High-performance liquid chromatographic separation of natural and synthetic desulphoglucosinolates and their chemical validation by UV, NMR and chemical ionisation-MS methods.

Methods are described for the optimised extraction, desulphation and HPLC separation of desulphoglucosinolates. These methods provide rapid separation, identification and quantitative measurements of glucosinolates extracted from Brassica napus L and related crops, of unusual glucosinolates found in crucifer weed species, and also of synthetic alkylglucosinolates. The desulphoglucosinolates used in these studies were either chemically synthesised (at least one example from each major structural class), or purified from various plant sources. Validation of the identities of the desulphoglucosinolates was by comparison of retention times with standards, and by UV, 1H- and 13C-NMR and chemical ionisation MS analysis. A list of useful species, and the specific tissues, from which high concentrations of standards can be extracted is included.

Evidence for autoregulation of cystathionine gamma-synthase mRNA stability in Arabidopsis.

Control of messenger RNA (mRNA) stability serves as an important mechanism for regulating gene expression. Analysis of Arabidopsis mutants that overaccumulate soluble methionine (Met) revealed that the gene for cystathionine gamma-synthase (CGS), the key enzyme in Met biosynthesis, is regulated at the level of mRNA stability. Transfection experiments with wild-type and mutant forms of the CGS gene suggest that an amino acid sequence encoded by the first exon of CGS acts in cis to destabilize its own mRNA in a process that is activated by Met or one of its metabolites.

Involvement of Cytochrome P450 in Glucosinolate Biosynthesis in White Mustard (A Biochemical Anomaly).

One of the first steps in glucosinolate biosynthesis is the conversion of amino acids to their aldoximes. The biochemistry of this process is controversial, and several very different enzyme systems have been described. The major glucosinolate in white mustard (Sinapis alba) is sinalbin, which is derived from tyrosine via its aldoxime, and this conversion is catalyzed by a cytochrome P450 (Cyt P450) monooxygenase. Phenylethyl- and alkenylglucosinolates are also present in white mustard leaves, as are the enzymes catalyzing the relevant aldoxime formation from homophenylalanine and methionine homologs, respectively. These enzymes are similar to those found in Brassica sp. and are distinct from the tyrosine-dependent enzyme in that they contain no heme and are unaffected by Cyt P450 inhibitors. They are instead inhibited by the flavoprotein inhibitor diphenylene iodonium and by Cu2+. In both white mustard and oilseed rape (Brassica napus) methyl jasmonate specifically stimulates indolylglucosinolate biosynthesis and yet has no effect on sinalbin accumulation in either cotyledons or leaves of white mustard. White mustard appears to be unique among crucifers in having a Cyt P450 aldoxime-forming enzyme for biosynthesis of one glucosinolate, although it also contains all of the non-Cyt P450 enzyme systems found in other members of the family. Sinalbin biosynthesis in white mustard is therefore an inappropriate model system for the synthesis of other glucosinolates in crucifers, including canola and oilseed rape.

Glucosinolate Biosynthesis (Further Characterization of the Aldoxime-Forming Microsomal Monooxygenases in Oilseed Rape Leaves).

The initial steps in glucosinolate biosynthesis are thought to proceed from amino acids, via N-hydroxy amino acids, to aldoximes. We showed previously that microsomes from green leaves of oilseed rape (Brassica napus cv Bienvenu) contain two distinct monooxygenases that catalyze the conversion of homophenylalanine and dihomomethionine to their respective aldoximes. Further characterization of these enzymes has now demonstrated that the latter enzyme catalyzes the NADPH-dependent oxidative decarboxylation of two higher homologs of methionine, in addition to dihomomethionine. No activity was found for either enzyme with L-methionine, DL-homomethionine, L-phenylalanine, L-tyrosine, or L-tryptophan. Both of these rape monooxygenase activities are dependent on O2, not requiring any other O2 species or radical. The presence of an unoxidized sulfur atom and its relative position in the side chain of the aliphatic substrates are important for binding to the active site of the methionine-homolog enzyme. Neither enzyme has any characteristics of a cytochrome P450-type enzyme, and antiserum raised against cytochrome P450 reductase did not significantly inhibit monooxygenase activity.

Purification and characterization of an acyclic monoterpene primary alcohol:NADP+ oxidoreductase from catmint (Nepeta racemosa).

A soluble monoterpene primary alcohol:NADP+ oxidoreductase has been purified to apparent homogeneity from leaves of the catmint, Nepeta racemosa. The purified enzyme consisted of two polypeptides, with molecular masses of 42,000 and 40,000 Da, and contained zinc ions. A number of monoterpene alcohols (geraniol, nerol, citronellol, and their hydroxylated derivatives) were substrates, but the enzyme was inactive toward ethanol. The enzyme required NADP(H) as cofactor, with NAD(H) ineffective. Gas chromatographic and coupled mass spectrometric analysis of the reaction products showed that 10-hydroxygeraniol and 10-hydroxynerol were oxidized by the enzyme in the presence of NADP+, at both C-1 and C-10. These results are consistent with a role for this enzyme in the biosynthesis of iridoid monoterpenes.

A Metabolic Control Analysis of the Glutamine Synthetase/Glutamate Synthase Cycle in Isolated Barley (Hordeum vulgare L.) Chloroplasts.

Ammonia assimilation in chloroplasts occurs via the glutamine synthetase/glutamate synthase (GS/GOGAT) cycle. To determine the extent to which these enzymes contribute to the control of ammonia assimilation, a metabolic control analysis was performed on isolated barley (Hordeum vulgare L.) leaf chloroplasts. Pathway flux was measured polarographically as ammonium-plus-2-oxoglutarate-plus-glutamine-dependent O2 evolution in illuminated chloroplasts. Enzyme activity was modulated by titration with specific, irreversible inhibitors of GS (phosphinothricin) and GOGAT (azaserine). Flux control coefficients (CJ0E0) were determined (a) by differentiation of best-fit hyperbolic curves of the data sets (flux versus enzyme activity), and (b) from estimates of the deviation indices (D/[prime]E0). Both analyses gave similar values for the coefficients. The control coefficient for GS was relatively high and the value did not change significantly with changes in 2-oxoglutarate concentration (C/0E0 = 0.58 at 5 mM 2-oxoglutarate and 0.40 at 20 mM 2-oxoglutarate). The control coefficient for GOGAT decreased with decreasing glutamine concentrations, from 0.76 at 20 mM glutamine to 0.19 at 10 mM glutamine. Thus, at high concentrations of glutamine, GOGAT exerts a major control over flux with a significant contribution also from GS. At lower concentrations of glutamine, however, GOGAT exerts far less control over pathway flux.

Synthesis of glucosinolate precursors and investigations into the biosynthesis of phenylalkyl- and methylthioalkylglucosinolates.

The alkenyl and aromatic glucosinolates in oilseed rape (Brassica napus) are biosynthesized from chain-extended homologues of protein amino acids, including methionine and phenylalanine. Homologues of these two amino acids, homophenylalanine (2-amino-4-phenylbutyric acid) and dihomomethionine (2-amino-6-methylthiohexanoic acid) were synthesized both with and without a 1-14C label. Microsomal preparations from oilseed rape leaves were shown to contain enzyme systems which metabolize these compounds, with loss of 14CO2, and produce the aldoxime intermediates possible in the biosynthetic pathway utilizing homophenylalanine. These were characterized by comparison with authenticated synthetic compounds. Potential intermediates on the pathway between homophenylalanine and its corresponding aldoxime, the N-hydroxyamino- and the oximino acids, were synthesized and their possible role in the pathway investigated.

Cloning and sequence analysis of a cDNA for barley ferredoxin-dependent glutamate synthase and molecular analysis of photorespiratory mutants deficient in the enzyme.

The NH2-terminal sequences of ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) purified from barley (Hordeum vulgare L.) and Chlamydomonas reinhardtii (Dangeard), and of a barley peptide, were determined and the barley sequences were used to design oligonucleotide primers for the polymerase chain reaction. A specific 1.3-kilobase (kb) cDNA fragment specifying the NH2-terminal one-third of the mature barley polypeptide, was amplified, cloned and sequenced. The NH2-terminus of plant Fd-GOGAT is highly conserved and homologous to the NH2-terminus of the heavy subunit of Escherichia coli NADPH-GOGAT. Based on sequence homologies, we tentatively identified the NH2-terminal region of Fd-GOGAT as the glutamine-amidotransferase domain, which is related to the corresponding domain of the purF-type amidotransferases. The Fd-GOGAT cDNA clone, and polyclonal antibodies raised against the barley enzyme, were used to analyse four Fd-GOGAT-deficient photorespiratory mutants. Three mutants (RPr 82/1, RPr 82/9 and RPr 84/82) had no detectable Fd-GOGAT protein in leaves, while the fourth (RPr 84/42) had a small amount of cross-reacting material. Hybridization to Northern blots of total leaf RNA revealed that both RPr 82/9 and RPr 84/82 were indistinguishable from the parental line (Maris Mink), having normal amounts of a 5.7-kb mRNA species. On the other hand, RPr 82/2 and RPr 84/42 each contained two distinct hybridizing RNA species, one of which was larger than 5.7 kb, the other smaller. Using a set of wheat-barley telosomic addition lines we have assigned the Fd-GOGAT structural locus to the short arm of chromosome 2.

Interactions of Avocado (Persea americana) Cytochrome P-450 with Monoterpenoids.

The microsomal fraction of avocado (Persea americana) mesocarp is a rich source of cytochrome P-450 active in the demethylation of xenobiotics. Cytochrome P-450 from this tissue has been purified and well characterized at the molecular level (DP O'Keefe, KJ Leto [1989] Plant Physiol 89: 1141-1149; KR Bozak, H Yu, R Sirevag, RE Christoffersen [1990] Proc Natl Acad Sci USA 87: 3904-3908). Despite this extensive characterization, the role of the enzyme in vivo was not established. Optical and electron paramagnetic resonance binding studies described here suggest that the monoterpenoids, nerol and geraniol, are substrates of avocado cytochrome P-450 (spectral dissociation constant of 7.2 and 35 micromolar, respectively). Avocado microsomes have been shown to catalyze the hydroxylation of these monoterpenoids, and both nerol and geraniol have been shown to inhibit the activity of avocado cytochrome P-450 toward the artificial substrate 7-ethoxycoumarin, with nerol a competitive inhibitor of this activity.

Molecular analysis of barley mutants deficient in chloroplast glutamine synthetase.

A barley leaf cDNA library has been screened with two oligonucleotide probes designed to hybridize to conserved sequences in glutamine synthetase (GS) genes from higher plants. Two GS cDNA clones were identified as hybridizing strongly to one or both probes. The larger clone (pcHvGS6) contained a 1.6 kb insert which was shown by primer extension analysis to be an almost full-length cDNA. Both clones were more closely related to cDNAs for the chloroplast form of GS (GS2) from pea and Phaseolus vulgaris than to cDNAs for the cytosolic form (GS1). A sequence identical to an N-terminal sequence determined from a purified preparation of the mature GS2 polypeptide (NH2-XLGPETTGVIQRMQQ) was found in the pcHvGS6-encoded polypeptide at residues 46-61, indicating a pre-sequence of at least 45 amino acids. The pre-sequence has only limited sequence homology to the pre-sequences of pea and P. vulgaris GS2 subunits, but is similarly rich in basic residues and possesses some of the structural features common to the targeting sequences of other chloroplast proteins. The molecular lesions responsible for the GS2-deficient phenotypes of eight photorespiratory mutants of barley were investigated using a gene-specific probe from pcHvGS6 to assay for GS2 mRNA, and an anti-GS antiserum to assay for GS2 protein. Three classes of mutants were identified: class I, in which absence of cross-reacting material was correlated with low or undetectable levels of GS2 mRNA; class II, which had normal or increased levels of GS2 mRNA but very little GS2 protein; and class III, which had significant amounts of GS2 protein but little or no GS2 activity.

The value of mutants unable to carry out photorespiration.

Manipulation of the CO2 concentration of the atmosphere allows the selection of photorespiratory mutants from populations of seeds treated with powerful mutagens such as sodium azide. So far, barley lines deficient in activity of phosphoglycolate phosphatase, catalase, the glycine to serine conversion, glutamine synthetase, glutamate synthase, 2-oxoglutarate uptake and serine: glyoxylate aminotransferase have been isolated. In addition one line of pea lacking glutamate synthase activity and one barley line containing reduced levels of Rubisco are available. The characteristics of these mutations are described and compared with similar mutants isolated from populations of Arabidopsis. As yet, no mutant lacking glutamine synthetase activity has been isolated from Arabidopsis and possible reasons for this difference between barley and Arabidopsis are discussed. The value of these mutant plants in the elucidation of the mechanism of photorespiration and its relationships with CO2 fixation and amino acid metabolism are highlighted.

Barley mutants lacking chloroplast glutamine synthetase-biochemical and genetic analysis.

Eight mutants of barley (Hordeum vulgare cv Maris Mink) lacking the chloroplast isozyme of glutamine synthetase (EC 6.3.1.2.) were isolated by their inability to grow under photorespiratory conditions. The cytoplasmic isozyme of glutamine synthetase was present in the leaves of all the mutants, with activities comparable to the wild-type (10-12 nanokatals per gram fresh weight). The mutant plants developed normally and were fully fertile under conditions that minimize photorespiration. In 1% O(2) the rate of CO(2) fixation in leaves of one of the mutants, RPr 83/32, was the same as the wild-type, but in air this rate declined to 60% of the wild-type after 30 minutes. During this time the ammonia concentration in leaves of the mutant rose from 1 to 50 micromoles per gram fresh weight. Such ammonia accumulation in air was found in all the mutant lines. In back-crosses with the parent line, F(1) plants were viable in air. In the F(2) generation, nonviability in air and the lack of chloroplast glutamine synthetase co-segregated, in both the lines tested. These two lines and four others proved to be allelic; we designate them gln 2a-f. The characteristics of these mutants conclusively demonstrate the major role of chloroplast glutamine synthetase in photorespiration and its associated nitrogen recycling.

Characteristics of a photorespiratory mutant of barley (Hordeum vulgare L.) deficient in phosphogly collate phosphatase.

A barley mutant RPr84/90 has been isolated by selecting for plants which grow poorly in natural air, but normally in air enriched to 0.8% CO2. After 5 minutes of photosynthesis in air containing(14)CO2 this mutant incorporated 26% of the(14)C carbon into phosphoglycollate, a compound not normally labelled in wild type (cv. Maris Mink) leaves.The activity of phosphoglycollate phosphatase (EC 3.1.1.18) was 1.2 nkat mg(-1) protein at 30°C in RPr 84/90 compared to 19.2 nkat mg(-1) protein in the wild-type leaves. Phosphoglycollate phosphatase activity was not detected after protein separation by electrophoresis of leaf extracts from the mutant on polyacrylamide gels; on linear 5% acrylamide gels three bands with enzyme activity were separated from extracts of wild type plants. Gradient gel electrophoresis followed by activity staining showed two bands in Maris Mink tracks of MW 86,000 and 96,000, but no bands in 84/90. This is the first report of isozymes of phosphoglycollate phosphatase in barley which were absent in the mutant extracts. Our results confirm an earlier report of isozymes of this phosphatase in Phaseolus vulgaris [18].The photosynthetic rate of RPr 84/90 in 1% O2, 350 µl CO2 l(-1) was 9-12 mg CO2 dm(-2) h(-1) at 20°C, whereas the wild-type rate was 27-29 mg CO2 dm(-2) h(-1) at 20°C. In 21% O2, 350 µl CO2 l(-1) the rate was 2-3 mg CO2 dm(-2) h(-1) in the mutant and 20 mg CO2 dm(-2) h(-1) in the wild type.Genetic analysis has shown that the mutation segregates as a single recessive nuclear gene.

Carbon and nitrogen metabolism in barley (Hordeum vulgare L.) mutants lacking ferredoxin-dependent glutamate synthase.

Five mutant lines of barley (Hordeum vulgare L.), which are only able to grow at elevated levels of CO2, contain less than 5% of the wild-type activity of ferredoxin-dependent glutamate synthase (EC 1.4.7.1). Two of these lines (RPr 82/1 and RPr 82/9) have been studied in detail. Leaves and roots of both lines contain normal activities of NADH-dependent glutamate synthase (EC 1.4.1.14) and the other enzymes of ammonia assimilation. Under conditions that minimise photorespiration, both mutants fix CO2 at normal rates; on transfer to air, the rates drop rapidly to 15% of the wild-type. Incorporation of (14)CO2 into sugar phosphates and glycollate is increased under such conditions, whilst incorporation of radioactivity into serine, glycine, glycerate and sucrose is decreased; continuous exposure to air leads to an accumulation of (14)C in malate. The concentrations of malate, glutamine, asparagine and ammonia are all high in air, whilst aspartate, alanine, glutamate, glycine and serine are low, by comparison with the wild-type parent line (cv. Maris Mink), under the same conditions. The metabolism of [(14)C]glutamate and [(14)C]glutamine by leaves of the mutants indicates a very much reduced ability to convert glutamine to glutamate. Genetic analysis has shown that the mutation in RPr 82/9 segregates as a single recessive nuclear gene.

Carbon and nitrogen metabolism in a barley (Hordeum vulgare L.) mutant with impaired chloroplast dicarboxylate transport.

A mutant line, RPr79/2, of barley (Hordeum vulgare L. cv. Maris Mink) has been isolated that has an apparent defect in photorespiratory nitrogen metabolism. The metabolism of (14)C-labelled glutamine, glutamate and 2-oxoglutarate indicates that the mutant has a greatly reduced ability to synthesise glutamate, especially in air, although in-vitro enzyme analysis indicates the presence of wild-type activities of glutamine synthetase (EC 6.3.1.2) glutamate synthase (EC 1.4.7.1 and EC 1.4.1.14) and glutamate dehydrogenase (EC 1.4.1.2). Several characteristics of RPr79/2 are very similar to those described for glutamate-synthase-deficient barley and Arabidopsis thaliana mutants, including the pattern of labelling following fixation of (14)CO2, and the rapid rise in glutamine content and fall in glutamate in leaves on transfer to air. The CO2-fixation rate in RPr79/2 declines much more slowly on transfer from 1% O2 to air than do the rates in glutamate-synthase-deficient plants, and RPr79/2 plants do not die in air unless the temperature and irradiance are high. Analysis of (glutamine+NH3+2-oxoglutarate)-dependent O2 evolution by isolated chloroplasts shows that chloroplasts from RPr79/2 require a fivefold greater concentration of 2-oxoglutarate than does the wild-type for maximum activity. The levels of 2-oxoglutarate in illuminated leaves of RPr79/2 in air are sevenfold higher than in Maris Mink. It is suggested that RPr79/2 is defective in chloroplast dicarboxylate transport.

Altered feedback sensitivity of acetohydroxyacid synthase from valine-resistant mutants of tobacco (Nicotiana tabacum L.).

Acetohydroxyacid synthase (EC 4.1.3.18) has been extracted from leaves of three valine-resistant (Val(r)) tobacco (Nicotiana tabacum) mutants, and compared with the enzyme from the wild-type. The enzyme from all three mutants is appreciably less sensitive to inhibition by leucine and valine than the wild-type. Two of the mutants, Val(r)-1 and Val(r)-6, have very similar enzymes, which under all conditions are inhibited by less than half that found for the wild-type. The other mutant, Val(r)-7, has an enzyme that only displays appreciably different characteristics from the wild-type at high pyruvate or inhibitor concentrations. Enzyme from Val(r)-7 also has a higher apparent Km for pyruvate, threefold greater than the value determined for the wild-type and the other mutants. The sulphonylurea herbicides strongly inhibit the enzyme from all the lines, though the concentrations required for half-maximal inhibition of enzyme from Val(r)-1 and Val(r)-6 are higher than for Val(r)-7 or the wildtype. No evidence has been found for multiple isoforms of acetohydroxyacid synthase, and it is suggested that the valine-resistance of these mutant lines is the result of two different mutations affecting a single enzyme, possibly involving different subunits.

Biochemical characterisation of Nicotiana plumbaginifolia auxotrophs that require branched-chain amino acids.

The biochemical lesions in six amino acid-requiring auxotrophic lines of Nicotiana plumbaginifolia have been investigated, by means of feeding experiments with [(14)C] and unlabelled substrates, and enzyme analysis. Three of the lines require isoleucine for growth, are unable to synthesise 2-oxobutyrate in vivo and have no detectable threonine dehydratase (E.C.4.2.1.16) in vitro. The other three lines require (isoleucine + valine), accumulate [(14)C] dihydroxymethylvalerate when fed [(14)C]-L-threonine, and contain no detectable dihydroxyacid dehydratase (E.C.4.2.1.9).

Intracellular localization of aspartate kinase and the enzymes of threonine and methionine biosynthesis in green leaves.

The intracellular localization of several aspartate pathway enzymes has been studied in pea (Pisum sativum cv Feltham First) and barley (Hordeum vulgare cv Julia) leaves. Protoplast lysates were fractionated by differential or sucrose density gradient centrifugation, in media optimized for each enzyme. The results show that aspartate kinase, homoserine kinase, threonine synthase, and cystathionine gamma-synthase are confined to the chloroplast. Cystathionine beta-lyase appears to be present in several fractions, though more than 50% of the total activity is associated with the chloroplasts. In contrast, neither methionine synthase nor methionine adenosyl-transferase were significantly associated with chloroplasts, and only a small proportion of the methionine synthase was associated with the mitochondrial fraction. Methionine adenosyltransferase, and hence S-adenosylmethionine synthesis, is not found in any organelle fraction. The conclusion is that whereas threonine, like lysine, is synthesized only in the chloroplast, the last step in methionine biosynthesis occurs largely in the cytoplasm.

The development of NAD(P)H-dependent and ferredoxin-dependent glutamate synthase in greening barley and pea leaves.

The activity of NAD(P)H-dependent glutamate synthase (E.C. 1.4.1.14) has been demonstrated in extracts from etiolated shoots of pea (Pisum sativum L.) and barley (Hordeum vulgare L.). This activity does not significantly alter upon greening of the etiolated shoots, and is at a similar level in light-grown material. Ferredoxin-dependent glutamate synthase (E.C. 1.4.7.1) has low activity in etiolated shoots but increases rapidly on greening. In light grown leaves ferredoxin-dependent activity is 30-40-fold higher than NAD(P)H-dependent activity. It is not considered that the NAD(P)H-dependent glutamate synthase plays an important role in ammonia assimilation in the photosynthetic tissue of higher plants.