Seasonal induction of alternative principal pathway for rose flower scent

Ecological adaptations to seasonal changes are often observed in the phenotypic traits of plants and animals, and these adaptations are usually expressed through the production of different biochemical end products. In this study, ecological adaptations are observed in a biochemical pathway without alteration of the end products. We present an alternative principal pathway to the characteristic floral scent compound 2-phenylethanol (2PE) in roses. The new pathway is seasonally induced in summer as a heat adaptation that uses rose phenylpyruvate decarboxylase (RyPPDC) as a novel enzyme. RyPPDC transcript levels and the resulting production of 2PE are increased time-dependently under high temperatures. The novel summer pathway produces levels of 2PE that are several orders of magnitude higher than those produced by the previously known pathway. Our results indicate that the alternative principal pathway identified here is a seasonal adaptation for managing the weakened volatility of summer roses.


Purification of RyPPDC
Rose petals were ground in liquid nitrogen. The powder (approximately 100 g) was extracted with 1 l of buffer A [50 mM citrate buffer, pH 6.0, containing 1% polyoxyethylene (10) octylphenyl ether (Tryton X-100)] (Wako Pure Chemicals) in the presence of 100 g of PVPP (Polyclar 10, ISP Japan) and stirred for 2 h at 4°C. The buffer extracts were prepared from rose petals. After filtering with gauze, the filtrate was centrifuged (10,000 ×g, 30 min, 4°C). The supernatant (crude enzyme extract) was fractionated with ammonium sulfate. Proteins that precipitated in 20%-60% saturated ammonium sulfate were re-dissolved in approximately 40 ml of buffer B (20 mM potassium phosphate buffer, pH 7.5). This fraction was dialyzed in buffer B for 4 h at 4°C. The dialyzates were further purified by chromatography using a HiTrap DEAE FF column (5 ml, GE Healthcare) equilibrated with buffer C (20 mM potassium phosphate buffer, pH 7.5, containing 1 mM DTT and 1 mM TPP) and eluted with a linear 0-1.5 M NaCl gradient in the same buffer. Ammonium sulfate was added to the eluted fraction to a concentration of 20%, and the solution was applied to a HiTrap Phenyl HP column (5 ml, GE Healthcare) equilibrated with buffer D (50 mM potassium phosphate buffer, pH 7.0, containing 1 M ammonium sulfate, 1 mM DTT, and 1 mM TPP). The proteins were eluted in buffer D without ammonium sulfate in a stepwise manner: 20% buffer D for eight column volumes (CV), 35% buffer D for 5 CV, 50% buffer D for 10 CV, and 100% buffer D for 5 CV. Further purifications were performed using Superdex 200 14/350 column chromatography (CV 70 ml, GE Healthcare) equilibrated with buffer E (50 mM potassium phosphate buffer, pH 7.0, containing 0.15 M NaCl, 1 mM DTT, and 1 mM TPP). The resulting proteins were separated by SDS-PAGE (7.5% polyacrylamide gel) and visualized by Ag staining (Sil-Best Stain One, Nacalai Tesque, Kyoto, Japan). Peptide sequences were analyzed by Nano-LC-MS/MS.

Preparation and determination of peptide sequences by Nano-LC-MS/MS
Purified proteins were separated by SDS-PAGE, and the major bands were excised from the gel and destained with wash solution [25 mM NH 4 HCO 3 /acetonitrile (1:1 v/v)].
Proteins in the gel pieces were reduced and alkylated by respective treatment with 10 mM dithiothreitol/50 mM NH 4 HCO 3 (45 min at 56°C) and 55 mM iodoacetoamide/50 mM NH 4 HCO 3 (30 min at room temperature). After sequential washings with wash solution and acetonitrile, the proteins were digested with trypsin (sequencing grade modified, Promega) at 37°C overnight. The tryptic peptides were extracted from the gel pieces with 50% acetonitrile containing 1% formic acid, and the extracts were pooled and concentrated in a vacuum centrifuge. The dissolved sample was centrifuged at 20,000 ×g for 10 min at room temperature, and the supernatant was subjected to LC-MS/MS analysis. Peptide assignments were performed using an LC-ESI-LIT-q-TOF mass spectrometer equipped with a Nano Frontier eLD System (Hitachi High-Technologies, Tokyo, Japan) and a nano-flow HPLC, NanoFrontier nLC (Hitachi High-Technologies). The LIT-TOF and CID modes were used for MS detection and peptide fragmentation, respectively. The trypsin-treated sample (10 l) was injected, and the peptides were trapped on a C18 column, Monolith Trap (50 m × 150 mm, Hitachi High Technologies). Peptide separation was achieved using a packed nano-capillary column (capillary-Ex nano mono cap, 0.05 × 150 mm, GL Science, Japan) at a flow rate of 200 nl/min. The separated peptides were then ionized with a capillary voltage of 1,700 V. The ionized peptides were detected at a detector potential TOF of 1,850 V. The peptides were eluted using an acetonitrile gradient (A: 2% acetonitrile containing 0.1% formic acid; B: 98% acetonitrile containing 0.1% formic acid; 0 min with A = 98%, B = 2%, followed by 60 min with A = 60%, B = 40%). All peptide mass data were analyzed by using Peaks software (Bioinformatics Solutions Inc.) and the MASCOT database (Matrix Science).

Analyses of gene expression by qRT-PCR
Total RNA was extracted from rose petals using the RNeasy Plant Mini Kit (Qiagen) according to the manufacturer's instructions. For qRT-PCR, cDNA was synthesized using the Quanti-Tect Reverse Transcription Kit (Qiagen). Averaged mRNA expression was normalized to -actin expression. Serial dilutions of a standard solution were included for each gene to generate a standard curve and allow calculation of the input amount of cDNA for each gene. A LightCycler 480 system was incubated at 95°C for 10 min to activate the FastStart Taq DNA polymerase. The run conditions were 60 cycles at 95°C for 10 s, 55°C for 10 s, and 72°C for 10 s. Melting curves of each amplified gene were created to obtain PCR efficiency. Each gene was quantified on the basis of three independent reverse transcription reactions.

SUPPLEMENTARY TABLE AND FIGURE LEGENDS
Supplementary Table 1 Mishima city (red line). There was significant difference (P<0.05) in the number of petal between W-flowers (51.0 ± 1.9, n=5, means ± SE) and S-flowers (57.7 ± 1.4, n=5, means ± SE). In contrast, the total petal weight is significantly lower in S-flower (7.13 ± 0.56 g, means ± SE, n=5, from June to October) than in W-flowers (11.90 ± 0.88 g, means ± SE, n=5, from November to April) (P<0.01). That means petal size was lower