Evolution of Novel O-methyltransferases from the Vanilla planifolia Caffeic Acid O-methyltransferase

The biosynthesis of many plant secondary compounds involves the methylation of one or more hydroxyl groups, catalyzed by O-methyltransferases (OMTs). Here, we report the characterization of two OMTs, Van OMT-2 and Van OMT-3, from the orchid Vanilla planifolia Andrews. These enzymes catalyze the methylation of a single outer hydroxyl group in substrates possessing a 1,2,3-trihydroxybenzene moiety, such as methyl gallate and myricetin. This is a substrate requirement not previously reported for any OMTs. Based on sequence analysis these enzymes are most similar to caffeic acid O-methyltransferases (COMTs), but they have negligible activity with typical COMT substrates. Seven of 12 conserved substrate-binding residues in COMTs are altered in Van OMT-2 and Van OMT-3. Phylogenetic analysis of the sequences suggests that Van OMT-2 and Van OMT-3 evolved from the V. planifolia COMT. These V. planifolia OMTs are new instances of COMT-like enzymes with novel substrate preferences.     

Substrate specificity of N-methyltransferase involved in purine alkaloids synthesis is dependent upon one amino acid residue of the enzyme

Caffeine (1,3,7-trimethylxanthine) and theobromine (3,7-dimethylxanthine) are the major purine alkaloids in plants. To investigate the diversity of N-methyltransferases involved in purine alkaloid biosynthesis, we isolated the genes homologous for caffeine synthase from theobromine-accumulating plants. The predicted amino acid sequences of N-methyltransferases in theobromine-accumulating species in Camellia were more than 80% identical to caffeine synthase in C. sinensis. However, there was a little homology among the N-methyltransferases between Camellia and Theobroma. The recombinant enzymes derived from theobromine-accumulating plants had only 3-N-methyltransferase activity. The accumulation of purine alkaloids was, therefore, dependent on the substrate specificity of N-methyltransferase determined by one amino acid residue in the central part of the protein.     

Biochemical and structural analysis of substrate promiscuity in plant Mg2+-dependent O-methyltransferases

Plant S-adenosyl-l-methionine-dependent class I natural product O-methyltransferases (OMTs), related to animal catechol OMTs, are dependent on bivalent cations and strictly specific for the meta position of aromatic vicinal dihydroxy groups. While the primary activity of these class I enzymes is methylation of caffeoyl coenzyme A OMTs, a distinct subset is able to methylate a wider range of substrates, characterized by the promiscuous phenylpropanoid and flavonoid OMT. The observed broad substrate specificity resides in two regions: the N-terminus and a variable insertion loop near the C-terminus, which displays the lowest degree of sequence conservation between the two subfamilies. Structural and biochemical data, based on site-directed mutagenesis and domain exchange between the two enzyme types, present evidence that only small topological changes among otherwise highly conserved 3-D structures are sufficient to differentiate between an enzymatic generalist and an enzymatic specialist in plant natural product methylation.     

A distinctly regulated protein repair L-isoaspartylmethyltransferase from Arabidopsis thaliana

Protein-L-isoaspartate (D-aspartate) O-methyltransferases (EC 2.1.1.77) that catalyze the transfer of methyl groups from S-adenosylmethionine to abnormal L-isoaspartyl and D-aspartyl residues in a variety of peptides and proteins are widely distributed in procaryotes and eucaryotes. These enzymes participate in the repair of spontaneous protein damage by facilitating the conversion of L-isoaspartyl and D-aspartyl residues to normal L-aspartyl residues. In this work, we have identified an L-isoaspartyl methyltransferase activity in Arabidopsis thaliana, a dicotyledonous plant of the mustard family. The highest levels of activity were detected in seeds. Using degenerate oligonucleotides corresponding to two highly conserved amino acid regions shared among the Escherichia coli, wheat, and human enzymes, we isolated and sequenced a full-length genomic clone encoding the A. thaliana methyltransferase. Several methyltransferase cDNAs were also characterized, including ones that would encode full-length polypeptides of 230 amino acid residues. Messenger RNAs for the A. thaliana enzyme were found in a variety of tissues that did not contain significant amounts of active enzyme suggesting the possibility of translational or posttranslational controls on methyltransferase levels. We have identified a putative abscisic acid-response element (ABRE) in the 5-untranslated region of the A. thaliana L-isoaspartyl methyltransferase gene and have shown that the expression of the mRNA is responsive to exogenous abscisic acid (ABA), but not to the environmental stresses of salt or drought. The expression of the A. thaliana enzyme appears to be regulated in a distinct fashion from that seen in wheat or in animal tissues.     

Paraquat-resistant lines in Pisum sativum cv. Alaska: biochemical and phenotypic characterization

In plants, the oxygen generated by photosynthesis can be excited to form reactive oxygen species (ROS) under excessive sunlight. Excess ROS including singlet oxygen (¹O₂) inhibit the growth, development and photosynthesis of plants. To isolate ROS-resistant crop plants, we used paraquat (PQ), a generator of O₂ ^·− as a source of screening and mutagen, and obtained two PQ-resistant lines in Pisum sativum, namely R3-1 and R3-2. Both lines showed greater resistance to PQ than their wild type (WT) siblings with respect to germination, root growth, and shoot growth. Biochemical analysis showed differences in these lines, in which ROS-scavenging enzymes undergo changes with a distinguishable increase in Mn-SOD. We further observed that the cytosolic catalases (CATs) in leaves in both lines were shifted in a native-PAGE analysis compared with that of the WT, indicating that the release of bound ¹O₂ was enhanced. Phenotypic analysis revealed distinguishable differences in leaf development, and in flowering time and position. In addition, R3-1 and R3-2 showed shorter individual internode lengths, dwarf plant height, and stronger branching compared with the WT. These results suggested that PQ-induced ROS-resistant Pisum have the potential pleiotropic effects on flowering time and stem branching, and that ROS including ¹O₂ plays not only important roles in plant growth and development as a signal transducer, but also appears as a strong inhibitor for crop yield. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Biological and molecular comparison between localized and systemic acquired resistance induced in tobacco by a Phytophthora megasperma glycoprotein elicitin

We have compared localized (LAR) and systemic (SAR) acquired resistance induced in tobacco by a hypersensitive response (HR) inducing Phytophthora megasperma glycoprotein elicitin. Three different zones were taken into account: LAR, SAR_T and SAR_S. The LAR zone was 5–10 mm wide and surrounded the HR lesion. SAR_T was the tissue of the elicitor-treated leaf immediately beyond the LAR zone. The systemic leaf was called SAR_S. Glycoprotein-treated plants showed enhanced resistance to challenge infection by tobacco mosaic virus (TMV). Disease resistance was similar in SAR_T and SAR_S, and higher in LAR. The expression pattern, in glycoprotein-treated plants, of acidic and basic PR1, PR2, PR3 and PR5 proteins and of O-methyltransferases (OMT), enzymes of the phenylpropanoid pathway, was similar to that in TMV-infected plants. OMT was stimulated in LAR but not in SAR_T and SAR_S. The four classes of acidic and basic PR proteins accumulated strongly in LAR. Reduced amounts of acidic PR1, PR2, PR3 and only minute amounts of basic PR2 and PR3 accumulated in SAR_T and SAR_S. In glycoprotein-treated plants, expression of the acidic and basic PR proteins in LAR and SAR of transgenic NahG and ETR tobacco plants and in LAR of plants treated with inhibitors of salicylic acid accumulation and of ethylene biosynthesis indicated a salicylic acid-dependent signalling pathway for acidic isoform activation and an ethylene-dependent signalling pathway for basic isoform activation.     

A portfolio of plasmids for identification and analysis of carotenoid pathway enzymes: <Emphasis Type="Italic">Adonis aestivalis</Emphasis> as a case study

Carotenoids are indispensable pigments of the photosynthetic apparatus in plants, algae, and cyanobacteria and are produced, as well, by many bacteria and fungi. Elucidation of biochemical pathways leading to the carotenoids that function in the photosynthetic membranes of land plants has been greatly aided by the use of carotenoid-accumulating strains of Escherichia coli as heterologous hosts for functional assays, in vivo, of the otherwise difficult to study membrane-associated pathway enzymes. This same experimental approach is uniquely well-suited to the discovery and characterization of yet-to-be identified enzymes that lead to carotenoids of the photosynthetic membranes in algal cells, to the multitude of carotenoids found in nongreen plant tissues, and to the myriad flavor and aroma compounds that are derived from carotenoids in plant tissues. A portfolio of plasmids suitable for the production in E. coli of a variety of carotenoids is presented herein. The use of these carotenoid-producing E. coli for the identification of cDNAs encoding enzymes of carotenoid and isoprenoid biosynthesis, for characterization of the enzymes these cDNAs encode, and for the production of specific carotenoids for use as enzyme substrates and reference standards, is described using the flowering plant Adonis aestivalis to provide examples. cDNAs encoding nine different A. aestivalis enzymes of carotenoid and isoprenoid synthesis were identified and the enzymatic activity of their products verified. Those cDNAs newly described include ones that encode phytoene synthase, β-carotene hydroxylase, deoxyxylulose-5-phosphate synthase, isopentenyl diphosphate isomerase, and geranylgeranyl diphosphate synthase.     

Tissue-specific and constitutive expression of a hypothetical gene At2g37610 in transgenic Arabidopsis

Hypothetical genes should play important roles in plant growth and development, although their biological functions await elucidation. One of these genes, namely At2g37610, caught our attention during the gene cloning of several salt-tolerant mutants. Promoter-GUS fusion analysis indicated a unique tissue-specific expression pattern of At2g37610 in Arabidopsis. Constitutive expression of the gene under 35S promoter caused obvious morphological changes in transgenic Arabidopsis plants, such as curled rosette leaves and bushy phenotype at maturity. Phenotypic characterization revealed that the cause of the bushy phenotype was the enhanced lateral bud outgrowth at the bottom region of the primary inflorescence, which is different from that of reported mutant plants (bushy or branched) such as max, axr1, and bus mutants. Together, these data suggest that At2g37610 is a possible novel gene related to the regulation of leaf development and shoot patterning.     

Comparative macroarray analysis of morphine containing Papaver somniferum and eight morphine free Papaver species identifies an O-methyltransferase involved in benzylisoquinoline biosynthesis

Benzylisoquinoline alkaloids constitute a group of about 2,500 structures and are mainly produced by plants of the order Ranunculales. But only the opium poppy, Papaver somniferum, and Papaver setigerum are able to produce morphine. In this study, we started to investigate by gene expression analysis the molecular basis for this exceptional biosynthetic ability. A sequencing project from P. somniferum seedlings was initiated using a method based on the amplified fragment length polymorphism technique that resulted in 849 UniGenes. These cDNAs were analysed on macroarrays for differential expression between morphine-containing P. somniferum plants and eight other Papaver species, which accumulate other benzylisoquinolines instead of morphine. Three cDNAs showing increased expression in P. somniferum compared to all the other Papaver species were identified. Whereas two showed no significant homology to any known protein, one putatively encoded an O-methyltransferase. Analysis of substrate specificity of the heterologously expressed protein and mass spectrometric identification of the enzymatic products identified this protein as S-adenosyl-L-methionine:(R,S)-3-hydroxy-N-methylcoclaurine 4-O-methyltransferase (EC 2.1.1.116). Unlike other O-methyltransferases of different positional specificities implicated in benzylisoquinoline metabolism, the enzyme only accepted tetrahydroxylated tetrahydrobenzylisoquinolines as substrates; methylation was tolerated only at the 6-hydroxy position.     

A cost-effective colorimetric assay for phenolic O-methyltransferases and characterization of caffeate 3-O-methyltransferases from Populus trichocarpa

S-Adenosyl-l-methionine (AdoMet)-dependent O-methyltransferases (OMTs) catalyze the transmethylation of a variety of phenolics in bacteria, plants, and humans. To rapidly characterize phenolic OMT activities, we adapted Gibbs’ reagent, the dye originally used for detecting phenols, to develop a convenient assay method for measuring the catalytic properties of enzymatic transmethylation of phenolics. We demonstrated that Gibbs’ reagent reacted with phenolics yielding distinct absorptive characters that we used to further develop the assay to monitor the reactivities of phenolic OMTs. To validate the method, we identified two caffeate/5-hydroxyferulate 3/5-O-methyltransferases (COMTs) from the black cottonwood, Populus trichocarpa. Together with a few other plant type I OMTs, we demonstrated that our Gibbs’ reagent-mediated colorimetric assay could reliably determine the functions and kinetic parameters of phenolic OMTs. Because Gibbs’ reagent reacting with different regioselectively modified phenolics displays different colorimetric properties, the assay method can be used to monitor both substrate specificity and the regioselectivity of phenolic OMTs.     

Tri-trophic consequences of UV-B exposure: plants,herbivores and parasitoids

In this paper we demonstrate a UV-B-mediated link between host plants, herbivores and their parasitoids, using a model system consisting of a host plant Brassica oleracea, a herbivore Plutella xylostella and its parasitoid Cotesia plutellae. Ultraviolet-B radiation (UV-B) is a potent elicitor of a variety of changes in the chemistry, morphology and physiology of plants and animals. Recent studies have demonstrated that common signals, such as jasmonic acid (JA), play important roles in the mechanisms by which plants respond to UV-B and to damage by herbivores. Plant responses elicited by UV-B radiation can affect the choices of ovipositing female insects and the fitness of their offspring. This leads to the prediction that, in plants, the changes induced as a consequence of UV damage will be similar to those elicited in response to insect damage, including knock-on effects upon the next trophic level, predators. In our trials female P. xylostella oviposited preferentially on host plants grown in depleted UV-B conditions, while their larvae preferred to feed on tissues from UV-depleted regimes over those from UV-supplemented ones. Larval feeding patterns on UV-supplemented tissues met the predictions of models which propose that induced defences in plants should disperse herbivory; feeding scars were significantly smaller and more numerous – though not significantly so – than those on host plant leaves grown in UV-depleted conditions. Most importantly, female parasitoids also showed a clear pattern of preference when given the choice between host plants and attendant larvae from the different UV regimes; however, in the case of the female parasitoids, the choice was in favour of potential hosts foraging on UV-supplemented tissues. This study demonstrates the potential for UV-B to elicit a variety of interactions between trophic levels, most likely mediated through effects upon host plant chemistry.     

A Bacterial Indole-3-acetyl-L-aspartic Acid Hydrolase Inhibits Mung Bean (Vigna radiata L.) Seed Germination Through Arginine-rich Intracellular Delivery

Indole-3-acetyl-L-aspartic acid (IAA-Asp) is a natural product in many plant species and plays many important roles in auxin metabolism and plant physiology. IAA-Asp hydrolysis activity is, therefore, believed to affect plant physiology through changes in IAA metabolism in plants. We applied a newly discovered technique, arginine-rich intracellular delivery (AID), to deliver a bacterial IAA-Asp hydrolase into cells of mung bean (Vigna radiata) seeds and measured its effects on mung bean seed germination. IAA-Asp hydrolase inhibited seed germination about 12 h after the enzyme was delivered into cells of mung bean seeds both covalently and noncovalently. Mung bean seed germination was delayed by 36 h when the enzyme protein was noncovalently attached to the AID peptide and longer than 60 h when the enzyme protein was covalently attached to the AID peptide. Root elongation of mung bean plants was inhibited as much as 90% or 80%, respectively, when the IAA-Asp hydrolase was delivered with the AID peptide by covalent or noncovalent association. Further thin-layer chromatography analysis of plant extracts indicated that the levels of IAA increased about 12 h after treatment and reached their peak at 24 h. This result suggests that IAA-Asp hydrolase may increase IAA levels and inhibit seed germination of mung bean plants and that the AID peptide is a new, rapid, and efficient experimental tool to study the in vivo activity of enzymes of interest in plant cells.     

Plant growth and development influenced by transgenic insertion of bacterial chitinolytic enzymes

The role of the chitinolytic enzymes in plants is not necessarilyrestricted to plant defense. Tomato plants transformed with an endochitinaseand a chitobiosidase gene from Streptomyces albidoflavus andgrowth under greenhouse conditions showed a significant reduction in plantheight, and reduced time to flowering compared with the control(non-transformed) plants. The levels of chitobiosidase and endochitinaseactivity in the transgenic tomato plants were positively correlated with earlyflowering, and negatively correlated with plant height. We have not determinedwhether these effects are exclusively due to the expression of the transgenesof endochitinase and chitobiosidase from S. albidoflavus orthe additive effect of these 2 enzymes combined with the endogenouschitinolytic enzymes produced by the plants. However, when control plants were trimmed,early flowering was observed compared with the controls that were not trimmed, whichindicates that wound induced proteins such as chitinolytic enzymes affect thetime of flowering. In addition, the expression of the endochitinase andchitobiosidase genes significantly increased the number of flowers and fruit onthe plants, resulting in an increase in yield of fruit. One of the primarygoals of crop breeding programs is to increase the productivity of plants. These twogenes were directly associated with plant productivity, and should be studied further.     

O-methyltransferase(s)-suppressed plants produce lower amounts of phenolic vir inducers and are less susceptible to Agrobacterium tumefaciens infection

The first step of Agrobacterium tumefaciens/plant interaction corresponds to the activation of a transduction pathway of the bacterium by plant exudate. Phenolic compounds rapidly secreted by wounded plant cells induce the expression of bacterial virulence (vir) genes; however, little is known about their biosynthesis in plant. Here we show that inoculation of an Agrobacterium tumefaciens virulent strain on orthodiphenol-O-methyltransferases-suppressed tobacco plants leads to significantly smaller tumors compared to control plants. These transgenic plants are inhibited for caffeic acid O-methyltransferase class I or II (OMT; EC 2.1.1.6) and/or caffeoyl-coenzyme A O-methyltransferase (CCoAOMT; EC 2.1.1.104) that are involved in monolignol biosynthesis. The significant decrease of tumor size could be suppressed by the pre-activation of bacterial virulence, before inoculation, using acetosyringone a known vir inducer. Total soluble phenolic amounts and cell wall composition analyzed by FT-IR analysis did not show significant differences between transgenic and control plants. The potential of phenolic extracts from control and OMT-suppressed plants to induce virulence was evaluated using an Agrobacterium tumefaciens reporter strain carrying a vir::LacZ gene fusion plasmid. Lower vir-inducing activities were recorded for plants that show inhibition to caffeic acid O-methyltransferase activity. HPLC analysis confirmed that the levels of several phenolic compounds were differently affected by wounding and/or by bacterial inoculation. Statistical correlations were established between tumor sizes, vir-inducing activities, O-methyltransferases proteins accumulations and the levels of various soluble phenolic compounds such as acetosyringone. These results demonstrate the role of the O-methyltransferases of the phenylpropanoid pathway in the early production of soluble Agrobacterium tumefaciens vir inducers.     

Antioxidant Enzyme Isoforms on Gels in Two Poplar Clones Differing in Sensitivity After Exposure to Ozone

The effect of acute ozone (O₃) fumigation on isozyme patterns of superoxide dismutase (SOD), peroxidase (POD) and ascorbate peroxidase (APX) in mature (ML) and young leaves (YL) of two poplar clones, contrasting in O₃-sensitivity was analysed. Untreated leaves of both the O₃-sensitive (O₃-S) clone Eridano of Populus deltoides×P. maximowiczii and the O₃-resistant (O₃-R) clone I-214 of P.×euramericana showed four distinct SOD isoforms with a relative mobility (R_f) of 0.54 (MnSOD), 0.60 (Cu/ZnSOD), 0.65 (unidentified), and 0.71 (Cu/ZnSOD). After O₃-fumigation the activity of the SOD isoforms showed only quantitative variations with respect to control plants. In ML of untreated O₃-R plants seven POD isoforms (R_f= 0.13, 0.19, 0.34, 0.59, 0.64, 0.70 and 0.75) were found, while in YL one isoform (R_f= 0.34) was undetected. Only three POD isoforms in both ML and YL of untreated O₃-S plants were resolved. The electrophoretic pattern of POD in O₃-S leaves was greatly modified by acute O₃-fumigation with the appearance of new isoforms in both YL and ML and the disappearance of an isoform (R_f= 0.13) in YL. Additionally, O₃-exposure induced the appearance of two APX isoforms in YL (R_f= 0.66 and 0.70), and one isoform in ML (R_f= 0.70) of the O₃-S clone. By contrast, the activity of the three APX isoformes (R_f= 0.64, 0.70 and 0.76) detected in O₃-R leaves showed only quantitative variation with respect to untreated plants. From these data it is concluded that: 1) in these poplar hybrids antioxidant enzyme activity is developmentally regulated and greatly affected by acute O₃ stress treatments and 2) the different enzymes activity displayed by the two poplar clones, especially for POD and APX isoformes, could partly explain their distinct O₃-sensitivity.