Down-Regulation of Phosphoenolpyruvate Carboxylase Kinase in Grapevine Cell Cultures and Leaves Is Linked to Enhanced Resveratrol Biosynthesis

Abstract

In grapevine, trans-Resveratrol (tR) is produced as a defence mechanism against stress or infection. tR is also considered to be important for human health, which increases its interest to the scientific community. Transcriptomic analysis in grapevine cell cultures treated with the defence response elicitor methyl-β-cyclodextrin (CD) revealed that both copies of PHOSPHOENOLPYRUVATE CARBOXYLASE KINASE (PPCK) were down-regulated significantly. A role for PPCK in the defence response pathway has not been proposed previously. We therefore analysed the control of PPCK transcript levels in grapevine cell cultures and leaves elicited with CD. Moreover, phosphoenolpyruvate carboxylase (PPC), stilbene synthase (STS), and the transcription factors MYB14 and WRKY24, which are involved in the activation of STS transcription, were also analysed by RT-qPCR. The results revealed that under CD elicitation conditions PPCK down-regulation, increased stilbene production and loss of PPC activity occurs in both tissues. Moreover, STS transcripts were co-induced with MYB14 and WRKY24 in cell cultures and leaves. These genes have not previously been reported to respond to CD in grape leaves. Our findings thus support the hypothesis that PPCK is involved in diverting metabolism towards stilbene biosynthesis, both for in vitro cell culture and whole leaves. We thus provide new evidence for PEP being redirected between primary and secondary metabolism to support tR production and the stress response.

Keywords: Gamay; PEP carboxylase kinase; Vitis vinifera; cell culture; elicitation; resveratrol biosynthesis regulation.

Figure 1

Analysis of grapevine PPCK and PPC genes expression in Gamay cell cultures in response to elicitation. (A) RT-qPCR analysis of VvPPCK1 and VvPPCK2 in cell cultures treated with elicitors CD, MJ, and CDMJ for 24 h. (B) RT-qPCR analysis of VvPPC1 and VvPPC2 in cell cultures treated with elicitors CD, MJ, and CDMJ for 24 h. Transcript levels were normalized to the reference gene VvEF1-alpha, and values are presented as relative expression to the control treatment. (* p-value < 0.05; ** p-value < 0.01).

Figure 2

Determination of total extractable activity and changes in the phosphorylation levels of PPC in response to elicitation with the combined treatment CDMJ. (a) Specific activity (U/mg) of PPC in native extracts of grapevine Gamay cell cultures upon elicitation with CDMJ at 48 and 72 h. Each bar represents the result of one biological replicate (A, B, C) with triplicate assays normalized to its respective control. One biological replicate is an independent culture flask. (b,c) Relative abundance of PPC surrogate tryptic peptides in native extracts of grapevine Gamay cell cultures upon elicitation with CDMJ at 72 h. MASIDAQLR contains the conserved PPCK phosphorylation target Ser residue [14]. The two other peptides serve as internal reference. Peptides were recovered from an excised 110 kDa SDS-PAGE band digested in-gel with trypsin and spiked with a mixture of the SIL version of the targeted peptides. Each bar is the average of three biological replicates. (b): Abundance of each native peptide relative to its SIL version measured as the peak area ratio acquired in MRM mode. Differences between peptides can be due to the inaccuracy in quantification of the SIL peptides of “SpikeTidesTM L” quality. (c): To highlight the differences in the abundance of peptides between the control and CDMJ, data were normalized versus the control condition for each peptide. Significance of differences between control and CDMJ were determined using Student’s t-test (* p < 0.05).

Figure 3

Analysis of grapevine PPCK and PPC genes expression intact Gamay leaf in response to CD elicitation. (A) RT-qPCR analysis of VvPPCK1 and VvPPCK2 and (B) RT-qPCR analysis of VvPPC1 and VvPPC2 in a leaf treated with CD, up to 96 h. Transcript levels were normalized to the reference gene VvEF1-alpha and are presented as values relative to the level of transcript quantified for the control treatment. (* p-value <0.05; ** p-value < 0.01).

Figure 4

CD elicitation of intact Gamay leaves induced stilbene accumulation. Time-course of stilbenes accumulation in Gamay leaf treated with 50 mM of CD. The stilbenes tR (a), tPiceid (b) and ε-Viniferin (c) were detected in elicited leaves of three independent Gamay plants (A, B, and C). For a better comparison, the abundance of each compound was normalized to the highest value in each plant: % compound X in plant Y = mg/g FW of compound X in plant Y × 100/highest value of mg/g FW of compound X in plant Y. Error bars correspond to MRM analysis of three technical replicates.

Figure 5

Analysis of expression of two grapevine STS paralogs and the TFs VvMYB14 and VvWRKY24 in Gamay cell cultures in response to CD elicitation. RT-qPCR analysis of VvSTS36, VvSTS29, VvMYB14, and VvWRKY24 in Gamay cell cultures treated with 50 mM of CD at 24 and 72 h of incubation. Expression levels were normalized to VvEF1-alpha, and represented as relative expression to control treatment. (* p-value < 0.05; ** p-value < 0.01).

Figure 6

Analysis of expression two grapevine STS paralogs and the TFs VvMYB14 and VvWRKY24 intact Gamay leaf in response to CD elicitation. RT-qPCR analysis of VvSTS36, VvSTS29, VvMYB14, and VvWRKY24 in Gamay leaves treated with 50 mM of CD up to 96 h. Expression levels were normalized to VvEF1-alpha, and represented as relative expression to control treatment. (* p-value < 0.05; ** p-value < 0.01).