A BAHD acyltransferase contributes to the biosynthesis of both ethyl benzoate and methyl benzoate in the flowers of Lilium oriental hybrid 'Siberia'

Abstract

Lily is a popular flower worldwide due to its elegant appearance and pleasant fragrance. Floral volatiles of lily are predominated by monoterpenes and benzenoids. While a number of genes for monoterpene biosynthesis have been characterized, the molecular mechanism underlying floral benzenoid formation in lily remains unclear. Here, we report on the identification and characterization of a novel BAHD acyltransferase gene that contributes to the biosynthesis of two related floral scent benzoate esters, ethyl benzoate and methyl benzoate, in the scented Lilium oriental hybrid 'Siberia'. The emission of both methyl benzoate and ethyl benzoate in L. 'Siberia' was found to be tepal-specific, floral development-regulated and rhythmic. Through transcriptome profiling and bioinformatic analysis, a BAHD acyltransferase gene designated LoAAT1 was identified as the top candidate gene for the production of ethyl benzoate. In vitro enzyme assays and substrate feeding assays provide substantial evidence that LoAAT1 is responsible for the biosynthesis of ethyl benzoate. It was interesting to note that in in vitro enzyme assay, LoAAT1 can also catalyze the formation of methyl benzoate, which is typically formed by the action of benzoic acid methyltransferase (BAMT). The lack of an expressed putative BAMT gene in the flower transcriptome of L. 'Siberia', together with biochemical and expression evidence, led us to conclude that LoAAT1 is also responsible for, or at least contributes to, the biosynthesis of the floral scent compound methyl benzoate. This is the first report that a member of the plant BAHD acyltransferase family contributes to the production of both ethyl benzoate and methyl benzoate, presenting a new mechanism for the biosynthesis of benzoate esters.

Keywords: BAHD acyltransferase; biosynthesis; ethyl benzoate; floral scent; lily; methyl benzoate.

Figure 1

Tepal-specific and floral development-regulated emission of methyl benzoate and ethyl benzoate in L. ‘Siberia’. (A, B) Emission of methyl benzoate (A) and ethyl benzoate (B) in different tissues of L. ‘Siberia’. (C, D) Changes in floral methyl benzoate (C) and ethyl benzoate (D) emissions during flower development. (E) Representative images of flowers in different floral developmental stages. Error bars indicate the standard deviation of three biological replicates. Different lowercase letters labelled on bars indicate statistically significant differences at the level of P < 0.05.

Figure 2

Rhythmic emission of methyl benzoate and ethyl benzoate in L. ‘Siberia’ flowers. (A) Changes in floral volatile methyl benzoate (A) and ethyl benzoate (B) during two consecutive light/dark (12-h/12-h) cycles on the day after full-opening. White and grey areas correspond to light and dark, respectively. Error bars indicate standard deviation of three biological replicates. Different lowercase letters labeled on bars indicate the statistically significant differences at the level of P < 0.05.

Figure 3

Expression profiles of genes encoding enzymes possibly involved in the biosynthesis of methyl benzoate and ethyl benzoate in L. ‘Siberia’. The enzyme’s abbreviated name for each catalytic step is shown in bold. Broken arrows represent proposed catalytic steps not yet described in plants. Gene expression levels (RPKM+1) with log₂ transformation are represented by color gradation. The three color blocks in the row signify the tepal samples in the bud stage, the blooming stage at 16:00 (BM_L) and the blooming stage at 4:00 (BM_D). The color blocks in the column represent different homologue genes that are termed in Arabic numerical order from top to bottom. AAT, alcohol acyltransferase; BALD, benzaldehyde dehydrogenase; BSMT, benzoic acid/salicylic acid carboxyl methyltransferase; CHD, cinnamoyl-CoA hydratase-dehydrogenase; CNL, cinnamoyl-CoA ligase; PAL, phenylalanine ammonialyase; KAT, 3-ketoacyl CoA thiolase.

Figure 4

Phylogenetic analysis of BAHD members in L. ‘Siberia’. The phylogenetic tree was constructed based on protein sequences of functionally characterized members in the BAHD family using the neighbor-joining method. AAT orthologues in clade Va are shaded in grey. The lily BAHD members are displayed with the prefix “Unigene”. The numbers at each branch indicate bootstrap percentages from 1000 replicates. GenBank accession numbers are shown behind their corresponding enzyme name. Ac, Actinidia chinensis; At, Arabidopsis thaliana; Ca, Capsicum annuum; Cb, Clarkia breweri; Cm, Cucumis melo; Cr, Catharanthus roseus; Dc, Dianthus caryophyllus; Dv, Dahlia variabilis; Fa, Fragaria ananassa; Fv, F. vesca; Gh, Glandularia hybrida; Gm, Glycine max; Gt, Gentiana trifloral; Hv, Hordeum vulgare; Ih, Iris hollandica; Lo, Lilium oriental hybrid; Lp, Lamium purpureum; Lt, Larrea tridentate; Md, Malus domestica; Ms, Musa sapientum; Mt, Medicago truncatula; Nt, Nicotiana tabacum; Ob, Ocimum basilicum; Os, Oryza sativa; Pf, Perilla frutescens; Ph, Petunia hybrid; Pm, Prunus mume; Pp, P. persica; Ps, Papaver somniferum; Rh, Rosa hybrida; Rs, Rauvolfia serpentine; Sb, Sorghum bicolor; Sc, Senecio cruentus; Sl, Solanum lycopersicum; Ss, Salvia splendens; Vl, Vitis labrusca; Zm, Zea mays.

Figure 5

Characterization of LoAAT1 in vitro. (A-D) Total ion chromatogram (TIC) of the products generated by recombinant LoAAT1 (C, D) and empty vector control (A, B) incubated with the alcohol substrates ethanol (A, C) or methanol (B, D) using benzoyl-CoA as the acyl donor. (E, F) TIC of ethyl benzoate (E) and methyl benzoate (F) authentic standards. Insets in panels (C-F) represent the mass spectra of the corresponding main peaks.

Figure 6

Tepal-specific, floral development-regulated and rhythmic expression of the LoAAT1 gene in L. ‘Siberia’. (A) The expression pattern of LoAAT1 in different tissues. (B, C) Expression analysis of LoAAT1 in the outer (B) and inner tepals (C) at different floral developmental stages. (D) Changes in LoAAT1 transcripts in tepals during two consecutive light/dark (12-h/12-h) cycles on the day after full-opening. White and grey areas correspond to light and dark, respectively. Error bars indicate the calculated maximum and minimum expression quantity of three replicates. Different lowercase letters labelled on bars indicate statistically significant differences at the level of P < 0.05.

Figure 7

Floral benzoate ester emission (A) and LoAAT1 orthologous expression (B) in scented and nonscented Lilium species. Relative expression level of LoAAT1 in tepals of L. ‘Siberia’ was set to 1 (100%). Different lowercase letters labelled on bars indicate statistically significant differences at the level of P < 0.05 (n=3).