The results described here show that the combined addition of MeJA and CD to V. vinifera
cv. Monastrell albino cell cultures yields a much higher resveratrol accumulation than the sum of the individual additions. In our experiments, the combined treatment increased seven times the yield of resveratrol when compared to CDs alone. This final resveratrol level (1600 μmole gDW-1
) represents an increase between 10- and more than 1000-fold with respect to previous reports [8
]. The expression analysis of this response shows that both elicitors stimulated the expression of PAL
independently of the anthocyanins/isoflavonoids (CHS) and lignins (CCR) pathways and therefore both induce stilbene biosynthetic genes in a highly specific way, in agreement to results reported by Saigne-Soulard et al.[17
]. Furthermore, the synergistic interaction of both elicitors on resveratrol production (Figure ) seems to be the result of their synergistic effect on the expression of biosynthetic genes (Figure ).
In MeJA-treated cells, a significant reduction in cell growth was observed (Figure ) in parallel with a strong induction of the general phenylpropanoid pathway (Figure ), as recently reported for Arabidopsis
cell suspension cultures [18
]. However, although STS
expression was highly induced (Figure ), no significant amounts of resveratrol were detected in the spent media (Figure ). Such discrepancy could be due to either post-transcriptional and/or post-translational regulatory mechanisms [12
CD elicited cells effectively produced significant amounts of resveratrol (Figure ) in correlation with a transient expression of the central phenylpropanoid enzymes and STS
genes (Figure ). Furthermore, most (or all) the resveratrol synthesized up to 72 h remained in the culture medium until the end of the assay (Figure ). CDs are able to form inclusion complexes with stilbene compounds, such as trans-
resveratrol and diethylstilbestrol [13
], which could protect resveratrol from oxidation or glucosylation. This could explain the observation that, although STS
expression dropped after 72 h in the CD treatment (Figure ), the amount of resveratrol stayed constant (Figure ). In contrast to the effect of MeJA, CD treated cells were not altered in their growth, displaying a similar biomass growth curve as control cells (Figure ). Since both complexed and uncomplexed CD molecules remain in the culture medium during the whole assay, the transient gene expression and the high but limited production of resveratrol must be the result of additional regulatory mechanisms. Given the regular growth curve of the CD-treated cell cultures it is tentative to propose that engagement of cells in active division could somehow compete with further production of resveratrol after a transient elicitation response. In fact, Naill & Roberts [21
] observed that most metabolite productive cells in Taxus cuspidata
suspension cultures were in the G0
phase of the cell cycle (i.e. non-cycling cells) and this stage was suggested as the most specialised for accumulation of secondary metabolites.
In agreement with the previous hypothesis, when both CD and MeJA were simultaneously added to the culture medium, they caused a significant reduction in cell growth (Figure ) as well as a sustainable maximum expression of STS
and central phenylpropanoid genes, even after 168 h (Figure ), which was paralleled by a maximum resveratrol accumulation in the medium (Figure ). We believe that the blockage in cell division and metabolic rearrangement likely caused by MeJA [18
] could place the cells in a non-cycling state [21
] allowing a sustained elicitation by CD.
It has been suggested that MeJA may induce a subset of secondary metabolite biosynthetic genes which could modulate expression of genes and accumulation of compounds induced by elicitors [22
]. Although we cannot completely discard this possibility, we show that the synergistic effect observed on resveratrol production is related with a synergistic effect on the expression of the same set of stilbene biosynthetic genes induced by CD (Figure ). The observed effects of MeJA on cell suspension growth and the recent characterization of MeJA effects on Arabidopsis
cell cultures open the possibility to propose an alternative hypothesis to explain this synergy based on the combined effect of MeJA on cell cycle together with a true and strong elicitor like CD. Further experiments will be required to confirm this possibility on the interaction between cell cycle and secondary metabolite biosynthetic gene expression.