Phenylpyruvate Contributes to the Synthesis of Fragrant Benzenoid-Phenylpropanoids in Petunia × hybrida Flowers

Abstract

Phenylalanine (Phe) is a precursor for a large group of plant specialized metabolites, including the fragrant volatile benzenoid-phenylpropanoids (BPs). In plants, the main pathway leading to production of Phe is via arogenate, while the pathway via phenylpyruvate (PPY) is considered merely an alternative route. Unlike plants, in most microorganisms the only pathway leading to the synthesis of Phe is via PPY. Here we studied the effect of increased PPY production in petunia on the formation of BPs volatiles and other specialized metabolites originating from Phe both in flowers and leaves. Stimulation of the pathway via PPY was achieved by transforming petunia with PheA^∗ , a gene encoding a bacterial feedback insensitive bi-functional chorismate mutase/prephenate dehydratase enzyme. PheA^∗ overexpression caused dramatic increase in the levels of flower BP volatiles such as phenylacetaldehyde, benzaldehyde, benzyl acetate, vanillin, and eugenol. All three BP pathways characterized in petunia flowers were stimulated in PheA^∗ flowers. In contrast, PheA^∗ overexpression had only a minor effect on the levels of amino acids and non-volatile metabolites both in the leaves and flowers. The one exception is a dramatic increase in the level of rosmarinate, a conjugate between Phe-derived caffeate and Tyr-derived 3,4-dihydroxyphenylacetate, in PheA^∗ leaves. PheA^∗ petunia flowers may serve as an excellent system for revealing the role of PPY in the production of BPs, including possible routes directly converting PPY to the fragrant volatiles. This study emphasizes the potential of the PPY route in achieving fragrance enhancement in flowering plants.

Keywords: PheA∗; benzenoid–phenylpropanoid; fragrant volatiles; phenylpyruvate; specialized metabolism.

FIGURE 1

A schematic diagram of the AAA and BP metabolic networks in petunia flowers. The enzymatic steps performed by the bi-functional feedback insensitive PheA^∗ enzyme are marked in red. Enzymes are marked in bold capital letters. The volatile compounds are marked by colored bold capital letters. Multiple arrows mark several biochemical reactions. The three BP volatile metabolic pathways originating from Phe in petunia flowers are marked in orange (1), blue (2), and purple (3). ADH, arogenate dehydrogenase; ADT, arogenate dehydratase; CM, chorismate mutase; HPPY-AT, hydroxyphenylpyruvate aminotransferase; PAAS, phenylacetaldehyde synthase; PAL, phenylalanine ammonia lyase; PAT, prephenate aminotransferases; PDH, prephenate dehydrogenase; PDT, prephenate dehydratase; PPY-AT, phenylpyruvate aminotransferase.

FIGURE 2

Effect of PheA^∗ protein abundance on the AAAs and shikimate pathway intermediates in the leaves. (A) Accumulation of PheA^∗ protein in the leaves of the five transgenic plants. Immunoblot analysis was performed using anti-HA antibody (1:1000). Lower panel indicates similar protein loading by Amido-black staining. (B) AAA levels in the leaves of the PheA^∗ lines. Results are presented as fold change enhancement (n = 3). Asterisks indicate p ≤ 0.05 statistically significant difference between the PheA^∗ line and control, using ANOVA followed by Dunnett’s post hoc test. Bars on top of the histograms indicate standard errors.

FIGURE 3

Effect of PheA^∗ protein abundance on the levels of the AAAs in PheA^∗ flowers. (A) Accumulation of PheA^∗ protein in the flower petals of the five transgenic plants. Immunoblot analysis was performed using anti-HA antibody (1:1000). Lower panel indicates similar protein loading by Amido-black staining. (B) Levels of the AAAs in flowers of control and the five PheA^∗ transgenic lines. Results are presented as fold change in transgenic vs. control flowers (n = 3). Tyrosine was not identified. Asterisks indicate a statistically significant difference between the PheA^∗ lines and control, using t-test with p ≤ 0.05. Bars on top of the histograms indicate standard errors.

FIGURE 4

Effect of PheA^∗ transgene on the internal pools of volatile metabolites in the petals. Results are presented by heat map hierarchical clustering of the volatiles. Analysis was performed in Expander (Ulitsky et al., 2010). Each column represents a sample from either PheA^∗ or control (Empty 21) flowers (n = 3). Each row represents a volatile. Values of volatiles’ internal pools were centered and scaled for the analysis and are presented by virtual colors as shown in the color key. Supplementary Table S2 presents the original absolute volatile internal pool levels. All volatiles were coded for their groups by key color. Benzenoid–phenylpropanoid volatiles marked by pink circles, short-chain fatty acids and terpenoids were marked by blue and yellow circle, respectively.

FIGURE 5

Effect of the PheA^∗ transgene on the Benzenoid–Phenylpropanoid volatiles in the petals. (A) Fold enhancement of total BP volatiles in PheA^∗ lines in comparison to control (n = 3). (B–M) Fold enhancement of specific BP volatiles. Results are presented according to their biosynthesis in the three BP pathways in petunia petals. Volatiles names are colored according to their pathway as presented in Figure 1 (orange, blue, and purple). Volatiles synthesized directly from Phe (B,C), volatiles synthesized from cinnamate (D–I), and volatiles synthesized in the pathway begins with ferulate (J,K). (L,M) BPs whose biosynthetic pathways have not been identified in petunia. Black and red asterisks indicate statistically significant differences (p ≤ 0.05 and p ≤ 0.1, respectively) between the PheA^∗ lines and the control, using ANOVA followed by Dunnett’s post hoc test after log transformation. Bars on top of the histograms indicate standard errors.

FIGURE 6

A metabolic map describing the effect of overexpression of PheA^∗ on the metabolic profile of petunia flowers and leaves in the two transgenic lines 5 and 8. The BP volatile metabolic pathways originating from Phe in petunia flowers are marked by orange (1), blue (2), and purple (3). The volatile compounds are marked by colored bold capital letters. Enzymatic steps performed by the bi-functional feedback insensitive PheA^∗ enzyme are marked in red. Enzymes are marked in bold capital letters. Multiple arrows mark several biochemical reactions. Dotted gray arrows mark reactions that have been shown in other organisms (production of phenylacetaldehyde from PPY in rose, and production of benzaldehyde from PPY in lactic acid bacteria or spontaneously) and may yet be revealed in petunia. A dashed line separates between primary to specialized metabolites. ADH, arogenate dehydrogenase; ADT, arogenate dehydratase; CM, chorismate mutase; HPPY-AT, hydroxyphenylpyruvate aminotransferase; PAAS, phenylacetaldehyde synthase; PAL, phenylalanine ammonia lyase; PAT, prephenate aminotransferases; PDH, prephenate dehydrogenase; PDT, prephenate dehydratase; PPY-AT, phenylpyruvate aminotransferase.