Cloning and characterisation of a prenyltransferase from the aphid Myzus persicae with potential involvement in alarm pheromone biosynthesis.

The majority of aphid species release an alarm pheromone with the most common component being the sesquiterpene (E)-beta-farnesene, sometimes accompanied by other sesquiterpenes or monoterpenes. The genes/enzymes involved in the production of these compounds have not been identified in aphids although some components of isoprenoid biosynthesis have been identified in other insect species. Here we report the cloning, expression and characterisation of a prenyltransferase from the aphid Myzus persicae which can act as a farnesyl pyrophosphate synthase or a geranyl pyrophosphate synthase to produce both sesquiterpenes and monoterpenes and hence could be responsible for the biosynthesis of the observed components of the alarm pheromones. In addition, the enzyme can utilise geranyl pyrophosphate to produce farnesyl pyrophosphate showing that the synthesis of the latter involves the sequential condensation of isoprenyl pyrophosphate units.

Rapid disruption of nitrogen metabolism and nitrate transport in spinach plants deprived of sulphate.

Hydroponically grown spinach plants were deprived of an external source of sulphate after an initial period when the S-supply was sufficient. The time-course of events following this treatment was monitored. The first responses were found in the uptake and translocation of NO(3)(-) and the uptake of SO(4)(2-). The former declined by approximately 50%, the effect being most significant at higher [NO(3)(-)](ext.) while the latter increased 6-fold over a 4 d period. Growth in the absence of external SO(4)(2-) resulted in exhaustion of internal SO(4)(2-) pools, the effect being seen first in roots, then in young leaves and, after a marked delay, in mature leaves. In young leaves, there were dramatic increases in the [NO(3)(-)] and the content of arginine in the first 2 d of S-deprivation. The concentration of glutamine, the most abundant amino acid in S-sufficient conditions, also more than doubled in S-deficient young leaves. The changes in arginine levels were also found in older leaves, but the change in glutamine level was not seen. Assays of nitrate reductase activity (NRA) and nitrate reductase (NR) mRNA from young leaves of S-replete and S-deprived plants revealed a divergence in activity and content only late in the experiments (between days 4 and 8) when results were expressed on a unit leaf basis. However, there were also time-dependent changes in the protein content that kept the specific activities (NRA:protein and RNA:protein) more or less unchanged. The results imply that the impact of S-deficiency on N-utilization are more sensitively monitored by simple measurements of the chemical composition of young leaves than by measurements of NRA or NR transcript abundance. They also suggest that protein synthesis in young leaves is strongly dependent on a continuous supply of SO(4)(2-) from outside the plant.

Spectroscopic and kinetic properties of a recombinant form of the flavin domain of spinach NADH: nitrate reductase.

The C-terminal 268 residues of the spinach assimilatory NADH:nitrate reductase amino acid sequence that correspond to the flavin-containing domain of the enzyme have been selectively amplified and expressed as a recombinant protein in Escherichia coli. The recombinant protein, which was produced in both soluble and insoluble forms, was purified to homogeneity using a combination of ammonium sulfate precipitation, affinity chromatography on 5'-ADP-agarose and FPLC gel filtration. The purified domain exhibited a molecular weight of approximately 30 kDa, estimated by polyacrylamide gel electrophoresis, and a molecular mass of 30,169 for the apoprotein determined by mass spectrometry, which also confirmed the presence of FAD. The UV/visible spectrum was typical of a flavoprotein, with maxima at 272, 386, and 461 nm in the oxidized form while CD spectroscopy yielded both positive and negative maxima at 313 and 382 nm and 461 and 484 nm, respectively. The purified domain showed immunological cross-reactivity with anti-spinach nitrate reductase polyclonal antibodies while both N-terminal and internal amino acid sequencing of isolated peptides confirmed the fidelity of the domain's primary sequence. The protein retained NADH-ferricyanide reductase activity (Vmax=84 micromol NADH consumer/min/nmol FAD) with Km's of 17 and 34 microM for NADH and ferricyanide, respectively, with a pH optimum of approximately 6.5 A variety of NADH-analogs could also function as electron donors, though with decreased efficiency, the most effective being reduced nicotinamide hypoxanthine dinucleotide (V(max) = 35 micromol NHDH consumer/min/nmol FAD) and Km = 22 microM). NAD+ was demonstrated to be a competitive inhibitor (Ki = 1.9 mM) while analysis of inhibition by a variety of NAD+-analogs indicated the most efficient inhibitor to be ADP (Ki = 0.2 mM), with analogs devoid of either the phosphate, ribose, or adenine moieties proving to be markedly less-efficient inhibitors. The isolated domain was also capable of reducing cytochrome b5 directly (V(max) = 1.2 micromol NADH consumed/min/nmol FAD, Km (cyt. b5) = 6 microM), supporting the FAD -> b557 -> Mo electron transfer sequence in spinach nitrate reductase.

Molecular cloning and characterisation of asparagine synthetase from Lotus japonicus: dynamics of asparagine synthesis in N-sufficient conditions.

Two cDNA clones, LJAS1 and LJAS2, encoding different asparagine synthetases (AS) have been identified and sequenced and their expression in Lotus japonicus characterised. Analysis of predicted amino acid sequences indicted a high level of identity with other plant AS sequences. No other AS genes were detected in the L. japonicus genome. LJAS1 gene expression was found to be root-enhanced and lower levels of transcript were also identified in photosynthetic tissues. In contrast, LJAS2 gene expression was root-specific. These patterns of AS gene expression are different from those seen in pea. AS gene expression was monitored throughout a 16 h light/8 h dark day, under nitrate-sufficient conditions. Neither transcript showed the dark-enhanced accumulation patterns previously reported for other plant AS genes. To evaluate AS activity, the molecular dynamics of asparagine synthesis were examined in vivo using 15N-ammonium labelling. A constant rate of asparagine synthesis in the roots was observed. Asparagine was the most predominant amino-component of the xylem sap and became labelled at a slightly slower rate than the asparagine in the roots, indicating that most root asparagine was located in a cytoplasmic 'transport' pool rather than in a vacuolar 'storage' pool. The steady-state mRNA levels and the 15N-labelling data suggest that light regulation of AS gene expression is not a factor controlling N-assimilation in L. japonicus roots during stable growth in N-sufficient conditions.

Isolation of a cDNA from Saccharomyces cerevisiae that encodes a high affinity sulphate transporter at the plasma membrane.

Resistance to selenate and chromate, toxic analogues of sulphate, was used to isolate a mutant of Saccharomyces cerevisiae deficient in the capacity to transport sulphate into the cells. A clone which complements this mutation was isolated from a cDNA library prepared from S. cerevisiae poly(A)+ RNA. This clone contains an insert which is 2775 bp in length and has a single open reading frame that encodes a 859 amino acid polypeptide with a molecular mass of 96 kDa. Sequence motifs within the deduced amino acid sequence of this cDNA (SUL1) show homology with conserved areas of sulphate transport proteins from other organisms. Sequence analysis predicts the position of 12 putative membrane spanning domains in SUL1. When the cDNA for SUL1 was expressed in S. cerevisiae, a high affinity sulphate uptake activity (Km = 7.5 +/- 0.6 microM for SO2-4) was observed. A genomic mutant of S. cerevisiae in which 1096 bp were deleted from the SUL1 coding region was constructed. This mutant was unable to grow on media containing less than 5 mM sulphate unless complemented with a plasmid containing the SUL1 cDNA. We conclude that the SUL1 cDNA encodes a S. cerevisiae high affinity sulphate transporter that is responsible for the transfer of sulphate across the plasma membrane from the external medium.