.

The fadD gene of Escherichia coli K12 was cloned and sequenced. The gene was identified by its ability to complement the corresponding mutant and by measuring the enzymatic activity after its expression in this mutant. The deduced polypeptide sequence exhibits similarity to other long chain acyl-CoA (coenzyme A) synthetases and a variety of other proteins, which together form a family of AMP-binding proteins. This family is extended by several new members and subdivided into four groups. fadD is assigned to a subgroup that does not include long chain acyl-CoA synthetases from eukaryotic organisms.

A UDP glucosyltransferase from Bacillus subtilis successively transfers up to four glucose residues to 1,2-diacylglycerol: expression of ypfP in Escherichia coli and structural analysis of its reaction products.

We have isolated the ypfP gene (accession number P54166) from genomic DNA of Bacillus subtilis Marburg strain 60015 (Freese and Fortnagel, 1967) using PCR. After cloning and expression in E. coli, SDS-PAGE showed strong expression of a protein that had the predicted size of 43.6 kDa. Chromatographic analysis of the lipids extracted from the transformed E. coli revealed several new glycolipids. These glycolipids were isolated and their structures determined by nuclear magnetic resonance (NMR) and mass spectrometry. They were identified as 3-[O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl]-1,2-diacylgl ycerol, 3-[O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl-(1-->6)-O-bet a-D-glucopyranosyl]-1,2-diacylglycerol and 3-[O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl-(1-->6)-O-bet a-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl]-1,2-diacylglycerol. The enzymatic activity expected to catalyse the synthesis of these compounds was confirmed by in vitro assays with radioactive substrates. In these assays, one additional glycolipid was formed and tentatively identified as 3-[O-beta-D-glucopyranosyl]-1,2-diacylglycerol, which was not detected in the lipid extract of transformed cells. Experiments with some of the above-described glycolipids as 14C-labelled sugar acceptors and unlabelled UDP-glucose as glucose donor suggest that the ypfP gene codes for a new processive UDP-glucose: 1,2-diacylglycerol-3-beta-D-glucosyl transferase. This glucosyltransferase can use diacylglycerol, monoglucosyl-diacylglycerol, diglucosyl diacylglycerol or triglucosyl diacylglycerol as sugar acceptor, which, apart from the first member, are formed by repetitive addition of a glucopyranosyl residue in beta (1-->6) linkage to the product of the preceding reaction.

rbcS genes in Solanum tuberosum: conservation of transit peptide and exon shuffling during evolution.

Five genes of the rbcS gene family of Solanum tuberosum (potato) were studied. One of these is a cDNA clone; the other four are located on two genomic clones representing two different chromosomal loci containing one (locus 1) and three genes (locus 2), respectively. The intron/exon structure of the three genes in locus 2 is highly conserved with respect to size and position. These genes contain two introns, whereas the gene from locus 1 contains three introns. Although in most cases the amino acid sequences in the transit peptide part of different rbcS genes from the same species varied considerably more than the corresponding mature amino acid sequences, one exception found in tomato and potato indicates that the transit peptide of rbcS could have a special function. A comparison of the rbcS genes of higher plants with those of prokaryotes offers suggestive evidence that introns first served as spacer material in the process of exon shuffling and then were removed stepwise during the evolution of higher plants.

Reduced steady-state levels of rbcS mRNA in plants kept in the dark are due to differential degradation.

When plants are placed in the dark, the level of the abundant mRNA encoding the small subunit of ribulose-1,5-bisphosphate carboxylase (rbcS) declines rapidly. We present evidence demonstrating an active degradation of rbcS mRNA in the dark. Detailed analysis shows that transcripts originating from different members of the rbcS gene family are differentially affected by this degradation. This phenomenon is not common to all light-regulated plant genes since the mRNA for ST-LS1, another leaf-specific and light-induced gene, is not degraded in the dark within the same time scale.

Purification and cDNA sequencing of an oleate-selective acyl-ACP:sn-glycerol-3-phosphate acyltransferase from pea chloroplasts.

The soluble acyl-ACP:sn-glycerol-3-phosphate acyltransferase from chloroplasts of chilling-sensitive and -resistant plants differ in their fatty acid selectivity. Enzymes from resistant plants discriminate against non-fluid palmitic acid and select oleic acid whereas the acyltransferase from sensitive plants accepts both fatty acids. To use this difference for improving plant chilling resistance by biotechnology the gene for an oleate-selective enzyme is required. Therefore, the oleate-selective enzyme from pea seedlings was purified to apparent homogeneity. Tryptic peptides of internal origin were sequenced. Polyclonal antibodies raised in rabbits were used for an immunological screening of a pea leaf cDNA expression library in lambda gt11. A positive clone of 1800 bp was selected showing an open reading frame which codes for 457 amino acids. The deduced amino acid sequence coincides perfectly with the tryptic sequences. A tentative assignment of the processing site was made which divides the preprotein into a mature protein of 41 kDa in accordance with experimental findings and a transit peptide of 88 amino acids. At present the comparison between a selective (pea) and an unselective (squash) acyltransferase sequence does not provide a clue for recognizing the structural differences resulting in different selectivities.

UDP-glucose:sterol glucosyltransferase: cloning and functional expression in Escherichia coli.

Steryl glucosides are characteristic lipids of plant membranes. The biosynthesis of these lipids is catalyzed by the membrane-bound UDP-glucose:sterol glucosyltransferase (EC 2.4.1.173). The purified enzyme (Warnecke and Heinz, Plant Physiol 105 (1994): 1067-1073) has been used for the cloning of a corresponding cDNA from oat (Avena sativa L.). Amino acid sequences derived from the amino terminus of the purified protein and from peptides of a trypsin digestion were used to construct oligonucleotide primers for polymerase chain reaction experiments. Screening of oat and Arabidopsis cDNA libraries with amplified labeled DNA fragments resulted in the isolation of sterol glucosyltransferase-specific cDNAs with insert lengths of ca. 2.3 kb for both plants. These cDNAs encode polypeptides of 608 (oat) and 637 (Arabidopsis) amino acid residues with molecular masses of 66 kDa and 69 kDa, respectively. The first amino acid of the purified oat protein corresponds to the amino acid 133 of the deduced polypeptide. The absence of these N-terminal amino acids reduces the molecular mass to 52 kDa, which is similar to the apparent molecular mass of 56 kDa determined for the purified protein. Different fragments of these cDNAs were expressed in Escherichia coli. Enzyme assays with homogenates of the transformed cells exhibited sterol glucosyltransferase activity.

A plant acyltransferase involved in triacylglycerol biosynthesis complements an Escherichia coli sn-1-acylglycerol-3-phosphate acyltransferase mutant.

The second acylation reaction in glycerolipid biosynthesis is catalyzed by an sn-1-acylglycerol-3-phosphate acyltransferase. The enzyme of Limnanthes douglasii involved in triacylglycerol synthesis has an unusual specificity for very long chain acyl groups in both of its substrates, namely acyl-CoA and sn-1-acylglycerol-3-phosphate, and causes the enrichment of erucoyl groups in the sn-2 position of the seed oil of this plant species. We have isolated a cDNA clone encoding this embryo-specific, microsomal acyltransferase via heterologous complementation of an Escherichia coli mutant deficient in sn-1-acylglycerol-3-phosphate acyltransferase activity. The open reading frame of the cDNA insert encodes a protein with a length of 281 amino acids, with three predicted membrane-spanning domains and of about 31.7 kDa. The sequence exhibits substantial sequence similarity to the sn-1-acylglycerol-3-phosphate acyltransferase of E. coli. The corresponding transcript was detectable in developing embryos but not in leaves of L. douglasii, and expression of the open reading frame in E. coli caused sn-1-acylglycerol-3-phosphate acyltransferase activity which showed properties different from those of the bacterial acyltransferase but typical of the L. douglasii enzyme involved in triacylglycerol biosynthesis.

Functional characterization of beta-ketoacyl-CoA synthase genes from Brassica napus L.

Seed-specifically expressed beta-ketoacyl-CoA synthase genes of Brassica napus (Bn-FAE1.1 genes) were cloned from two cultivars, namely Askari, a high-erucic-acid type, and Drakkar, a low-erucic-acid type. The genes from the two cultivars were found to be nearly identical. They encode proteins of 507 amino acids, the sequences of which differ only at position 282. The Bn-FAE1.1 gene of Askari, unlike that of Drakkar, was functionally expressed in yeast cells suggesting that the single amino acid exchange effects the low erucic acid phenotype at the E1 gene locus. In yeast cells the beta-ketoacyl-CoA synthase of Askari elongated not only oleoyl but also palmitoleoyl groups as well as saturated acyl groups in such a way that monounsaturated acyl groups of 22 carbons and saturated ones of 26 carbons were formed as main products. A reporter gene fused to the promoter region of the Bn-FAE1.1 gene from Askari showed seed-specific expression in transgenic rapeseed plants. Over-expression of the coding region of the Askari gene in developing seeds of transgenic Drakkar plants resulted in a significant increase in the levels of eicosenoic acid and erucic acid esterified in the seed oil. On the other hand, in transgenic high-erucic-acid rapeseed plants the increase in erucic acid level was at most 60% although the chimeric Bn-FAE1.1 gene was co-expressed with an erucoyl-CoA-specific lysophosphatidate acyltransferase gene enabling trierucoyl glycerol to accumulate in the seed oil.