Phenylalanine and tyrosine levels are rate-limiting factors in production of health promoting metabolites in Vitis vinifera cv. Gamay Red cell suspension

Abstract

Environmental stresses such as high light intensity and temperature cause induction of the shikimate pathway, aromatic amino acids (AAA) pathways, and of pathways downstream from AAAs. The induction leads to production of specialized metabolites that protect the cells from oxidative damage. The regulation of the diverse AAA derived pathways is still not well understood. To gain insight on that regulation, we increased AAA production in red grape Vitis vinifera cv. Gamay Red cell suspension, without inducing external stress on the cells, and characterized the metabolic effect of this induction. Increased AAA production was achieved by expressing a feedback-insensitive bacterial form of 3-deoxy- D-arabino-heptulosonate 7-phosphate synthase enzyme (AroG (*)) of the shikimate pathway under a constitutive promoter. The presence of AroG(*) protein led to elevated levels of primary metabolites in the shikimate and AAA pathways including phenylalanine and tyrosine, and to a dramatic increase in phenylpropanoids. The AroG (*) transformed lines accumulated up to 20 and 150 fold higher levels of resveratrol and dihydroquercetin, respectively. Quercetin, formed from dihydroquercetin, and resveratrol, are health promoting metabolites that are induced due to environmental stresses. Testing the expression level of key genes along the stilbenoids, benzenoids, and phenylpropanoid pathways showed that transcription was not affected by AroG (*). This suggests that concentrations of AAAs, and of phenylalanine in particular, are rate-limiting in production of these metabolites. In contrast, increased phenylalanine production did not lead to elevated concentrations of anthocyanins, even though they are also phenylpropanoid metabolites. This suggests a control mechanism of this pathway that is independent of AAA concentration. Interestingly, total anthocyanin concentrations were slightly lower in AroG(*) cells, and the relative frequencies of the different anthocyanins changed as well.

Keywords: DAHPS; Vitis vinifera; grape cell suspension; phenylalanine; phenylpropanoids; tyrosine.

Figure 1

Transformation of transgenic Vitis vinifera cv. Gamay cell culture. (A) kanamycin resistant cell culture colonies, from which the four AroG^* lines were chosen. (B) Typical AroG^* cell culture colonies, (C) Growth of the culture in liquid medium.

Figure 2

Accumulation of AroG^* protein in four transgenic Gamay cell culture lines. Immunoblot analysis was performed on protein extracts of the four AroG^* lines and two controls, using anti-HA antibody (1:1000). The major protein band at 43 kDa and the additional band slightly above it represent the mature and the unprocessed AroG^* protein, respectively. Ponceau staining of the gel indicates similar protein loading. The relative intensity of the 43 KDa band was 10.9:3.7:1:2.5 in lines 13, 3, 50, and 51 respectively.

Figure 3

Growth and morphology of AroG^* cell lines. (A) The growth curve of the cultures was determined by the increase in fresh weight from the day of reculturing (day 0) until beginning of cell death. (B) Microscopic photographs (taken by a Leica MZ FLII microscope) of line 13 and control cells during their growth. Bars on top of the histograms indicate standard errors (n = 3).

Figure 4

Effect of AroG^* transformation on total anthocyanin accumulation in the Gamay cell cultures. The increase in total anthocyanin concentration was determined as the increase in OD absorption at 530 nm. Bars indicate standard errors (n = 3).

Figure 5

Effect of AroG^* transformation on the levels of shikimate and AAA metabolites. Levels of metabolites [(A) shikimate, (B) phenylpyruvate, (C) tyrosine, and (D) phenylalanine] are presented as fold increase for each AroG^* line in comparison with the average of the two control lines. Samples were collected at day 9 of growth. Bars on top of the histograms indicate standard errors (In the control n = 6, including 3 samples from each of the two lines, while in the AroG^* lines n = 3). Statistical significance was analyzed by One-Way ANOVA, followed by Dunnett's post–hoc test. Asterisks indicate P ≤ 0.05.

Figure 6

Effect of AroG^* transformation on the levels of AAA-derived specialized metabolites. Levels of metabolites [(A) 3-hydroxy phenylacetate, (B) hydroquinone, (C) 4-hydroxyphenyl β glucopiranoside, (D) p-coumarate, (E) resveratrol, (F) 4-hydroxy benzoate, (G) dihydroquercetin, and (H) epicatechin] are presented as fold change of AroG^* line in comparison with the control line. Bars on top of the histograms indicate standard errors (In the control n = 6, including 3 samples from two lines, while in the AroG^* lines, n = 3). Statistical significance was analyzed by One-Way ANOVA, followed by Dunnett's post-hoc test. Asterisks indicate P ≤ 0.05.

Figure 7

Effect of AroG^* abundance on the cell cultures' anthocyanin aglycon composition. (A) The distribution between the five anthocyanin aglycons in the transgenic lines. (B–F) The concentration of the five aglycons: (B,C) The two major aglycons, cyaniding and peonidin, (D–F) the three minor components. Bars on top of the histograms indicate standard errors (In the control n = 6, including 3 samples from two lines, while in the AroG^* lines, n = 3). Statistical significance was analyzed by One-Way ANOVA, followed by Dunnett's post hoc test. Asterisks indicate P ≤ 0.05.

Figure 8

Effect of AroG^* protein on the transcript levels of key genes related to specialized metabolites. (A) day 6, (B) day 8, and (C) day 9. Bars on top of the histograms indicate standard errors (n = 3). One of the two control lines was included (line 15). Statistical significance was analyzed by One-Way ANOVA, followed by Dunnett's post hoc test. Asterisks indicate P ≤ 0.05.

Figure 9

Summary of the metabolic and transcriptomic changes in the transgenic Gamay cell line with the highest AroG^* abundance, line 13. The scheme summarizes the fold change in the levels of AAA related metabolites in the AroG* lines in comparison to controls. In addition, the genes whose transcript levels were determined in line 13 are marked in red. None of these transcripts showed significant change.