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

MAIN CONCLUSION: 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 1.46 µM, 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.

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.

Identification and Characterization of Jasmonic Acid Methyltransferase Involved in the Formation of Floral Methyl Jasmonate in Hedychium coronarium.

Hedychium coronarium is a popular ornamental flower in tropical and subtropical areas due to its elegant appearance and inviting fragrance. Methyl jasmonate (MeJA) is one of the volatile compounds in the blooming flowers of H. coronarium. However, the molecular mechanism underlying floral MeJA formation is still unclear in H. coronarium. In this study, a total of 12 SABATH family genes were identified in the genome of H. coronarium, and their encoded proteins range from 366 to 387 amino acids. Phylogenetic analysis revealed seven clades in the SABATH family and a JMT ortholog clade, including two HcSABATH members. Combined with expression profiling of HcSABATH members, HcJMT1 was identified as the top candidate gene for floral MeJA biosynthesis. In vitro enzyme assays showed that HcJMT1 can catalyze the production of MeJA from jasmonic acid. Gene expression analysis indicated that HcJMT1 exhibited the highest expression in the labella and lateral petals, the major sites of MeJA emission. During flower development, the two MeJA isomers, major isomers in the products of the HcJMT1 protein, were released after anthesis, in which stage HcJMT1 displayed high expression. Our results indicated that HcJMT1 is involved in the formation of floral MeJA in H. coronarium.

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.

Biosynthesis of methyl (E)-cinnamate in the liverwort Conocephalum salebrosum and evolution of cinnamic acid methyltransferase.

Methyl (E)-cinnamate is a specialized metabolite that occurs in a variety of land plants. In flowering plants, it is synthesized by cinnamic acid methyltransferase (CAMT) that belongs to the SABATH family. While rarely reported in bryophytes, methyl (E)-cinnamate is produced by some liverworts of the Conocephalum conicum complex, including C. salebrosum. In axenically grown thalli of C. salebrosum, methyl (E)-cinnamate was detected as the dominant compound. To characterize its biosynthesis, six full-length SABATH genes, which were designated CsSABATH1-6, were cloned from C. salebrosum. These six genes showed different levels of expression in the thalli of C. salebrosum. Next, CsSABATH1-6 were expressed in Escherichia coli to produce recombinant proteins, which were tested for methyltransferase activity with cinnamic acid and a few related compounds as substrates. Among the six SABATH proteins, CsSABATH6 exhibited the highest level of activity with cinnamic acid. It was renamed CsCAMT. The apparent Km value of CsCAMT using (E)-cinnamic acid as substrate was determined to be 50.5 µM. In contrast, CsSABATH4 was demonstrated to function as salicylic acid methyltransferase and was renamed CsSAMT. Interestingly, the CsCAMT gene from a sabinene-dominant chemotype of C. salebrosum is identical to that of the methyl (E)-cinnamate-dominant chemotype. Structure models for CsCAMT, CsSAMT and one flowering plant CAMT (ObCCMT1) in complex with (E)-cinnamic acid and salicylic acid were built, which provided structural explanations to substrate specificity of these three enzymes. In phylogenetic analysis, CsCAMT and ObCCMT1 were in different clades, implying that methyl (E)-cinnamate biosynthesis in bryophytes and flowering plants originated through convergent evolution.

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.