Characterization of an acyltransferase capable of synthesizing benzylbenzoate and other volatile esters in flowers and damaged leaves of Clarkia breweri

Abstract

A cDNA encoding a protein with 456 amino acids whose sequence shows considerable similarity to plant acyltransferases was identified among 750 Clarkia breweri flower expressed sequence tags. The cDNA was expressed in Escherichia coli, and the protein produced was shown to encode the enzyme benzoyl-coenzyme A (CoA):benzyl alcohol benzoyl transferase (BEBT). BEBT catalyzes the formation of benzylbenzoate, a minor constituent of the C. breweri floral aroma, but it also has activity with a number of other alcohols and acyl CoAs. The BEBT gene is expressed in different parts of the flowers with maximal RNA transcript levels in the stigma, and no expression was observed in the leaves under normal conditions. However, BEBT expression was induced in damaged leaves, reaching a maximum 6 h after damage occurred. We also show here that a closely related tobacco (Nicotiana tabacum) gene previously shown to be induced in leaves after being challenged by phytopathogenic bacteria also has BEBT activity, whereas the most similar protein to BEBT in the Arabidopsis proteome does not use benzoyl CoA as a substrate and instead can use acetyl CoA to catalyze the formation of cis-3-hexen-1-yl acetate, a green-leaf volatile.

Figure 1

The reaction catalyzed by BEBT.

Figure 2

BEBT enzymatic activity in different floral tissues during the lifespan of the flower. Petal and stigma tissues were collected from C. breweri flowers daily starting 2 d before flower opening (d −2) and ending on d 4 postanthesis. For each data point, tissues from three different plants were combined for each assay, at least three independent assays were conducted, and the mean was obtained. ●, Petals; ▪, stigma. pkat, Picomoles of product per second.

Figure 3

Sequence comparisons of BEBT and BEAT from C. breweri, HSR201 from tobacco, and the two most similar proteins to BEBT from Arabidopsis. The BEBT gene accession number is AF500200, and the gene accession number of HSR201 is AF500202. The two Arabidopsis acyltransferase proteins in this figure are identified by their protein accession numbers and correspond to genes from two BAC clones (accession nos. AL391151 and AC009895, respectively). A, Protein sequence alignments of all five proteins using the ClustalX program. Amino acids shaded in black represent identical matches; gray shaded boxes represent conservative changes. The HXXXD motif is indicated with arrowheads; the DFGWG motif is indicated with asterisks. B, Maximum parsimony tree based on protein sequence alignments from A using the program PAUP*.

Figure 4

Purification of C. breweri BEBT produced in E. coli. Lane 1, M_r markers. Lane 2, Q-Superose fraction with the highest levels of BEBT-specific activity.

Figure 5

RNA gel-blot analysis of the relative abundance of BEBT mRNA transcripts in the stigma, stamen, style, sepals, petals, and leaves of C. breweri plants. Tissues were harvested from mature plants and floral tissue samples were taken from flowers on d 1 of anthesis. Lanes were loaded with 4 μg of total RNA. After probing with the BEBT probe and quantitation of the results, each blot was rehybridized with an 18S rDNA probe to normalize samples. A sample blot is shown above. The graphical representation below represents an average of three independent experiments.

Figure 6

Expression of BEBT in petal and stigma tissues of C. breweri flowers during floral development. A, RNA gel-blot analysis of the relative abundance of BEBT mRNA in stigma tissue. Stigma tissue was collected daily from flowers starting 2 d before flower opening and continuing until d 4 postanthesis. B, RNA gel-blot analysis of the relative abundance of BEBT mRNA in petal tissue. Petal tissue was collected at the same times as the tissues in A. For all experiments, each lane was loaded with 4 μg of total RNA. After hybridization with the BEBT probe and quantitation of the results, blots were stripped and reprobed with an 18S rDNA probe to normalize samples. In each panel, a sample blot is shown above, and the graphical representation below represents an average of two independent experiments.

Figure 7

Variation of the levels of BEBT protein over the lifespan of the stigma. Stigma tissue was collected daily from flowers starting 2 d before flower opening, and continuing until d 4 postanthesis. Samples were run on SDS-PAGE, and the gels were blotted onto filters. The filters were first probed with anti-BEBT antibodies, followed by incubation with secondary antibodies conjugated to alkaline phosphatase. Bands were visualized by chemiluminescence. A sample blot is shown above, and the graphical representation below represents an average of two independent experiments.

Figure 8

RNA gel-blot analysis of the relative expression of BEBT in wounded and non-wounded leaf tissues of C. breweri. Mature leaves were harvested either before (untreated) or after mechanical wounding. Leaves were wounded by making two parallel incisions approximately 8 mm long to each side of the midvein with a sterile razor blade, and tissue was collected at 0, 1, 2, 4, 6, 8, 12, and 24 h after wounding. RNA from stigma tissue (included for comparison) was collected from d 1 flowers. Total RNA (4 μg) was loaded onto each lane of the gel. After probing with the BEBT probe and quantitation of the results, the blots were then stripped and reprobed with an 18S rDNA probe to normalize samples. A sample blot is shown above, and the graphical representation below represents an average of two independent experiments.