SABATH Methyltransferases Research Articles

Molecular cloning and biochemical characterization of indole-3-acetic acid methyltransferase from Japanese star anise (Illicium anisatum).

SABATH proteins methylate the carboxyl groups or nitrogen atoms of small plant molecules and play important roles in many developmental processes and plant defense responses. Previous studies have shown that indole-3-acetic acid (IAA) carboxyl methyltransferase (IAMT), a member of the SABATH methyltransferase family, converts IAA into its methyl ester (Me-IAA). We used RNA-seq analysis to identify a putative IAMT gene, IaIAMT, in the ancient angiosperm Illicium anisatum. Functional characterization of the recombinant IaIAMT protein expressed in Escherichia coli showed the highest level of activity with IAA, whereas indole-3-propionic acid and indole-3-butyric acid were not used as substrates. The apparent Km value of IaIAMT using IAA as a substrate was determined to be 122 µM. Phylogenetic analysis and structural modeling of IaIAMT suggested that IaIAMT evolved independently from IAMTs isolated from other plant species, whereas strict substrate specificity toward IAA was conserved in Illicium species, as observed in other plants.

Biosynthesis of kratom opioids.

Mitragynine, an analgesic alkaloid from the plant Mitragyna speciosa (kratom), offers a safer alternative to clinical opioids such as morphine, owing to its more favorable side effect profile. Although kratom has been traditionally used for stimulation and pain management in Southeast Asia, the mitragynine biosynthesis pathway has remained elusive. We embarked on a search for mitragynine biosynthetic genes from the transcriptomes of kratom and other members of the Rubiaceae family. We studied their functions invitro and invivo. Our investigations led to the identification of several reductases and an enol methyltransferase that forms a new clade within the SABATH methyltransferase family. Furthermore, we discovered a methyltransferase from Hamelia patens (firebush), which catalyzes the final step. With the tryptamine 4-hydroxylase from the psychedelic mushroom Psilocybe cubensis, we accomplished the four-step biosynthesis for mitragynine and its stereoisomer, speciogynine in both yeast and Escherichia coli when supplied with tryptamine and secologanin. Although we have yet to pinpoint the authentic hydroxylase and methyltransferase in kratom, our discovery completes the mitragynine biosynthesis. Through these breakthroughs, we achieved the microbial biosynthesis of kratom opioids for the first time. The remarkable enzyme promiscuity suggests the possibility of generating derivatives and analogs of kratom opioids in heterologous systems.

A large conserved family of small-molecule carboxyl methyltransferases identified from microorganisms

Small-molecule carboxyl methyltransferases (CbMTs) constitute a small proportion of the reported methyltransferases, but they have received extensive attention due to their important physiological functions. Most of the small-molecule CbMTs isolated to date originate from plants and are members of the SABATH family. In this study, we identified a type of CbMT (OPCMT) from a group of Mycobacteria, which has a distinct catalytic mechanism from the SABATH methyltransferases. The enzyme contains a large hydrophobic substrate-binding pocket (~400Å3) and utilizes two conserved residues, Thr20 and Try194, to retain the substrate in a favorable orientation for catalytic transmethylation. The OPCMT_like MTs have a broad substrate scope and can accept diverse carboxylic acids enabling efficient production of methyl esters. They are widely (more than 10,000) distributed in microorganisms, including several well-known pathogens, whereas no related genes are found in humans. Invivo experiments implied that the OPCMT_like MTs was indispensable for M. neoaurum, suggesting that these proteins have important physiological functions.

Genome-wide identification and expression pattern analysis of the SABATH gene family in Neolamarckia cadamba.

Plant SABATH methyltransferases are a class of enzymes that catalyze the transfer of the methyl group from S-adenosyl-L-methionine (SAM) to the carboxyl group or the nitrogen group of the substrate to form small molecule methyl esters or N-methylated compounds, which are involved in various secondary metabolite biosynthesis and have important impacts on plant growth, development, and defense reactions. We previously reported the monoterpenoid indole alkaloids (MIAs) cadambine biosynthetic pathway in Neolamarckia cadamba, a woody tree species that provides an important traditional medicine widely used to treat diseases such as diabetes, leprosy, and cancer in Southeast Asia. However, the functions of NcSABATHs in cadambine biosynthesis remain unclear. In this study, 23 NcSABATHs were identified and found to be distributed on 12 of the total 22 chromosomes. Gene structure, conserved motifs, and phylogenetic analysis showed that NcSABATHs could be divided into three groups. According to cis-element analysis, the NcSABATH promoters contained a large number of elements involved in light, plant hormone, and environmental stress responses, as well as binding sites for the BBR-BPC, DOF, and MYB transcription factor families. Based on RNA-seq data and qRT-PCR analysis, the NcSABATH genes exhibited diverse tissue expression patterns. Furthermore, NcSABATH7/22, which clustered with LAMT in the same clade, were both up-regulated under MeJA treatment. The correlation analysis between gene expression and cadambine content showed that NcSABATH7 potentially participated in cadambine biosynthesis. Taken together, our study not only enhanced our understanding of SABATH in N. cadamba but also identified potential candidate genes involved in cadambine biosynthesis.

Discovery of specific catalytic activity toward IAA/FA by LaSABATHs based on genome-wide phylogenetic and enzymatic analysis of SABATH gene family from Larix kaempferi

The SABATH methyltransferases catalyze methylation of small-molecule metabolites, which participate in plant growth, development and defense response. Given lack of genome-wide studies on gymnosperms SABATH family, the formation and functional differentiation mechanism of the Larix kaempferi SABATH gene family was systematically and exhaustively explored by analyzing gene sequence characteristics, phylogenetic relationship, expression pattern, and enzyme activities. Phylogenetic analysis showed that 247 SABATH genes from 14 land plants were divided into 4 clades, and lineage-specific gene duplication events were important factors that contributed to the evolution of the SABATH gene family in gymnosperms and angiosperms. Substrate specificity analysis of 18 Larix SABATH proteins showed that LaSABATHs could catalyze O-methylation of indole-3-acetic acid (IAA) and farnesic acid (FA), N-methylation of theobromine, and S-methylation of thiobenzoic acid. Furthermore, only LaSABATH2 and LaSABATH29 could catalyze O-methylation of FA, and only LaSABATH30 could catalyze O-methylation of IAA. Homology modeling and molecular docking studies showed the hydrogen bond formed between the His188 of LaSABATH30 and IAA and the noticeable hydrophobic IAA-binding pocket may be helpful for IAA methylation. In this study, identification of proteins with significant specific catalytic activity toward FA and IAA provided high-quality candidate genes for forest genetics and breeding.

Evolution of substrate specificity in a maize anthranilate methyltransferase

Secondary metabolites play a key role in mediating pollination and plant defense against herbivory. One such metabolite, methyl anthranilate, is a volatile compound responsible for the recognizable scent of Concord grapes. Methyl anthranilate is commercially used as a bird repellent for golf courses and as a flavoring agent for grape‐flavored foods, beverages, and pharmaceuticals. A variety of plant species such as Zea mays (maize) and Vitis labrusca (fox grape) synthesize methyl anthranilate from anthranilate, an intermediate in tryptophan biosynthesis. Methyl anthranilate biosynthesis is a classic example of convergent evolution; in maize, this reaction is catalyzed by an anthranilate methyltransferase (AAMT1), while in grape, there is a two‐step pathway. Previous studies have found that the ZmAAMT1 SABATH methyltransferase likely evolved from methyltransferases that recognize the plant pathogen response hormone salicylic acid as a substrate. Using site‐directed mutagenesis, we introduced 12 independent missense mutations at 7 key active site residues with the intention of restoring substrate specificity for salicylic acid. This work will allow us to better trace and understand the evolution of anthranilate‐derived specialized metabolism in plants.

A Collaborative Classroom Investigation of the Evolution of SABATH Methyltransferase Substrate Preference Shifts over 120 My of Flowering Plant History.

Next-generation sequencing has resulted in an explosion of available data, much of which remains unstudied in terms of biochemical function; yet, experimental characterization of these sequences has the potential to provide unprecedented insight into the evolution of enzyme activity. One way to make inroads into the experimental study of the voluminous data available is to engage students by integrating teaching and research in a college classroom such that eventually hundreds or thousands of enzymes may be characterized. In this study, we capitalize on this potential to focus on SABATH methyltransferase enzymes that have been shown to methylate the important plant hormone, salicylic acid (SA), to form methyl salicylate. We analyze data from 76 enzymes of flowering plant species in 23 orders and 41 families to investigate how widely conserved substrate preference is for SA methyltransferase orthologs. We find a high degree of conservation of substrate preference for SA over the structurally similar metabolite, benzoic acid, with recent switches that appear to be associated with gene duplication and at least three cases of functional compensation by paralogous enzymes. The presence of Met in active site position 150 is a useful predictor of SA methylation preference in SABATH methyltransferases but enzymes with other residues in the homologous position show the same substrate preference. Although our dense and systematic sampling of SABATH enzymes across angiosperms has revealed novel insights, this is merely the “tip of the iceberg” since thousands of sequences remain uncharacterized in this enzyme family alone.

Specific methylation of (11R)-carlactonoic acid by an Arabidopsis SABATH methyltransferase.

An Arabidopsis S-adenosyl-L-methionine-dependent methyltransferase belonging to the SABATH family catalyzes the specific carboxymethylation of (11R)-carlactonoic acid. Methyl carlactonoate (MeCLA), found in Arabidopsis (Arabidopsis thaliana) as a non-canonical strigolactone (SL), may be a biosynthetic intermediate of various non-canonical SLs and biologically active as a plant hormone. MeCLA is formed from carlactonoic acid (CLA), but the methyltransferases (MTs) converting CLA to MeCLA remain unclear. Previous studies have demonstrated that the carboxymethylation of acidic plant hormones is catalyzed by the same protein family, the SABATH family (Wang et al. in Evol Bioinform 15:117693431986086. https://doi.org/10.1177/1176934319860864 , 2019). In the present study, we focused on the At4g36470 gene, an Arabidopsis SABATH MT gene co-expressed with the MAX1 gene responsible for CLA formation for biochemical characterization. The recombinant At4g36470 protein expressed in Escherichia coli exhibited exclusive activity against naturally occurring (11R)-CLA among the substrates, including CLA enantiomers and a variety of acidic plant hormones. The apparent Km value for (11R)-CLA was 1.46μM, which was relatively smaller than that of the other Arabidopsis SABATH MTs responsible for the carboxymethylation of acidic plant hormones. The strict substrate specificity and high affinity of At4g36470 suggested it is an (11R)-CLA MT. We also confirmed the function of the identified gene by reconstructing MeCLA biosynthesis using transient expression in Nicotiana benthamiana. Phylogenetic analysis demonstrated that At4g36470 and its orthologs in non-canonical SL-producing plants cluster together in an exclusive clade, suggesting that the SABATH MTs of this clade may be involved in the carboxymethylation of CLA and the biosynthesis of non-canonical SLs.

Biosynthesis and emission of methyl hexanoate, the major constituent of floral scent of a night-blooming water lily Victoriacruziana

Among the factors that have made flowering plants the most species-rich lineage of land plants is the interaction between flower and insect pollinators, for which floral scent plays a pivotal role. Water lilies belong to the ANA (Amborellales, Nymphaeales, and Austrobaileyales) grade of basal flowering plants. In this study, Victoria cruziana was investigated as a model night-blooming water lily for floral scent biosynthesis. Four volatile compounds, including three benzenoids and one fatty acid methyl ester methyl hexanoate, were detected from the flowers of V. cruziana during their first bloom, with methyl hexanoate accounting for 45 % of total floral volatile emission. Emission rates were largely constant before significant drop starting at the end of second bloom. To understand the molecular basis of floral scent biosynthesis in V. cruziana, particularly methyl hexanoate, a transcriptome from the whole flowers at the full-bloom stage was created and analyzed. Methyl hexanoate was hypothesized to be biosynthesized by SABATH methyltransferases. From the transcriptome, three full-length SABATH genes designated VcSABATH1-3 were identified. A full-length cDNA for each of the three VcSABATH genes was expressed in Escherichia coli to produce recombinant proteins. When tested in in vitro methyltransferase enzyme assays with different fatty acids, both VcSABATH1 and VcSABATH3 exhibited highest levels of activity with hexanoic acid to produce methyl hexanoate, with the specific activity of VcSABATH1 being about 15 % of that for VcSABATH3. VcSABATH1 and VcSABATH3 showed the highest levels of expression in stamen and pistil, respectively. In phylogenetic analysis, three VcSABATH genes clustered with other water lily SABATH methyltransferase genes including the one known for making other fatty acid methyl esters, implying both a common evolutionary origin and functional divergence. Fatty acid methyl esters are not frequent constituents of floral scents of mesangiosperms, pointing to the importance for the evolution of novel fatty acid methyltransferase for making fatty acid methyl esters in the pollination biology of water lilies.

Biochemical characterization of the jasmonic acid methyltransferase gene from wasabi (Eutrema japonicum).

Methyl jasmonate and jasmonic acid play important roles as signaling molecules in regulating plant development and stress-related responses. Previous studies have shown that jasmonic acid carboxyl methyltransferase (JMT), which belongs to the SABATH methyltransferase gene family, catalyzes the transfer of methyl groups from S-adenosyl-L-methionine to the carboxyl groups of jasmonic acid. In the present study, we used RNA-seq analysis to identify a putative JMT gene, EujJMT, in wasabi (Eutrema japonicum). The EujJMT proteins showed the highest similarity (89% identity) to JMT proteins of Brassica rapa. Functional characterization of a recombinant EujJMT protein expressed in Escherichia coli showed the highest level of activity with jasmonic acid among the different carboxylic acids tested. The apparent Km value of EujJMT using jasmonic acid as substrate was 62.6 µM, which is comparable to the values of known JMTs. Phylogenetic analysis suggested that EujJMT shares a common ancestor with the JMTs of Arabidopsis and Brassica species and that the strict substrate specificity toward jasmonic acid is conserved among Brassicaceae JMTs.

Genome-wide identification and expression analysis of SABATH methyltransferases in tea plant (Camellia sinensis): insights into their roles in plant defense responses

ABSTRACT SABATH methyltransferases convent plant small-molecule metabolites into volatile methyl esters, which play important roles in many biological processes and defense reactions in plants. In this study, a total of 32 SABATH genes were identified in the Camellia sinensis var. sinensis (CSS) genome, which were renamed CsSABATH1 to CsSABATH32. Genome location annotation suggested that tandem duplication was responsible for the expansion of SABATH genes in tea plant. Multiple sequence alignment and phylogenetic analysis showed that the CsSABATHs could be classified into three groups (I, II and III), which were also supported by gene structures and conserved motifs analysis. Group II contained only two CsSABATH proteins, which were closely related to PtIAMT, AtIAMT and OsIAMT. The group III SABATH genes of tea plant exhibited expansion on the CSS genome compared with Camellia sinensis var. assamica (CSA) genome. Based on RNA-seq data, the CsSABATHs exhibited tissue-specific expression patterns, and the members with high expression in buds and young leaves were also obviously upregulated after MeJA treatment. The expression of many transcription factors was significantly correlated with that of different members of the CsSABATH gene family, suggesting a potential regulatory relationship between them. Quantitative real-time PCR (qPCR) expression analysis showed that CsSABATHs could respond to exogenous JA, SA and MeSA treatments in tea plants. RNA-seq data analysis and qPCR validation suggested that CsSABATH8, 11, 16, 25, 29 and 32 might play a special role in plant defense against insect herbivory. These results provide references for evolutionary studies of the plant SABATH family and the exploration of the potential roles of CsSABATHs in tea plant defense responses.

5-Azacytidine downregulates the SABATH methyltransferase genes and augments bixin content in Bixa orellana L. leaves

Bixa orellana L. seeds are an important source of the liposoluble apocarotenoid bixin and the natural hydrosoluble dye norbixin. These natural dyes are used for food, pharmaceutical, and cosmetic applications. The participation of methyltransferases has already been demonstrated in the bixin biosynthesis pathway, and the alteration of these genes expression can lead to changes in the production of bixin. Treatment with demethylating agents, such as 5-azacytidine (5-AzaC), was previously related to an increased production of secondary compounds and serves for the study of secondary metabolism pathways in plants. Here, we evaluated for the first time the effect of 5-AzaC on the expression of SABATH methyltransferase genes as well as some bixin pathway candidate genes, global DNA methylation, and bixin, chlorophylls, and total carotenoid content in the leaves of B. orellana plants cultured in vitro. Exposing shoot apexes to 5-AzaC for 48 h affected global genome methylation but did not alter chlorophylls and total carotenoid content in the leaves of B. orellana. Importantly, plants submitted to this demethylating agent exhibited downregulation of the SABATH and CCD4 families of genes, upregulation of the ALDH3H1 gene, and increased bixin content in the leaves, suggesting that this methyltransferase inhibitor can selectively regulate the biosynthesis and production of bixin in B. orellana by an unknown mechanism. Moreover, here we demonstrate the possibility to use 5-AzaC to increase bixin contents in B. orellana plants. The methyltransferase inhibitor, 5-Azacytidine, can selectively regulate the biosynthesis and production of bixin in annatto plant leaves, by upregulating BoALDH3H1 gene and downregulating BoSABATH and BoCCD4 gene families.

Pyrethric acid of natural pyrethrin insecticide: complete pathway elucidation and reconstitution in Nicotiana benthamiana.

In the natural pesticides known as pyrethrins, which are esters produced in flowers of Tanacetum cinerariifolium (Asteraceae), the monoterpenoid acyl moiety is pyrethric acid or chrysanthemic acid. We show here that pyrethric acid is produced from chrysanthemol in six steps catalyzed by four enzymes, the first five steps occurring in the trichomes covering the ovaries and the last one occurring inside the ovary tissues. Three steps involve the successive oxidation of carbon 10 (C10) to a carboxylic group by TcCHH, a cytochrome P450 oxidoreductase. Two other steps involve the successive oxidation of the hydroxylated carbon 1 to give a carboxylic group by TcADH2 and TcALDH1, the same enzymes that catalyze these reactions in the formation of chrysanthemic acid. The ultimate result of the actions of these three enzymes is the formation of 10-carboxychrysanthemic acid in the trichomes. Finally, the carboxyl group at C10 is methylated by TcCCMT, a member of the SABATH methyltransferase family, to give pyrethric acid. This reaction occurs mostly in the ovaries. Expression in N.benthamiana plants of all four genes encoding aforementioned enzymes, together with TcCDS, a gene that encodes an enzyme that catalyzes the formation of chrysanthemol, led to the production of pyrethric acid.

Biochemical characterization in Norway spruce (Picea abies) of SABATH methyltransferases that methylate phytohormones

Indole-3-acetic acid (IAA), gibberellins (GAs), salicylic acid (SA) and jasmonic acid (JA) exist in methyl ester forms in plants in addition to their free acid forms. The enzymes that catalyze methylation of these carboxylic acid phytohormones belong to a same protein family, the SABATH methyltransferases. While the genes encoding these enzymes have been isolated from a small number of flowering plants, little is known about their occurrence and evolution in non-flowering plants. Here, we report the systematic characterization of the SABATH family from Norway spruce (Picea abies), a gymnosperm. The Norway spruce genome contains ten SABATH genes (PaSABATH1-10). Full-length cDNA for each of the ten PaSABATH genes was cloned and expressed in Escherichia coli. Recombinant PaSABATHs were tested for activity with IAA, GA, SA, and JA. Among the ten PaSABATHs, five had activity with one or more of the four substrates. PaSABATH1 and PaSABATH2 had the highest activities with IAA and SA, respectively. PaSABATH4, PaSABATH5 and PaSABATH10 all had JA as a preferred substrate but with notable differences in biochemical properties. The structural basis of PaSABATHs in discriminating various phytohormone substrates was inferred based on structural models of the enzyme-substrate complexes. The phylogeny of PaSABATHs with selected SABATHs from other plants implies that the enzymes methylating IAA are conserved in seed plants whereas the enzymes methylating JA and SA have independent evolution in gymnosperms and angiosperms.

Evolution and Function of the Populus SABATH Family Reveal That a Single Amino Acid Change Results in a Substrate Switch.

Evolutionary mechanisms of substrate specificities of enzyme families remain poorly understood. Plant SABATH methyltransferases catalyze methylation of the carboxyl group of various low molecular weight metabolites. Investigation of the functional diversification of the SABATH family in plants could shed light on the evolution of substrate specificities in this enzyme family. Previous studies identified 28 SABATH genes from the Populus trichocarpa genome. In this study, we re-annotated the Populus SABATH gene family, and performed molecular evolution, gene expression and biochemical analyses of this large gene family. Twenty-eight Populus SABATH genes were divided into three classes with distinct divergences in their gene structure, expression responses to abiotic stressors and enzymatic properties of encoded proteins. Populus class I SABATH proteins converted IAA to methyl-IAA, class II SABATH proteins converted benzoic acid (BA) and salicylic acid (SA) to methyl-BA and methyl-SA, while class III SABATH proteins converted farnesoic acid (FA) to methyl-FA. For Populus class II SABATH proteins, both forward and reverse mutagenesis studies showed that a single amino acid switch between PtSABATH4 and PtSABATH24 resulted in substrate switch. Our findings provide new insights into the evolution of substrate specificities of enzyme families.

Gene cloning and bioinformatics analysis of SABATH methyltransferase in Lonicera japonica var. chinensis

To clone SABATH methyltransferase (rLjSABATHMT) gene in Lonicera japonica var. chinensis, and compare the gene expression and intron sequence of SABATH methyltransferase orthologous in L. japonica with L. japonica var. chinensis. It provide a basis for gene regulate the formation of L. japonica floral scents. The cDNA and genome sequences of LjSABATHMT from L. japonica var. chinensis were cloned according to the gene fragments in cDNA library. The LjSABATHMT protein was characterized by bioinformatics analysis. SABATH family phylogenetic tree were built by MEGA 5.0. The transcripted level of SABATHMT orthologous were analyzed in different organs and different flower periods of L. japonica and L. japonica var. chinensis using RT-PCR analysis. Intron sequences of SABATHMT orthologous were also analyzied. The cDNA of LjSABATHMT was 1 251 bp, had a complete coding frame with 365 amino acids. The protein had the conservative SABATHMT domain, and phylogenetic tree showed that it may be a salicylic acid/benzoic acid methyltransferase. Higher expression of SABATH methyltransferase orthologous was found in flower. The intron sequence of L. japonica and L. japonica var. chinensis had rich polymorphism, and two SNP are unique genotype of L. japonica var. chinensis. The motif elements in two orthologous genes were significant differences. The intron difference of SABATH methyltransferase orthologous could be inducing to difference of gene expression between L. japonica and L. japonica var. chinensis. These results will provide important base on regulating active compounds of L. japonica.

A SABATH Methyltransferase from the moss Physcomitrella patens catalyzes S-methylation of thiols and has a role in detoxification

Known SABATH methyltransferases, all of which were identified from seed plants, catalyze methylation of either the carboxyl group of a variety of low molecular weight metabolites or the nitrogen moiety of precursors of caffeine. In this study, the SABATH family from the bryophyte Physcomitrella patens was identified and characterized. Four SABATH-like sequences (PpSABATH1, PpSABATH2, PpSABATH3, and PpSABATH4) were identified from the P. patens genome. Only PpSABATH1 and PpSABATH2 showed expression in the leafy gametophyte of P. patens. Full-length cDNAs of PpSABATH1 and PpSABATH2 were cloned and expressed in soluble form in Escherichia coli. Recombinant PpSABATH1 and PpSABATH2 were tested for methyltransferase activity with a total of 75 compounds. While showing no activity with carboxylic acids or nitrogen-containing compounds, PpSABATH1 displayed methyltransferase activity with a number of thiols. PpSABATH2 did not show activity with any of the compounds tested. Among the thiols analyzed, PpSABATH1 showed the highest level of activity with thiobenzoic acid with an apparent Km value of 95.5μM, which is comparable to those of known SABATHs. Using thiobenzoic acid as substrate, GC-MS analysis indicated that the methylation catalyzed by PpSABATH1 is on the sulfur atom. The mechanism for S-methylation of thiols catalyzed by PpSABATH1 was partially revealed by homology-based structural modeling. The expression of PpSABATH1 was induced by the treatment of thiobenzoic acid. Further transgenic studies showed that tobacco plants overexpressing PpSABATH1 exhibited enhanced tolerance to thiobenzoic acid, suggesting that PpSABATH1 have a role in the detoxification of xenobiotic thiols.

Methylation of phytohormones by the SABATH methyltransferases

In plants, one of the most common modifications of secondary metabolites is methylation catalyzed by various methyltransferases. Recently, a new class of methyltransferases, the SABATH family of methyltransferases, was found to modify phytohormones and other small molecules. The SABATH methyltransferases share little sequence similarity with other well characterized methyltransferases. Arabidopsis has 24 members of the SABATH methyltransferase genes, and a subset of them has been shown to catalyze the formation of methyl esters with phytohormones and other small molecules. Physiological and genetic analyses show that methylation of phytohormones plays important roles in regulating various biological processes in plants, including stress responses, leaf development, and seed maturation/germination. In this review, we focus on phytohormone methylation by the SABATH family methyltransferases and the implication of these modifications in plant development.

SABATH methyltransferases from white spruce (Picea glauca): gene cloning, functional characterization and structural analysis

Known members of the plant SABATH family of methyltransferases have important biological functions by methylating hormones, signalling molecules and other metabolites. While all previously characterized SABATH genes were isolated from angiosperms, in this article, we report on the isolation and functional characterization of SABATH genes from white spruce (Picea glauca [Moench] Voss), a gymnosperm. Through EST database search, three genes that encode proteins significantly homologous to known SABATH proteins were identified from white spruce. They were named PgSABATH1, PgSABATH2 and PgSABATH3, respectively. Full length cDNAs of these three genes were cloned and expressed in Escherichia coli. The E. coli-expressed recombinant proteins were tested for methyltransferase activity with a large number of compounds. While no activity was detected for PgSABATH2 and PgSABATH3, PgSABATH1 displayed the highest level of catalytic activity with indole-3-acetic acid (IAA). PgSABATH1 was, therefore, renamed PgIAMT1. Under steady-state conditions, PgIAMT1 exhibited apparent Km values of 18.2 microM for IAA. Homology-based structural modelling of PgIAMT1 revealed that the active site of PgIAMT1 is highly similar to other characterized IAMTs from angiosperms. PgIAMT1 showed expression in multiple tissues, with the highest level of expression detected in embryonic tissues. During somatic embryo maturation, a significant reduction in PgIAMT1 transcript levels was observed when developing cotyledons become apparent which is indicative of mature embryos. The biological roles of white spruce SABATH genes, especially those of PgIAMT1, and the evolution of the SABATH family are discussed.