Gramine Accumulation in Leaves of Barley Grown under High-Temperature Stress

The indole alkaloid gramine is toxic to animals and may play a defensive role in plants. Under certain conditions, shoots of barley cultivars such as `Arimar' and CI 12020 accumulate gramine (N,N-dimethyl-3-aminomethylindole) and lesser amounts of its precursors 3-aminomethylindole (AMI) and N-methyl-3-aminomethylindole (MAMI); other cultivars such as `Proctor' do not. When grown at optimal temperatures (21°C/16°C, day/night), Arimar contained a high level of gramine in the first leaf (approximately 6 milligrams per gram dry weight), but progressively less accumulated in successive leaves so that the gramine level in the shoot as a whole fell sharply with age. In Arimar and CI 12020 plants transferred at the two- to three-leaf stage from 21°C/16°C to supra-optimal temperatures (≥30°C/25°C), there was massive gramine accumulation in leaves which developed at high temperature, so that gramine level in the whole shoot remained high (about 3-8 milligrams per gram dry weight).

Proctor lacked both constitutive gramine accumulation in the first leaf and heat-induced gramine accumulation in later leaves. The following evidence indicates that this results from a lesion in the pathway of synthesis (tryptophan →→ AMI → MAMI → gramine) between tryptophan and AMI. (a) Proctor and Arimar leaves readily absorbed [¹⁴C]gramine, but neither cultivar degraded it extensively. (b) Arimar leaf tissue incorporated [¹⁴C]formate label into the N-methyl groups of gramine and MAMI, and converted [methylene-¹⁴C]tryptophan to AMI, MAMI, and gramine; Proctor leaf tissue did not, even when a trapping pool of unlabeled gramine was supplied. (c) Proctor converted [¹⁴C]MAMI to gramine as actively as Arimar. (d) Proctor incorporated [¹⁴C]formate label into gramine and MAMI when supplied with AMI; the ratio [¹⁴C]gramine/[¹⁴C]MAMI fell with leaf age, suggesting that the two N-methylations involve different enzymes. Inasmuch as Proctor leaf tissue did not methylate added tryptamine or tyramine, the N-methyltransferase(s) of gramine synthesis may be substrate specific.

In sterile culture at optimal temperatures, 10 millimolar gramine did not affect autotrophic growth of Arimar or Proctor plantlets or heterotrophic growth of callus. At supra-optimal temperature, plantlet growth was reduced by gramine although callus growth was not. We speculate that gramine-accumulating cultivars may suffer autotoxic effects at high leaf temperatures.

     

Induction of a Specific N-Methyltransferase Enzyme by Long-Term Heat Stress during Barley Leaf Growth

Previous work showed that the indole alkaloid gramine accumulates in the upper leaves (e.g. the fifth) of barley as a response to high growth temperatures. The biosynthesis of gramine proceeds from tryptophan to 3-aminomethylindole (AMI); sequential N-methylations of AMI then yield N-methyl-3-aminomethylindole (MAMI) and gramine.

To determine whether high-temperature stress increases the activity of gramine pathway enzymes, leaf tissue from plants grown at various temperatures was assayed for N-methyltransferase (NMT) activity using AMI and MAMI as substrates in both in vivo and in vitro assays. NMT activity in expanding fifth leaves was increased 8- to 20-fold by growth at high temperatures (35°C day/30°C night) compared to cool temperatures (15°C/10°C). Several days of high temperature were required for full induction of NMT activity. No induction of NMT activity occurred in leaves which had completed expansion in cool conditions before exposure to high temperature.

To investigate NMT induction at the protein level, NMT activity was purified to homogeneity and used to produce polyclonal antibodies. Throughout enzyme purification, relative NMT activities towards AMI and MAMI remained constant, consistent with a single NMT enzyme. Immunoblot analysis showed that a large increase in NMT polypeptide coincided with induction of NMT activity by heat stress. Our results point to a type of high-temperature regulation of gene expression that is quite distinct from heat shock.

     

Effect of gramine in the resistance of barley seedlings to the aphid Rhopalosiphum padi

Gramine (N,N-Dimethyl-3-aminomethylindole) content in various barley cultivars varied from 0 to 2.6 mmoles/kg fresh weight. Those cultivars which were lacking gramine were the most susceptible to the aphid Rhopalosiphum padi (L.). The population growth rate of R. padi negatively correlated with gramine content in leaves of barley seedlings. In addition, gramine incorporated in artificial diets decreased survival, amount of diet ingested and reproduction of aphids at concentrations similar to those found in plant leaves. Thus, it is suggested that gramine may be one of the factors responsible for the resistance of barley seedlings to R. padi.

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Résumé La teneur de gramine (N,N-dimethyl-3-aminomethyl-indole) dans différentes cultures de seigle présente des variations comprises entre 0 et 2,8 mmoles/Kg (poids frois). Les varietés dépourvues de gramine sont plus sensibles à l'attaque des pucerons. Le taux de croissance de la population des Rhopalosiphum padi a une correlation négative avec la teneur en gramine des feuilles de plantules de seigle. D'ailleurs, la gramine diminue les taux de nourrissement, de survie et de réproduction des pucerons alimentés avec des diètes artificielles contenant des concentrations du produit testé, similaire à celles trouvées pour les feuilles des plantes. Donc, on suggèrent que la gramine peut être un des facteurs responsables de la résistance des plantules de seigle contre l'attaque de Rhopalosiphum padi.

     

The pur3 gene from the pur cluster encodes a monophosphatase essential for puromycin biosynthesis in Streptomyces

The pur3 gene of the puromycin (pur) cluster from Streptomyces alboniger is essential for the biosynthesis of this antibiotic. Cell extracts from Streptomyces lividans containing pur3 had monophosphatase activity versus a variety of mononucleotides including 3'-amino-3'-dAMP (3'-N-3'-dAMP), (N6,N6)-dimethyl-3'-amino-3'-dAMP (PAN-5'-P) and AMP. This is in accordance with the high similarity of this protein to inositol monophosphatases from different sources. Pur3 was expressed in Escherichia coli as a recombinant protein and purified to apparent homogeneity. Similar to the intact protein in S. lividans, this recombinant enzyme dephosphorylated a wide variety of substrates for which the lowest Km values were obtained for the putative intermediates of the puromycin biosynthetic pathway 3'-N-3'-dAMP (Km = 1.37 mM) and PAN-5'-P (Km = 1.40 mM). The identification of this activity has allowed the revision of a previous proposal for the puromycin biosynthetic pathway.     

Biochemical,immunological and genetic characterization of natural gramine-free variants of Hordeum vulgare L.

Many barley cultivars (e.g. Arimar) contain the indole alkaloid gramine, but some do not. Among seven gramine-free cultivars tested, two phenotypic classes were found: those with a normal level of the N-methyltransferase (NMT) activity that catalyzes the last two steps of gramine synthesis (e.g. Proctor); and those having neither NMT activity nor protein recognized by polyclonal antibodies raised against purified NMT (e.g. Morex).A 3 × 3 diallel cross with reciprocals was made using cultivars Arimar, Proctor and Morex. The pattern of occurrence of gramine and NMT activity among the F₁ hybrids suggested that Proctor and Morex carried defective alleles of the same nuclear gene governing an early step in the indole alkaloid pathway, and that Morex also carried a recessive allele at a nuclear locus encoding NMT activity. However, no non-parental alkaloid phenotypes were found in the F₂ generation from an Arimar × Morex cross and the ratio of progeny with gramine to those with no alkaloids was 3 : 1. One explanation of these results is tight linkage between genes controlling two of the steps in gramine biosynthesis.     

Gramine increase associated with rapid and transient systemic resistance in barley seedlings induced by mechanical and biological stresses.

Systemic acquired resistance (SAR) is one of the intriguing issues for studying the mechanism in signal transduction system in a whole plant. We found that SAR and increase of an antifungal compound were induced rapidly and transiently in barley (Hordeum vulgare L. cv. Goseshikoku) by mechanical and biological stresses. One of the major antifungal compounds was identified as an indole alkaloid, gramine (N,N-dimethyl-3-aminomethylindole), by mass spectrum and NMR analyses. Gramine is well known as a constitutive compound of barley, but it increased significantly in the primary and secondary leaves of barley seedlings within 12 h after pruning or inoculating with the powdery mildew fungi of barley (Blumeria graminis f.sp. hordei) and wheat (B. graminis f.sp. tritici). However, in the leaf detached from unwounded seedlings or in the leaf inoculated with the barley powdery mildew fungus, gramine did not increase at all. In the water droplets contacted with barley leaves, the amount of leaked gramine increased dependently upon the time after the seedling was injured mechanically. We also found a tight correlation between gramine increase and enhancement of resistance to the barley powdery mildew fungus in barley leaves treated with an endogenous elicitor. Furthermore, such a systemic resistance was not observed in a barley cultivar Morex that lacks the biosynthetic pathway of gramine. From these results, we conclude that gramine is the excellent marker in rapid and transient systemic acquired resistance in barley.     

Mitochondrial intermediate peptidase: Expression in Escherichia coli and improvement of its enzymatic activity detection with FRET substrates

In the present study, soluble, functionally-active, recombinant human mitochondrial intermediate peptidase (hMIP), a mitochondrial metalloendoprotease, was expressed in a prokaryotic system. The hMIP fusion protein, with a poly-His-tag (6× His), was obtained by cloning the coding region of hMIP cDNA into the pET-28a expression vector, which was then used to transform Escherichia coli BL21 (DE3) pLysS. After isolation and purification of the fusion protein by affinity chromatography using Ni-Sepharose resin, the protein was purified further using ion exchange chromatography with a Hi-trap resource Q column. The recombinant hMIP was characterized by Western blotting using three distinct antibodies, circular dichroism, and enzymatic assays that used the first FRET substrates developed for MIP and a series of protease inhibitors. The successful expression of enzymatically-active hMIP in addition to the FRET substrates will contribute greatly to the determination of substrate specificity of this protease and to the development of specific inhibitors that are essential for a better understanding of the role of this protease in mitochondrial functioning.     

O-Methylation of benzaldehyde derivatives by "lignin specific" caffeic acid 3-O-methyltransferase

Although S-adenosyl-l-methionine (SAM) dependent caffeic acid/5-hydroxyferulic acid 3/5-O-methyltransferase (COMT) is one of the key enzymes in lignin biosynthesis, the present work demonstrates that alfalfa COMT methylates benzaldehyde derivatives more efficiently than lignin pathway intermediates. 3,4-Dihydroxy, 5-methoxybenzaldehyde and protocatechuic aldehyde were the best in vitro substrates for OMT activity in extracts from developing alfalfa stems, and these compounds were preferred over lignin pathway intermediates for 3-O-methylation by recombinant alfalfa COMT expressed in Escherichia coli. OMT activity with benzaldehydes was strongly reduced in extracts from stems of transgenic alfalfa down-regulated in COMT. However, although COMT down-regulation drastically affects lignin composition, it does not appear to significantly impact metabolism of benzaldehyde derivatives in alfalfa. Structurally designed site-directed mutants of COMT showed altered relative substrate preferences for lignin precursors and benzaldehyde derivatives. Taken together, these results indicate that COMT may have more than one role in phenylpropanoid metabolism (but probably not in alfalfa), and that engineered COMT enzymes could be useful for metabolic engineering of both lignin and benzaldehyde-derived flavors and fragrances.     

Role of an indole alkaloid in the resistance of barley seedlings to aphids

The content of the simple indole alkaloid gramine in barley leaves decreased with age. Conversely, susceptibility to aphids increased in older plants. Population growth rate of the greenbug Schizaphis graminum correlated with gramine content of leaves of several barley cultivars. Gramine decreased rate of feeding, survival and reproductive index of aphids feeding on artificial diets at concentrations similar to those found in plant leaves. Thus, it is suggested that gramine plays a role in the resistance of barley seedlings to S. graminum. Benzyl alcohol, a previously reported insect resistance factor from barley, was absent from all barley cultivars analysed.     

Assessment of genetic diversity in Brazilian barley using SSR markers

Barley is a major cereal grown widely and used in several food products, beverage production and animal fodder. Genetic diversity is a key component in breeding programs. We have analyzed the genetic diversity of barley accessions using microsatellite markers. The accessions were composed of wild and domesticated barley representing genotypes from six countries and three breeding programs in Brazil. A total of 280 alleles were detected, 36 unique to Brazilian barley. The marker Bmag120 showed the greatest polymorphism information content (PIC), with the highest mean value found on chromosome three, and the lowest on chromosomes four and six. The wild accessions presented the highest diversity followed by the foreign genotypes. Genetic analysis was performed using Principal Coordinates Analysis, UPGMA clustering, and Bayesian clustering analysis implemented in Structure. All results obtained by the different methods were similar. Loss of genetic diversity has occurred in Brazilian genotypes. The number of alleles detected in genotypes released in 1980s was higher, whereas most of the cultivars released thereafter showed lower PIC and clustered in separate subgroups from the older cultivars. The use of a more diverse panel of genotypes should be considered in order to exploit novel alleles in Brazilian barley breeding programs.     

Extracellular tyrosinase from the fungus Trichoderma reesei shows product inhibition and different inhibition mechanism from the intracellular tyrosinase from Agaricus bisporus

Tyrosinase (EC 1.14.18.1) is a widely distributed type 3 copper enzyme participating in essential biological functions. Tyrosinases are potential biotools as biosensors or protein crosslinkers. Understanding the reaction mechanism of tyrosinases is fundamental for developing tyrosinase-based applications. The reaction mechanisms of tyrosinases from Trichoderma reesei (TrT) and Agaricus bisporus (AbT) were analyzed using three diphenolic substrates: caffeic acid, L-DOPA (3,4-dihydroxy-l-phenylalanine), and catechol. With caffeic acid the oxidation rates of TrT and AbT were comparable; whereas with L-DOPA or catechol a fast decrease in the oxidation rates was observed in the TrT-catalyzed reactions only, suggesting end product inhibition of TrT. Dopachrome was the only reaction end product formed by TrT- or AbT-catalyzed oxidation of L-DOPA. We produced dopachrome by AbT-catalyzed oxidation of L-DOPA and analyzed the TrT end product (i.e. dopachrome) inhibition by oxygen consumption measurement. In the presence of 1.5 mM dopachrome the oxygen consumption rate of TrT on 8 mM L-DOPA was halved. The type of inhibition of potential inhibitors for TrT was studied using p-coumaric acid (monophenol) and caffeic acid (diphenol) as substrates. The strongest inhibitors were potassium cyanide for the TrT-monophenolase activity, and kojic acid for the TrT-diphenolase activity. The lag period related to the TrT-catalyzed oxidation of monophenol was prolonged by kojic acid, sodium azide and arbutin; contrary it was reduced by potassium cyanide. Furthermore, sodium azide slowed down the initial oxidation rate of TrT- and AbT-catalyzed oxidation of L-DOPA or catechol, but it also formed adducts with the reaction end products, i.e., dopachrome and o-benzoquinone.     

Monolignol biosynthesis in microsomal preparations from lignifying stems of alfalfa (Medicago sativa L.)

Microsomal preparations from lignifying stems of alfalfa (Medicago sativa L.) contained coniferaldehyde 5-hydroxylase activity and immunodetectable caffeic acid 3-O-methyltransferase (COMT), and catalyzed the S-adenosyl L-methionine (SAM) dependent methylation of caffeic acid, caffeyl aldehyde and caffeyl alcohol. When supplied with NADPH and SAM, the microsomes converted caffeyl aldehyde to coniferaldehyde, 5-hydroxyconiferaldehyde, and traces of sinapaldehyde. Coniferaldehyde was a better precursor of sinapaldehyde than was 5-hydroxyconiferaldehyde. The alfalfa microsomes could not metabolize 4-coumaric acid, 4-coumaraldehyde, 4-coumaroyl CoA, or ferulic acid. No metabolism of monolignol precursors was observed in microsomal preparations from transgenic alfalfa down-regulated in COMT expression. In most microsomal preparations, the level of the metabolic conversions was independent of added recombinant COMT. Taken together, the data provide only limited support for the concept of metabolic channeling in the biosynthesis of S monolignols via coniferaldehyde.     

Biosynthesis and Metabolism of Indol-3yl-acetic Acid: I. THE NATIVE INDOLES OF BARLEY AND TOMATO SHOOTS

Barley and tomato shoots were examined quantitatively for naturally-occurringindole compounds. Both tissues were found to contain detectablelevels of indol-3yl-acetic acid (IAA), indol-3yl-aldehyde, tryptamine,5-hydroxytryptamine and malonyltryptophan. Tomato shoots alsocontained small amounts of indol-3yl-lactic acid and tryptophol.These two compounds were absent in barley, but this tissue containedsignificant quantities of 3 -aminomethylindole, 3-methylaminomethylindole,gramine, N-methyltryptamine and 5-hydroxy-N-methyltryptamine.The possible importance of these compounds in the biosynthesisof IAA, or in the formation of alkaloids, is discussed.     

An enzyme-coupled continuous spectrophotometric assay for S-adenosylmethionine-dependent methyltransferases

Modification of small molecules and proteins by methyltransferases affects a wide range of biological processes. Here, we report an enzyme-coupled continuous spectrophotometric assay to quantitatively characterize S-adenosyl-L-methionine (AdoMet/SAM)-dependent methyltransferase activity. In this assay, S-adenosyl-L-homocysteine (AdoHcy/SAH), the transmethylation product of AdoMet-dependent methyltransferases, is hydrolyzed to S-ribosylhomocysteine and adenine by recombinant S-adenosylhomocysteine/5'-methylthioadenosine nucleosidase (SAHN/MTAN, EC 3.2.2.9). Subsequently, adenine generated from AdoHcy is further hydrolyzed to hypoxanthine and ammonia by recombinant adenine deaminase (EC 3.5.4.2). This deamination is associated with a decrease in absorbance at 265 nm that can be monitored continuously. Coupling enzymes are recombinant and easily purified. The utility of this assay was shown using recombinant rat protein arginine N-methyltransferase 1 (PRMT1, EC 2.1.1.125), which catalyzes the mono- and dimethylation of guanidino nitrogens of arginine residues in select proteins. Using this assay, the kinetic parameters of PRMT1 with three synthetic peptides were determined. An advantage of this assay is the destruction of AdoHcy by AdoHcy nucleosidase, which alleviates AdoHcy product feedback inhibition of S-adenosylmethionine-dependent methyltransferases. Finally, this method may be used to assay other enzymes that produce AdoHcy, 5'-methylthioadenosine, or compounds that can be cleaved by AdoHcy nucleosidase.     

Synthesis and bacterial expression of a gene encoding the heme domain of assimilatory nitrate reductase

Assimilatory NADH:nitrate reductase (EC 1.6.6.1), a complex Mo-pterin-, cytochrome b(557)-, and FAD-containing protein, catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by higher plants. A codon-optimized gene has been synthesized for expression of the central cytochrome b(557)-containing fragment, corresponding to residues A542-E658, of spinach assimilatory nitrate reductase. While expression of the full-length synthetic gene in Escherichia coli did not result in significant heme domain production, expression of a Y647* truncated form resulted in substantial heme domain production as evidenced by the generation of "pink" cells. The histidine-tagged heme domain was purified to homogeneity using a combination of NTA-agarose and size-exclusion FPLC, resulting in a single protein band following SDS-PAGE analysis with a molecular mass of approximately 13 kDa. MALDI-TOF mass spectrometry yielded an m/z ratio of 12,435 and confirmed the presence of the heme prosthetic group (m/z=622) while cofactor analysis indicated a 1:1 heme to protein stoichiometry. The oxidized heme domain exhibited spectroscopic properties typical of a b-type cytochrome with a visible Soret maximum at 413 nm together with epr g-values of 2.98, 2.26, and 1.49, consistent with low-spin bis-histidyl coordination. Oxidation-reduction titrations of the heme domain indicated a standard midpoint potential (E(o)') of -118 mV. The isolated heme domain formed a 1:1 complex with cytochrome c with a K(A) of 7 microM (micro=0.007) and reconstituted NADH:cytochrome c reductase activity in the presence of a recombinant form of the spinach nitrate reductase flavin domain, yielding a k(cat) of 1.4 s(-1) and a K(m app) for cytochrome c of 9 microM. These results indicate the efficient expression of a recombinant form of the heme domain of spinach nitrate reductase that retained the spectroscopic and thermodynamic properties characteristic of the corresponding domain in the native spinach enzyme.     

In vitro properties of a recombinant flavonol synthase from Arabidopsis thaliana

cDNA corresponding to a flavonol synthase gene from Arabidopsis thaliana was cloned and expressed in Escherichia coli. The recombinant protein was purified to near-homogeneity and the catalytic properties of the enzyme were studied in vitro. Together with kaempferol and apigenin the recombinant protein synthesised the (2R,3S)-cis- and (2S,3S)-trans-isomers of dihydrokaempferol from the (2S)- and (2R)-isomers of naringenin, respectively. Flavanones and dihydroflavanols differing in degree of A- or B-ring hydroxylation were also accepted as substrates.     

An ectotherm homologue of human predicted gene NAT16 encodes histidine N-acetyltransferase responsible for Nα-acetylhistidine synthesis

Background

Nα-Acetylhistidine (NAH) is present in very high concentrations exclusively in the brain and lens of ectothermic vertebrates, including ray-finned fishes, amphibians and reptiles, and not in those of endothermic birds and mammals. Although NAH is known to be synthesized from l-His and acetyl-CoA by histidine N-acetyltransferase (HISAT; EC 2.3.1.33), the gene encoding HISAT has remained unknown for any organism.

Methods

HISAT was purified from the blue mackerel brain, and its partial amino acid sequences were analyzed using mass spectrometry and Edman degradation. Using the sequence information, the corresponding gene was cloned and sequenced. Recombinant proteins encoded by the fish gene and its human homologue were expressed in a cell-free translation system.

Results

HISAT was identified to be a protein encoded by a fish homologue of the human predicted gene NAT16 (N-acetyltransferase 16). HISAT is an unstable enzyme that is rapidly and irreversibly inactivated during preincubation at 37 °C in the absence of acetyl-CoA. In fish brain, the HISAT gene is expressed as two splice variants containing an identical ORF but differing lengths of 5′-UTR. Both variants are expressed exclusively in the fish brain and lens. Interestingly, the recombinant human NAT16 protein, unlike the recombinant fish HISAT, has only trace enzyme activity for NAH synthesis.

Conclusions

These results propose that the function of mammalian NAT16 has been altered from l-His acetylation (NAH synthesis) to another different biological role.

General significance

The molecular identification of HISAT will allow progress in the understanding of the physiological function of NAH in ectothermic vertebrates.