Rose scent: genomics approach to discovering novel floral fragrance-related genes

Abstract

For centuries, rose has been the most important crop in the floriculture industry; its economic importance also lies in the use of its petals as a source of natural fragrances. Here, we used genomics approaches to identify novel scent-related genes, using rose flowers from tetraploid scented and nonscented cultivars. An annotated petal EST database of approximately 2100 unique genes from both cultivars was created, and DNA chips were prepared and used for expression analyses of selected clones. Detailed chemical analysis of volatile composition in the two cultivars, together with the identification of secondary metabolism-related genes whose expression coincides with scent production, led to the discovery of several novel flower scent-related candidate genes. The function of some of these genes, including a germacrene D synthase, was biochemically determined using an Escherichia coli expression system. This work demonstrates the advantages of using the high-throughput approaches of genomics to detail traits of interest expressed in a cultivar-specific manner in nonmodel plants. EST sequences were submitted to the GenBank database (accession numbers BQ 103855 to BQ 106728).

Figure 1.

Rose Flowers of Cultivars GG and FC at Different Developmental Stages.

Figure 2.

Evaluation of the Microarray Analysis. (A) Overlay image of the microarray hybridized with labeled cDNA originating from FC petals at stage 4 (Cy3) and GG petals at the same stage (Cy5). (B) Overlay image of the same microarray as in (A) hybridized with the same probes but with reverse labeling. (C) Scatterplot of average signal values of three replicates on the microarray. Poly(A)⁺ RNA was isolated from FC petals at stage 4 and from GG petals at the same stage, reverse transcribed, and labeled with Cy5 and Cy3. In the first experiment, FC RNA was labeled with Cy3 and GG RNA was labeled with Cy5, and in the second experiment, labeling was reversed. The ln values of the Cy5-to-Cy3 ratios were plotted.

Figure 3.

Expression Profiles of Selected Rose Genes. (A) Distribution of rose genes according to the ratio of expression in stage-4 FC versus stage-4 GG petals. The ratio for each clone is the average of six replicates taken from two independent experiments. Threshold values are indicated by vertical lines. (B) As in (A), but comparing expression in FC petals at stage 1 versus stage 4. (C) Interloping diagram of differentially expressed genes that exhibited increased expression during FC petal maturation and higher expression in mature (stage 4) FC versus GG petals. The threshold for increased expression was twofold. The number in the overlapping area indicates the number of unique genes that exhibited upregulation in both experiments.

Figure 4.

Alignment of the Deduced Amino Acid Sequence of FC0592 (the Putative Rose Germacrene D Synthase) with Those of Cotton (+)-δ-Cadinene Synthase and Tomato Germacrene C Synthase. Amino acids conserved in all three sequences are shaded black, and those conserved in only two sequences are shaded gray.

Figure 5.

RNA Gel Blot Analysis of the Putative Sesquiterpene Synthase. Total RNA was extracted from FC and GG young (YL) and mature (ML) leaves and from FC and GG petals at different developmental stages (1, 2, 4, and 6) and analyzed for FC0592 expression.

Figure 6.

Functional Analysis of Clone FC0592 in E. coli. Lysates extracted from E. coli overexpressing clone FC0592 were incubated with FPP in assay buffer, and the reaction product was assayed by GC-MS. (A) Single-ion GC-MS chromatogram of hexane extracts of bacterial lysates derived from cells overexpressing FC0592 or from cells overexpressing FC1018 used as a control. (B) The sesquiterpene produced had a mass spectrum corresponding to that of germacrene D (99% identity) compared with the computerized NIST98 library. The Kovac index of this compound also corresponded with that of authentic germacrene D. These properties were identical with those of authentic germacrene D present in the commercial essential oil of Cananga odorata. (C) Chemical structures of FPP and the reaction product germacrene D.

Figure 7.

Germacrene D Emission from Rose Petals during Development. Germacrene D levels were measured using the headspace technique described in Methods. FC and GG flowers were sampled at different stages of flower development. Each point represents an average of three to five individually sampled flowers. Germacrene D was identified by GC-MS.

Figure 8.

Rose Genes with a Putative Role in Secondary Metabolism Whose Expression Is Upregulated during FC Petal Maturation, as Revealed by Microarray and/or RNA Gel Blot Analysis. n.a., not analyzed. ^a Gene functionally identified as an alcohol acetyl-CoA:acetyltransferase. ^b Gene functionally identified as an S-adenosylmethionine:orcinol methyltransferase (OMT1). ^c Gene functionally identified as an S-adenosylmethionine:orcinol methyl ether methyltransferase (OMT2).