Synthesis of the flavour precursor, alliin, in garlic tissue cultures.

The path of synthesis of alkyl cysteine sulphoxides, or flavour precursors, in the Alliums is still speculative. There are two proposed routes for alliin biosynthesis, one is from serine and allyl thiol while the other is from glutathione and an allyl source via gamma glutamyl peptides. The routes have been investigated by exposing undifferentiated callus cultures of garlic and onion to potential pathway intermediates. After a period of incubation of 2 days the callus was extracted, and analysed for flavour precursors and related compounds by HPLC. Standards of alliin, isoallin and propiin were synthesised and their identity confirmed by HPLC and NMR. Putative intermediates selected included the amino acids serine and cysteine, as well as more complex intermediates such as allylthiol, allyl cysteine and glutathione. Both garlic and onion tissue cultures were able to synthesize alliin following incubation with allylthiol, and cysteine conjugates such as allyl cysteine. The ability of the tissue cultures to form alliin from intermediates was compatible with the proposed routes of synthesis of alliin.

Characterization of the ethanol-inducible alc gene-expression system in Arabidopsis thaliana.

Controlled expression of transgenes in plants is key to the characterization of gene function and the regulated manipulation of growth and development. The alc gene-expression system, derived from the filamentous fungus Aspergillus nidulans, has previously been used successfully in both tobacco and potato, and has potential for use in agriculture. Its value to fundamental research is largely dependent on its utility in Arabidopsis thaliana. We have undertaken a detailed function analysis of the alc regulon in A. thaliana. By linking the alcA promoter to beta-glucuronidase (GUS), luciferase (LUC) and green fluorescent protein (GFP) genes, we demonstrate that alcR-mediated expression occurs throughout the plant in a highly responsive manner. Induction occurs within one hour and is dose-dependent, with negligible activity in the absence of the exogenous inducer for soil-grown plants. Direct application of ethanol or exposure of whole plants to ethanol vapour are equally effective means of induction. Maximal expression using soil-grown plants occurred after 5 days of induction. In the majority of transgenics, expression is tightly regulated and reversible. We describe optimal strategies for utilizing the alc system in A. thaliana.

An ethanol inducible gene switch for plants used to manipulate carbon metabolism.

Many transgenic plant studies use constitutive promoters to express transgenes. For certain genes, deleterious effects arise from constant expression in all tissues throughout development. We describe a chemically inducible plant gene expression system, with negligible background activity, that obviates this problem. We demonstrate its potential by showing inducible manipulation of carbon metabolism in transgenic plants. Upon rapid induction of yeast cytosolic invertase, a marked phenotype appears in developing leaves that is absent from leaves that developed before induction or after it has ceased.

The isolation and characterization of type II metallothionein-like genes from tomato (Lycopersicon esculentum L.).

Two cDNAs encoding putative metallothionein (MT)-like peptides have been isolated from tomato (L. esculentum L.). The predicted protein products of these two cDNAs (LeMT(A) and LeMT(B)) are 72 and 83 amino acids respectively and both encode peptides with arrangements of cysteine residues characteristic of type II plant MTs. In other plants which possess more than one gene expressing MT proteins of the same type, the products are closely related or identical, but LeMT(A) and LeMT(B) constitute two different classes of message, and encode two different protein products. Northern blot analysis of LeMT(A) and LeMT(B) showed that transcripts of both MT-like genes were more abundant in leaves than roots in tomato plants grown without addition of extra metal ions, a characteristic of type II MTs. A genomic clone corresponding to LeMT(B) (LeMT(B)) was isolated and sequenced. The 5'-flanking region of LeMT(B) was shown to contain a putative metal regulatory element (MRE) which suggests the possibility of metal-regulated transcription. In addition, the upstream region also contains a G-box like motif (CACGTG) and an 8 bp sequence (AATTCAAA) found within the promoters of genes shown to be ethylene-responsive.

Site-directed mutagenesis of nitrate reductase from Aspergillus nidulans. Identification of some essential and some nonessential amino acids among conserved residues.

Nitrate reductase is a multiredox enzyme possessing three functional domains associated with the prosthetic groups FAD, heme iron, and molybdopterin. In Aspergillus nidulans, it is encoded by the niaD gene. A homologous transformation system has been used whereby a major deletion at the niiAniaD locus of the host was repaired by gene replacement. Employing site-directed mutagenesis and this transformation system, nine niaD mutants were generated carrying specific amino acid substitutions. Mutants in which alanine replaced cysteine 150, which is thought to bind the molybdenum atom of the molybdenum-pterin, and in which alanine replaced histidine 547, which putatively binds heme iron, had no detectable nitrate reductase (NAR) activity. This clearly establishes an essential catalytic role for these residues. Of the remaining mutants, all altered in the NADPH/FAD domain, two were temperature-sensitive for NAR activity, two had reduced NAR activity levels, and three had normal levels. Since some of these mutants change residues conserved between homologous nitrate reductases from a wide range of species, it is clear that such amino acid identities do not necessarily signify essential roles for the activity of the enzyme. These findings are considered in the light of predicted structural/functional roles for the altered amino acids.

Metallothionein genes from the flowering plant Mimulus guttatus.

In response to excess metal, higher plants produce metal-binding peptides [( gamma EC]nG) whose biosynthesis is believed to be mediated by enzymes involved in glutathione (gamma ECG) metabolism. In contrast, animals synthesize metallothioneins, gene-encoded low molecular weight cysteine-rich metal-binding proteins. In an investigation of copper-regulated genes in the copper-tolerant flowering plant Mimulus guttatus, we have isolated a series of cDNA clones identifying two genes which encode a protein with class I metallothionein domains. This represents the first description of a metallothionein gene in a flowering plant.

Glutamate synthase levels in Neurospora crassa mutants altered with respect to nitrogen metabolism.

Glutamate synthase catalyzes glutamate formation from 2-oxoglutarate plus glutamine and plays an essential role when glutamate biosynthesis by glutamate dehydrogenase is not possible. Glutamate synthase activity has been determined in a number of Neurospora crassa mutant strains with various defects in nitrogen metabolism. Of particular interest were two mutants phenotypically mute except in an am (biosynthetic nicotinamide adenine dinucleotide phosphate-glutamate dehydrogenase deficient, glutamate requiring) background. These mutants, i and en-am, are so-called enhancers of am; they have been redesignated herein as en(am)-1 and en(am)-2, respectively. Although glutamate synthase levels in en(am)-1 were essentially wild type, the en(am)-2 strain was devoid of glutamate synthase activity under all conditions examined, suggesting that en(am)-2 may be the structural locus for glutamate synthase. Regulation of glutamate synthase occurred to some extent, presumably in response to glutamate requirements. Glutamate starvation, as in am mutants, led to enhanced activity. In contrast, glutamine limitation, as in gln-1 mutants, depressed glutamate synthase levels.

The regulation of nitrate assimilation in Neurospora crassa: the isolation and genetic analysis of nmr-1 mutants.

Four mutants of Neurospora crassa have been isolated which have altered regulation of nitrate reductase. They each carry a mutation which results in derepressed synthesis of nitrate reductase even in the presence of glutamine. They map to a single locus which has been designated nmr-1 and which is located between am and gln on linkage group VR. The mutations appear to affect only nitrate assimilation. The nit-2, nit-3 and nit-4/5 mutations are epistatic to nmr-1 since the double mutants have the single nit mutant phenotype. For nitrate reductase synthesis, the nmr-1 mutation is epistatic to am such that the double mutant is derepressed even in the presence of glutamate or glutamine. In all other respects however, the double mutant exhibits the am phenotype. We suggest therefore that the nmr-1 mutations do not directly affect the regulation of nitrate reductase at the level of transcription but instead act post-transcriptionally.

The regulation of nitrate assimilation in Neurospora crassa: biochemical analysis of the nmr-1 mutants.

Neurospora crassa nmr-1 mutants, selected on the basis of their sensitivity to chlorate in the presence of glutamine, have elevated levels of the nitrate assimilation enzymes, NADPH-nitrate reductase and NAD(P)H-nitrite reductase. Immunoelectrophoretic determinations show that the higher nitrate reductase activities in nmr-1 mutants are due to greater enzyme concentrations. The half-life of nitrate reductase in these mutants is unaltered. As in wild-type, expression of nitrate assimilation in nmr-1 mutants is dependent on induction by nitrate. Reduced nitrogen metabolites like ammonium and glutamine still repress this expression in nmr-1 mutants, but not as effectively as in wild-type. Enzymatic activity measurements in double mutant strains confirm that the nit regulatory loci, nit-2 and nit-4/5, are epistatic to nmr-1, but nmr-1 is epistatic to nit-3, the nitrate reductase structural gene. The results imply that nmr-1 is involved in post-transcriptional control of nitrate assimilation.

Biochemical analysis of mutants defective in nitrate assimilation in Neurospora crassa: evidence for autogenous control by nitrate reductase.

A biochemical analysis of mutants altered for nitrate assimilation in Neurospora crassa is described. Mutant alleles at each of the nine nit (nitrate-nonutilizing) loci were assayed for nitrite reductase activity, for three partial activities of nitrate reductase, and for nitrite reductase activity. In each case, the enzyme deficiency was consistent with data obtained from growth tests and complementation tests in previous studies. The mutant strains at these nit loci were also examined for altered regulation of enzyme synthesis. Such experiments revealed that mutations which affect the structural integrity of the native nitrate reductase molecule can result in constitutive synthesis of this enzyme protein and of nitrite reductase. These results provide very strong evidence that, as in Aspergillus nidulans, nitrate reductase autogenously regulates the pathway of nitrate assimilation. However, only mutants at the nit-2 locus affect the regulation of this pathway by nitrogen metabolite repression.

The isolation and characterization of mutants defective in nitrate assimilation in Neurospora crassa.

The isolation and characterization of mutants altered for nitrate assimilation in Neurospora crassa is described. The mutants isolated can be subdivided into five classes on the basis of growth test that correspond to the growth patterns of existing mutants at six distinct loci. Mutants with growth characteristics like those of nit-2, nit-3 and nit-6 are assigned to those loci on the basis of noncomplementation and lack of recombination. Mutants that, from their growth patterns, appear to lack the molybdenum-containing cofactor for both nitrate reductase and xanthine dehydrogenase subdivide into three loci (nit-y, nit-8 and nit-9), all of which are gentically distinct from nit-1. nit-9 is a complex locus consisting of three complementation groups and thus appears similar ao the cnxABC locus of Asperillus nidulans. Extensive complementational and recombinational analyses reveal that nit-4 and nit-5 are alleles of the same locus, and two new alleles of that locus have been isolated. The results indicate that, as in A. nidulans, nitrate assimilation in N. crassa requires at least four loci (nit-1, 7, 8 and 9) to produce the molybdenum co-factor for nitrate reductase (and xanthine dehydrogenase), one locus (nit-3) to code for the nitrate reductase apoprotein, one locus (nit-6) to code for the nitrite reductase approtein and only one lous (nit-4/5) for the regulation of induction of the pathway by nitrate and nitrite.

Nitrogen metabolite repression of nitrate reductase in Neurospora crassa: effect of the gln-1a locus.

Nicotinamide adenine dinucleotide phosphate (reduced form)-nitrate reductase was freed from ammonium repression in a Neurospora crassa mutant having drastically lowered glutamine synthetase activity, gln-1a. The general phenomenon of nitrogen metabolite repression required glutamine or some aspect of glutamine metabolism.