Role of petal-specific orcinol O-methyltransferases in the evolution of rose scent

Abstract

Orcinol O-methyltransferase (OOMT) 1 and 2 catalyze the last two steps of the biosynthetic pathway leading to the phenolic methyl ether 3,5-dimethoxytoluene (DMT), the major scent compound of many rose (Rosa x hybrida) varieties. Modern roses are descended from both European and Chinese species, the latter being producers of phenolic methyl ethers but not the former. Here we investigated why phenolic methyl ether production occurs in some but not all rose varieties. In DMT-producing varieties, OOMTs were shown to be localized specifically in the petal, predominantly in the adaxial epidermal cells. In these cells, OOMTs become increasingly associated with membranes during petal development, suggesting that the scent biosynthesis pathway catalyzed by these enzymes may be directly linked to the cells' secretory machinery. OOMT gene sequences were detected in two non-DMT-producing rose species of European origin, but no mRNA transcripts were detected, and these varieties lacked both OOMT protein and enzyme activity. These data indicate that up-regulation of OOMT gene expression may have been a critical step in the evolution of scent production in roses.

Figure 1.

Absence of OOMT activity in European roses. TLC analysis of OOMT activity in European and Chinese rose petal extracts (20 μg of total protein), incubated with orcinol (1 mm) in the presence of S-adenosyl-l-[methyl-¹⁴C]Met (50 μm). R.g., R. gallica; R.d., Damask rose; R.c., R. chinensis cv Old Blush. Arrows indicate the positions of the origin (Ori) and the reaction product MHT.

Figure 2.

Western-blot analysis of OOMT expression. A, Western-blot analysis of OOMT expression in different organs of R. chinensis cv Old Blush. L, Leaves; Se, sepals; P3, petals at stage 3; P4, petals at stage 4; P5, petals at stage 5; St, stamens; Pi, pistils. Each lane was loaded with 10 μg of total protein. B, Western-blot analysis of OOMT expression in European roses. Total protein extracts from flower organs and leaves of Damask rose and R. gallica were analyzed by western blot, using anti-OOMT antibody. Old Blush (OB) petals were used as positive control. L, Leaves; Se, sepals; P, petals (stage 4); St/Pi, stamens + pistils. Each lane was loaded with 10 μg of total protein.

Figure 3.

Tissue and cellular localization of OOMT proteins. A to C, Immunolocalization of OOMTs in Lady Hillingdon petals. A, Cross sections of young petals (stage 3) were incubated with anti-OOMT polyclonal antibody. B, Higher magnification of adaxial epidermis cells from A. C, Control incubated with preimmune serum. OOMTs were visualized using goat anti-rabbit secondary antibodies coupled to alkaline phosphatase and NBT/BCIP as substrate. D, Ultrastructure of a rose petal adaxial epidermal cell in transverse section (transmission electron microscopy). E to L, Transient expression of GFP fusion proteins following biolistic transformation of rose petal epidermal cells. E to G, Optical sections in a rose epidermal cell expressing GFP. E, Projection of 10× 1 μm Z-series optical sections through the apex of the conical cell. F, One micrometer optical section through the middle of the same cell. G, One micrometer optical section through the base of the same cell. H, Expression of GFP targeted to plastids (projection of 25× 1 μm optical sections). I, Expression of GFP targeted to the ER (projection of 25× 1 μm optical sections). J, Expression of GFP-OOMT fusion protein (projection of 10× 1 μm optical sections through the apex of the conical cell). K, Expression of OOMT-GFP fusion protein, single 1 μm optical section through the labeled structure at the apex of the cell. L, Projection of 25× 1 μm optical sections of the basal region of another cell expressing OOMT-GFP fusion protein. Bars = 10 μm.

Figure 4.

Association of OOMT with rose petal microsomes. A, R. x hybrida cv Anna petals were sampled at development stages 3, 4, and 5. Cell-free extracts (C) from these petals were centrifuged at 150,000g for 1 h and supernatants (S) and pellets (P) were analyzed by western-blot using the anti-OOMT antibody. B, Microsomal membranes, prepared from petals at stage 4, were incubated for 30 min with buffer (1), 2 m NaCl (2), 0.1% Triton X-100 (3), 0.1 m Na₂CO₃ (4), 0.1 m NaOH (5), or 6.8 m urea (6). The membranes were recovered by a centrifugation at 150,000g for 1 h and analyzed by western blot using anti-OOMT antibody.

Figure 5.

Presence of OOMT-like genes in European roses. Agarose gel analysis of the products of PCR reactions using OOMT-specific primers and genomic DNA from different rose varieties as a template. R.c., R. chinensis cv Old Blush; R.d., Damask rose; R.g., R. gallica; R.gig., R. gigantea; R.h., R. x hybrida cv Lady Hillingdon; M, size marker.

Figure 6.

Alignment of proteins deduced from OOMT genes. The complete amino acid sequence of OOMT1 from Old Blush (OB OOMT1, accession no. AJ439741) is shown, and only the variant amino acid residues from the remaining 14 sequences are indicated. Other OOMTs and putative OOMTs are deduced from OOMT genes in Old Blush (OB OOMT2, AJ439742), R. gigantea (RgiOOMT1, AJ786313; RgiOOMT2A, AJ786314; RgiOOMT2B, AJ786315; RgiOOMT2C, AJ786316), Lady Hillingdon (LH OOMT3A, AJ786309; LH OOMT3B, AJ786310; LH OOMT4A, AJ786311; LH OOMT4B, AJ786312), R. gallica (RgaOOMT2A, AJ786304; RgaOOMT2B, AJ786305), and Damask rose (RdaOOMT2A, AJ786306; RdaOOMT2B, AJ786307; RdaOOMT2C, AJ786308). The percentage identity of the different OOMT proteins, compared to OOMT1 from Old Blush, is indicated.

Figure 7.

RT-PCR analysis of OOMT gene expression in different organs of Damask rose, R. gallica, and R. chinensis cv Old Blush. Shown is agarose gel analysis of the RT-PCR reaction products using OOMT-specific primers (top section) and RNA extracted from leaves (L), sepals (Se), petals (P; stage 4), and stamens + pistils (St/Pi). Glyceraldehyde-3-P dehydrogenase was used as a control (bottom section). The positions of DNA size markers are shown to the left in kilobases (kb).

Figure 8.

Characterization of OOMT-GFP fusion proteins expressed in tobacco. A, TLC analysis of OOMT activity in tobacco leaves transiently expressing GFP and GFP fusion proteins. Tobacco leaf sectors expressing GFP, or GFP fusion proteins, were excised 48 h after Agrobacterium-mediated transformation. Purified recombinant R. chinensis cv Old Blush OOMT1 (1) and cell-free extracts (20 μg proteins) of leaf sectors expressing GFP (2), Old Blush GFP-OOMT (3), or Old Blush OOMT-GFP (4) were incubated with orcinol in the presence of S-adenosyl-l-[methyl-¹⁴C]Met. Arrows indicate the positions of the origin (Ori) and the reaction product MHT. B, Western-blot analysis of OOMT-GFP fusion proteins transiently expressed in tobacco. Cell-free extracts (20 μg proteins) of leaf sectors expressing GFP (1), Old Blush OOMT-GFP (2), R. gallica OOMT2A-GFP (3), and R. gallica OOMT2B-GFP (4) were subjected to SDS-PAGE and western blot using anti-OOMT antibody. C, OOMT activity in cell-free extracts (20 μg proteins) of leaf sectors expressing GFP (1), Old Blush OOMT-GFP (2), R. gallica OOMT2A-GFP (3), and R. gallica OOMT2B-GFP (4) incubated with orcinol in the presence of S-adenosyl-l-[methyl-¹⁴C]Met. Bars indicate ±ses.