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

Abstract

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.

Keywords: Hedychium coronarium; JMT; SABATH methyltransferase; floral volatiles; methyl jasmonate.

Figure 1

Gene structure and chromosome distribution of SABATH members in H. coronarium. (A) Exon–intron structure of HcSABATHs. The frames and bold lines represent the position of the exon and intron, respectively. The numerals in the frames indicate the length of the exon. The gene length is labeled under the last frame of each gene; (B) The distribution of HcSABATHs on H. coronarium chromosomes.

Figure 2

Phylogenetic analysis of HcSABATHs with Arabidopsis and rice SABATHs and other functionally characterized SABATHs. The SABATH members in H. coronarium are labeled with red triangles. The JMT clade is indicated in blue. Ab, Atropa belladonna; Al, Arabidopsis lyrata; Am, Antirrhinum majus; At, Arabidopsis thaliana; Bc, Brassica campestris; Ca, Coffea arabica; Cb, Clarkia breweri; Cc, Coffea canephora; Ce, Cymbidium ensifolium; Cf, C. faberi; Cs, Camellia sinensis; Fv, Fragaria vesca; Na, Nicotiana alata; Ns, N. suaveolens; Ob, Ocimum basilicum; Os, Oryza sativa; Pg, Picea glauca; Ph, Petunia hybrid; Pt, Populus trichocarpa; Sf, Stephanotis floribunda; Sl, Solanum lycopersium; Zm, Zea mays.

Figure 3

Expression profiles of HcSABATH genes in different tissues based on transcriptomic data. P, petals; L, leaves; R, rhizomes.

Figure 4

Alignment of amino acid sequences of HcJMT1 with other plant functionally characterized SABATHs. Amino acid residues shaded in black, gray, and light gray represent 100, 80, and 60% conserved identity, respectively. Dashes indicate gaps inserted for optimal alignment. Residues with “#” below indicate SAM binding sites. The amino acids with “&” below indicate substrate binding sites.

Figure 5

Characterization of HcJMT1 in vitro. (A) Total ion chromatogram of the products generated by incubating the crude protein extracts of the empty vector control with jasmonic acid (JA); (B) Total ion chromatogram of reaction products formed by recombinant HcJMT1 with the substrate JA; (C) Total ion chromatogram of the methyl jasmonate (MeJA) authentic standard. The chromatogram peaks indicated by arrows are different stereoisomers of MeJA; (D) Mass spectra of the reaction products in (B).

Figure 6

Expression of HcJMT1 and emission of MeJA in H. coronarium. (A) Expression analysis of the HcJMT1 gene in different tissues. SS, styles and stigmas; A, anthers; La, labella; LP, lateral petals; Se, sepals; Pe, pedicels; Br, bracts; Le, leaves; AS, aerial stems; Rh, rhizomes; Ro, roots; (B) Expression analysis of the HcJMT1 gene in petals at different floral developmental stages; (C) Emission of floral MeJA isomers at different flower developmental stages. S1, flower bud stage; S2, full-opening stage; S3, 8 h after full-opening; S4, 16 h after full-opening; S5, senescence stage. Different lowercase letters labelled on bars indicate statistically significant differences at the level of p < 0.05.