Seasonal induction of alternative principal pathway for rose flower scent

Abstract

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.

Figure 1. Two alternative principal pathways leading to synthesis of the major floral scent compound 2PE in roses and their activities in winter and summer flowers.

[²H₈]-2PE and [²H₇]-2PE from L-[²H₈]Phe in rose petals, and a comparison of activities in winter and summer flowers. (a) Photo of a representative W-flower and S-flower (left) and individual petals (right). (b) Two alternative principal pathways leading to 2PE. In W-flowers (route a), [²H₈]PAld is synthesized by AADC after a Schiff base formation, the release of carbon dioxide, and the retention of the α-deuterium of L-[²H₈]Phe. In S-flowers (route b), RyAAAT3 forms a Schiff base and releases α-deuterium prior to decarboxylation; [²H₇]PPA is then converted to [²H₇]PAld by PPDC. In both routes, [²H_n, n = 7, 8]PAld is converted to 2PE by PAR. (c) Typical mass spectra of [²H_n, n = 7, 8]-2PE produced by rose protoplasts of W-flowers harvested in January (upper) and S-flowers harvested in August (lower). (d) Ratios of [²H_n, n = 7, 8]-2PE synthesized in the L-[²H₈]Phe feeding experiments in protoplasts prepared in W-flower (left) and S-flower (right). The production ratios of [²H₈]- and [²H₇]-2PE were calculated according to the intensities of the molecular ions at m/z 130 and m/z 129 (upper diagram). The ratios for the benzyl cations (lower diagram) at m/z 98 and m/z 97 were used to determine the chemical structure of each isotopolog. Error bars represent the standard deviation (SD) (n = 3). (e) Kinetic parameters of RyPPDC. Mean ± SD (n = 5). PPA and PA indicate phenylpyruvic acid and pyruvic acid, respectively. (f) Comparison of transcript levels involved in the 2PE pathway in W-flowers and S-flowers. Transcript analysis of the biosynthetic enzyme genes for 2PE in W-flowers (white bars) and S-flowers (gray bars) was performed by real-time PCR. All of the genetic data were standardized to β-actin transcript levels and represent the relative transcript level. The transcript levels of W-flowers were set to 1.0. Error bars represent the standard error (SE) (n = 5); *indicates P < 0.05.

Figure 2. Seasonal changes in the production of [2Hn, n = 7, 8]-2PE and its. 11 biosynthetic enzymes.

(a,b) Seasonal changes in the [2H8]- and [2H7]-2PE ratios (a) and amounts (b) in protoplasts collected each month for 15 months (month 1 is January). Each month, three flowers were used to prepare three independent protoplast samples. The protoplasts were fed with L-[2H8]Phe as described in the Methods. Error bars represent the SD (n = 3). The rose used was R. x hybrida ‘Yves Piaget’. Note that the y-axis scale is different for [2H8]- and [2H7]-2PE in b. (c) Seasonal changes in the average temperature in Mishima City. All of the data were obtained from the Mishim Local Meteorological Observatory. (d) Relationship between the [2H7]-2PE ratio and average temperature. (e) Production of [2Hn, n = 7, 8]-2PE at 4 °C, and at 30 °C. (f) Changes in 2PE-related gene transcript levels in temperature-treated rose petals. All of the genetic data were standardized to β-actin transcript levels and represent the relative transcript amount. Each transcript was quantified from three independent reverse transcription reactions. The transcript levels before incubation were set to 1.0. Closed circles and open circles depict low-temperature (4 °C) and high-temperature (30 °C) treatment, respectively. Error bars represent the SD (n = 3). **P < 0.01 and ***P < 0.001 indicate values that were significantly different.