Functional characterization of a bark-specific monoterpene synthase potentially involved in wounding- and methyl jasmonate-induced linalool emission in rubber (Hevea brasiliensis).

Rubber (Hevea brasiliensis) is a latex-producing plant that often encounters mechanical wounding, as well as pathogen and pest attacks through wound sites during and after tapping. Terpenoids play an important role in the ecological interactions of many plant species, and their diversity is mainly generated by enzymes known as terpene synthases (TPS). In this study, one cDNA sequence encoding a putative terpene synthase, HbTPS20, was obtained from the bark tissues of H. brasiliensis. The encoded protein contains 610 amino acids with a putative N-terminal plastid transit peptide of approximately 70 residues. It belongs to the TPS-b subfamily. Further phylogenetic analysis showed that HbTPS20 formed a separate branch that diverged from the progenitor of all other potentially functional terpene synthases of the rubber TPS-b subfamily. The truncated HbTPS20 without the signal peptide coding sequence was successfully expressed in E. coli and in vitro enzymatic assays with geranyl diphosphate (GPP) or neryl diphosphate (NPP) as a substrate defined HbTPS20 as an active linalool synthase (HbLIS) with the ability to produce linalool as the principal product. RT-qPCR analysis showed that the highest transcript levels of HbTPS20 were found in barks, and this gene was expressed at 2.26- and 250-fold greater levels in the bark tissues of wounded and MeJA-treated plants, respectively, than in those of the control plants. This indicates that this gene may be involved in the induced stress responses of rubber.

Identification and Functional Characterization of a Soybean (Glycine max) Thioesterase that Acts on Intermediates of Fatty Acid Biosynthesis.

(1) Background: Plants possess many acyl-acyl carrier protein (acyl-ACP) thioesterases (TEs) with unique specificity. One such TE is methylketone synthase 2 (MKS2), an enzyme with a single-hotdog-fold structure found in several tomato species that hydrolyzes 3-ketoacyl-ACPs to give free 3-ketoacids. (2) Methods: In this study, we identified and characterized a tomato MKS2 homolog gene, namely, GmMKS2, in the genome of soybean (Glycine max). (3) Results: GmMKS2 underwent alternative splicing to produce three alternative transcripts, but only one encodes a protein with thioesterase activity when recombinantly expressed in Escherichia coli. Heterologous expression of the main transcript of GmMKS2, GmMKS2-X2, in E. coli generated various types of fatty acids, including 3-ketoacids-with 3-ketotetradecenoic acid (14:1) being the most abundant-cis-Δ5-dodecanoic acid, and 3-hydroxyacids, suggesting that GmMKS2 acts as an acyl-ACP thioesterase. In plants, the GmMKS2-X2 transcript level was found to be higher in the roots compared to other examined organs. In silico analysis revealed that there is a substantial enrichment of putative cis-regulatory elements related to disease-resistance responses and abiotic stress responses in the promoter of this gene. (4) Conclusions: GmMKS2 showed broad substrate specificities toward a wide range of acyl-ACPs that varied in terms of chain length, oxidation state, and saturation degree. Our results suggest that GmMKS2 might have a stress-related physiological function in G. max.

Characterization of Solanum melongena Thioesterases Related to Tomato Methylketone Synthase 2.

2-Methylketones are involved in plant defense and fragrance and have industrial applications as flavor additives and for biofuel production. We isolated three genes from the crop plant Solanum melongena (eggplant) and investigated these as candidates for methylketone production. The wild tomato methylketone synthase 2 (ShMKS2), which hydrolyzes ß-ketoacyl-acyl carrier proteins (ACP) to release ß-ketoacids in the penultimate step of methylketone synthesis, was used as a query to identify three homologs from S. melongena: SmMKS2-1, SmMKS2-2, and SmMKS2-3. Expression and functional characterization of SmMKS2s in E. coli showed that SmMKS2-1 and SmMKS2-2 exhibited the thioesterase activity against different ß-ketoacyl-ACP substrates to generate the corresponding saturated and unsaturated ß-ketoacids, which can undergo decarboxylation to form their respective 2-methylketone products, whereas SmMKS2-3 showed no activity. SmMKS2-1 was expressed at high level in leaves, stems, roots, flowers, and fruits, whereas expression of SmMKS2-2 and SmMKS2-3 was mainly in flowers and fruits, respectively. Expression of SmMKS2-1 was induced in leaves by mechanical wounding, and by methyl jasmonate or methyl salicylate, but SmMKS2-2 and SmMKS2-3 genes were not induced. SmMKS2-1 is a candidate for methylketone-based defense in eggplant, and both SmMKS2-1 and SmMKS2-2 are novel MKS2 enzymes for biosynthesis of methylketones as feedstocks to biofuel production.