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Biosynthesis of monoterpene scent compounds in roses

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Science  03 Jul 2015:
Vol. 349, Issue 6243, pp. 81-83
DOI: 10.1126/science.aab0696

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  1. Fig. 1 Correlation map of the expression of RhNUDX1 and quantity of scent compounds found in 10 rose cultivars.

    A nonparametric Spearman correlation test was used. Strengths of correlations are depicted by colors. Dark blue squares with minus signs indicate a significant negative correlation with correlation coefficient r close to –1 (P < 0.05). Dark red squares with plus signs indicate a significant positive correlation with r close to +1 (P < 0.05). BAL, benzyl alcohol and benzaldehyde; CIT, citronellol; DMT, 3,5-dimethoxytoluene; EUG, eugenol and methyleugenol; FAD, hexanal, E-2-hexenal, Z-3-hexenol, E-2-hexenol, 1-hexanol, Z-3-hexenyl acetate, hexyl acetate, and nonanal; FAR, E-α-farnesene, farnesol, farnesal, and farnesyl acetate; GEM, germacrene D, germacrene D-4-ol, and bicyclogermacrene; GER, geraniol, geranial, geranic acid, and geranyl acetate; ION, 3,4-dihydro-α-ionone and dihydro-α-ionol; MON, β-myrcene, Z-β-ocimene, and E-β-ocimene; NER, nerol and neral; PHE, 2-phenylethanol and phenylacetaldehyde; SES, δ-cadinene, elemol, α-cadinol, τ-cadinol, and τ-muurolol; TMB, 1,3,5-trimethoxybenzene.

  2. Fig. 2 Analysis of RhNUDX1 expression in Rosa x hybrida.

    Flower developmental stages were defined as in (16). SE values are indicated by vertical bars (n = 6 replicates). Means with different letters (a, b, c) are significantly different (Tukey’s HSD test, P < 0.05). (A) Expression in young leaves and floral organs at stage 4 (S4), analyzed by quantitative polymerase chain reaction (qPCR). (B) Expression in petals during development (from S1 to S5), analyzed by qPCR. (C) Western blot analysis of RhNUDX1 protein in Rosa x hybrida petals. M, Molecular weight marker; R, recombinant protein produced in E. coli.

  3. Fig. 3 Characterization of RNAi-RhNUDX1 transgenic rose lines.

    (A) Real-time quantitative reverse transcription PCR analyses of RhNUDX1 expression in petals of nontransformed plants, in plants transformed with the Gus reporter gene under the control of the 35S promoter (35S::GUS), and in three transgenic RNAi-RhNUDX1 lines. SE values are indicated by vertical bars (n = 6 replicates). (B) GC-MS analyses of the petal volatile monoterpenes in RNAi-RhNUDX1 transgenic rose lines. Amounts are expressed in micrograms per gram of fresh leaf weight (μg/g FW). SE values are indicated by vertical bars (n = 8 to 12 replicates). For both panels, means with different letters (a and b) are significantly different (Tukey’s HSD test, P < 0.05).

  4. Fig. 4 Functional characterization of RhNUDX1 in vitro and in planta.

    (A) Decrease in GPP and concomitant increase in GP after incubation with recombinant NusA-RhNUDX1 fusion protein. NusA-RhNUDX1 (250 ng) was incubated in the presence of 1 μM GPP at 30°C. At the indicated time points, GPP and GP were quantified by liquid chromatography–mass spectrometry (LC-MS). (B) SDS–polyacrylamide gel electrophoresis analysis of recombinant NusA-RhNUDX1 protein. 1, molecular weight marker; 2, soluble protein extract from NusA-RhNUDX1–expressing E. coli; 3 and 4, purified NusA-RhNUDX1 protein. Molecular weights (in kilodaltons) are indicated. (C) Accumulation of geranyl glycosides after transient expression of RhNUDX1 in N. benthamiana. GES from basil (9) was expressed as a full-length protein including its transit peptide (plastidic GES, 35S::plaGES) and as a truncated protein without transit peptide (cytosolic GES, 35S::cytGES). This latter construct was aimed at comparing GES and RhNUDX1 activity in a cytosolic context. GFP was used as a control. Geranyl glycosides were quantified by LC-MS 96 hours after transformation. Amounts are expressed in micrograms per gram of fresh leaf weight (μg/g FW) of geranyl glucoside equivalent, as means of triplicate assays. Bars indicate SE.

  5. Fig. 5 Hypothetical biogenetic scheme for the formation of free geraniol in rose petals.

    1, dimethylallyl diphosphate; 2, isopentenyl diphosphate; 3, GPP; 4, GP; 5, geraniol.