John L. Wray

Molecular genetics of sulphate assimilation

The importance of sulphur in promoting yield, quality and stress resistance parameters in plants has been highlighted by the recent increased problems of S-deficiency in agriculture. These deficiencies are in part a consequence of reduced atmospheric emissions from industry and the subsequent decreased deposition on agricultural land. Contributions from several laboratories, worldwide, have resulted in the cloning of almost all of the genes responsible for uptake, transport and assimilation of sulphate. This has led directly to the resolution of many outstanding questions regarding the control of uptake and of the pathways and intermediates involved in assimilation. Furthermore, the ability to manipulate these pathways is now possible and will allow the engineering of crops with improved sulphur acquisition and utilization traits. This paper reviews the present status of the molecular genetics of sulphate assimilation in plants.

Cysteine biosynthesis in higher plants: a new member of the Arabidopsis thaliana serine acetyltransferase small gene-family obtained by functional complementation of an Escherichia coli cysteine auxotroph

A cDNA clone, Sat-52, encoding a novel isoform of serine acetyltransferase (EC 2.3.1.30) was isolated by functional complementation of an Escherichia coli cysE mutant defective in serine acetyltransferase. The 1158 base pair clone contains a full-length open reading frame encoding a deduced protein of 312 amino acids with an M(r) of 32.77 kDa. Northern analysis revealed a single transcript of ca 1.19 kb that did not increase in abundance under sulfate limitation. Genomic Southern hybridization suggests the presence of a single copy of the Sat-52 gene.

A conditional-lethal cnx-type nitrate reductase-deficient barley mutant

SummarySelection for resistance to chlorate in an M2 population of barley (Hordeum vulgare cv. Maris Mink, M1 seeds treated with sodium azide) yielded nine green plants with zero or very low in vivo nitrate reductase activity. One of these plants, R9401, was crossed with the wild-type cv. Golden Promise and analysis of the F2 progeny showed that both loss of nitrate reductase activity and resistance to chlorate was due to a recessive mutation in a single nuclear gene. Homozygous mutant plants did not grow on nitrate as sole nitrogen source, in vermiculite or in sterile culture. They grew aseptically on ammonium nitrate, ammonium citrate or urea, although glutamine was the best nitrogen source. Homozygous mutant plants possessd <1% of wild-type in vitro NADH-nitrate reductase activity and lacked detectable xanthine dehydrogenase activity. The plants possessed normal nitrite reductase activity and an inducible NADH-cytochrome c reductase activity. We conclude that loss of nitrate reductase activity is due to a cnx-type mutation affecting the formation of a functional cofactor.

Cloning and characterisation of an Arabidopsis thaliana cDNA clone encoding an organellar isoform of serine acetyltransferase

We have cloned an Arabidopsis thaliana cDNA encoding serine acetyltransferase (EC 2.3.1.30) by functional complementation of the Escherichia coli cysE mutant JM15. The cDNA clone Sat-1 conferred serine acetyltransferase activity (with apparent Km for the two substrates acetyl CoA and L-serine of 0.043 and 3.47 mmol/dm3 respectively) on the cysE mutant. The 1515 bp full-length cDNA encodes a deduced protein of 391 amino acids which includes a putative chloroplastic targeting presequence. Northern analysis revealed a single message of 1.5 kb, while Southern hybridisation suggests a small multigene family of related sequences.

Inhibition of NADH-nitrate reductase degradation in barley leaf extracts by leupeptin

Abstract NADH-nitrate reductase (NR) is unstable in extracts prepared from 6-day-old primary leaves of barley and breaks down to NADH-cytochrome c reductase (CR) species of 3.1 S and 3.8 S. Leupeptin, an inhibitor of trypsinlike enzymes, both stabilises NADH-NR activity in extracts and prevents conversion to the 3.1 S and 3.8 S NADH-CR species. We conclude that the leupeptin inhibited thiol-dependent acid endoproteinase described by Huffaker and Miller [6,7] is mainly responsible for degradation of NADH NR to smaller NADH-CR species in barley primary leaf extracts and that it cleaves peptide bonds on the carboxyl side of arginine/lysine in the enzyme. We suggest that these susceptible peptide bonds are located in interdomain hinge regions.

Isolation of molybdenum cofactor defective cell lines of Nicotiana tabacum

SummaryThirty-nine chlorate resistant cell lines were isolated after plating ethylmethane sulphonate treated allodihaploid cells of Nicotiana tabacum cv. Xanthi on agar medium containing 20 mM chlorate. Thirty-two of these cell lines grew as well on nitrate medium as on amino acid medium and three other cell lines grew well on amino acid medium but poorly on nitrate medium. Four other cell lines, 042, P12, P31 and P47 which could grow on amino acid medium, but not on nitrate medium, were examined further. They lacked in vitro nitrate reductase activity but were able to accumulate nitrate. All lines possessed nitrite reductase activity. Lines 042, P12, and P31 had a cytochrome c reductase species which was the same size as the wild type nitrate reductase associated cytochrome c reductase species, whilst the cytochrome c reductase species in line P47 was slightly smaller. All four lines lacked xanthine dehydrogenase activity and neither nitrate reductase nor xanthine dehydrogenase activity was restored by subculture of the four lines into either nitrate medium or glutamine medium supplemented with 1 mM sodium molybdate. These four lines are different from other molybdenum cofactor defective cell lines so far described in N. tabacum and possess similar properties to certain other cnx mutants described in Aspergillus nidulans.

The Molecular Genetics of Higher Plant Nitrate Assimilation

The nitrate assimilation pathway is the major point of entry of inorganic nitrogen into organic combination in most crop plants growing in well-aerated soils. This pathway converts nitrate to ammonium via a nitrate uptake mechanism and two enzymes, nitrate reductase (NR) and nitrite reductase (NiR), thereby making nitrogen available for a variety of biosyntheses, of which the most important quantitatively is protein synthesis.

Purification of barley nitrate reductase and demonstration of nicked subunits

Abstract Nitrate reductase was purified from 90-hr-old, nitrate-treated barley shoots by the same four-step procedure under four sets of conditions (A, B, C, D)

nir1, a conditional-lethal mutation in barley causing a defect in nitrite reduction

SummaryEleven green individuals were isolated when 95000 M2 plants of barley (Hordeum vulgare L.), mutagenised with azide in the M1, were screened for nitrite accumulation in their leaves after nitrate treatment in the light. The selected plants were maintained in aerated liquid culture solution containing glutamine as sole nitrogen source. Not all plants survived to flowering and some others that did were not fertile. One of the selected plants, STA3999, from the cultivar Tweed could be crossed to the wild-type cultivar and analysis of the F2 progeny showed that leaf nitrite accumulation was due to a recessive mutation in a single nuclear gene, which has been designated Nir1. The homozygous nir1 mutant could be maintained to flowering in liquid culture with either glutamine or ammonium as sole nitrogen source, but died within 14 days after transfer to compost. The nitrite reductase cross-reacting material seen in nitrate-treated wild-type plants could not be detected in either the leaf or the root of the homozygous nir1 mutant. Nitrite reductase activity, measured with dithionite-reduced methyl viologen as electron donor, of the nitrate-treated homozygous nir1 mutant was much reduced but NADH-nitrate reductase activity was elevated compared to wild-type plants. We conclude that the Nir1 locus determines the formation of nitrite reductase apoprotein in both the leaf and root of barley and speculate that it represents either the nitrite reductase apoprotein gene locus or, less likely, a regulatory locus whose product is required for the synthesis of nitrite reductase, but not nitrate reductase. Elevation of NADH-nitrate reductase activity in the nir1 mutant suggests a regulatory perturbation in the expression of the Narl gene.

Assay of molybdenum cofactor of barley

Abstract Conditions for assay of molybdenum cofactor in barley shoot extracts in the presence of molybdate (25 mM N 2 MoO 4 ) and the sulphydryl-group protector, reduced glutathione (5 mM) were optimized. Both total Mo-cofactor (assayed after heat-treatment of cell-free extracts) and ‘free’ Mo-cofactor (assayed in untreated cell-free extracts) were assayed. Compared to control plants grown in the absence of an exogenous nitrogen source total Mo-cofactor levels increased around 70 % when plants were grown for 4 days in the presence of either 15 mM KNO 3 or 15 mM NH 4 NO 3 . Growth in the presence of 15 mM (NH 4 ) 2 SO 4 did not affect the Mo-cofactor level. Very similar results were seen when plants were transferred to these nitrogen sources for 24 hr after previous growth in the absence of an exogenous nitrogen source. In contrast ‘free’ Mo-cofactor levels of both KNO 3 and NH 4 NO 3 -treated plants were increased 2-3-fold over untreated controls. Growth in the presence of (NH 4 ) 2 SO 4 did not affect the ‘free’ Mo-cofactor level.