Flavonoids and darkness lower PCD in senescing Vitis vinifera suspension cell cultures

Abstract

Background: Senescence is a key developmental process occurring during the life cycle of plants that can be induced also by environmental conditions, such as starvation and/or darkness. During senescence, strict control of genes regulates ordered degradation and dismantling events, the most remarkable of which are genetically programmed cell death (PCD) and, in most cases, an upregulation of flavonoid biosynthesis in the presence of light. Flavonoids are secondary metabolites that play multiple essential roles in development, reproduction and defence of plants, partly due to their well-known antioxidant properties, which could affect also the same cell death machinery. To understand further the effect of endogenously-produced flavonoids and their interplay with different environment (light or dark) conditions, two portions (red and green) of a senescing grapevine callus were used to obtain suspension cell cultures. Red Suspension cell Cultures (RSC) and Green Suspension cell Cultures (GSC) were finally grown under either dark or light conditions for 6 days.

Results: Darkness enhanced cell death (mainly necrosis) in suspension cell culture, when compared to those grown under light condition. Furthermore, RSC with high flavonoid content showed a higher viability compared to GSC and were more protected toward PCD, in accordance to their high content in flavonoids, which might quench ROS, thus limiting the relative signalling cascade. Conversely, PCD was mainly occurring in GSC and further increased by light, as it was shown by cytochrome c release and TUNEL assays.

Conclusions: Endogenous flavonoids were shown to be good candidates for exploiting an efficient protection against oxidative stress and PCD induction. Light seemed to be an important environmental factor able to induce PCD, especially in GSC, which lacking of flavonoids were not capable of preventing oxidative damage and signalling leading to senescence.

Keywords: Cell cultures; Flavonoids; PCD; Senescence; Vitis vinifera.

Fig. 1

RP-HPLC analysis of anthocyanins (a) and polyphenolic profile (b) from alcoholic extracts obtained by V. vinifera (cv. Limberger) suspension cell cultures. GSC (dotted line) and RSC (solid line) were grown under light conditions for 6 days. Their metabolite content was determined at day 6. Chromatographic profiles of anthocyanidin glucosides, cyanidin and malvidin, as well as their respective substituted derivatives, are presented. Malvidin glucoside (dashed line) was used as a standard (Panel a). The identification was obtained by mass spectrometry analysis on chromatographic peaks detected at 520 nm. Similar analysis was performed at 320 nm (Panel b) and the retrieved flavonoids were identified as follows: 1) gallic acid; 2) quercetin-diglucoside; 3) quercetin-glucoside; 4) quercetin-glucuronide methyl ester; 5) kaempferol-glucoside; 6) quercetin; 7) kaempferol. Data are representative of three different experiments

Fig. 2

Cell death in V. vinifera (cv. Limberger) suspension cell cultures evaluated by FDA staining. Panel a GSC (b, d) and RSC (a, c) were grown under light conditions for 6 days. After staining with FDA, they were analyzed under fluorescent (c, d) or visible (a, b) light. Panel b time-course of total cell death in GSC and RSC, grown under light or dark conditions, and sampled at day 0, 3 and 6, respectively. The percentage of dead cells was calculated by the ratio between FDA-stained and total cell number. Bars are means ± S.D. of at least three different experiments. Different letters indicate a significant difference (P ≤ 0.05), evaluated by ANOVA test

Fig. 3

PCD in V. vinifera (cv. Limberger) suspension cell cultures, evaluated by TUNEL assay. Cells at day 0 were observed under visible light (a, d); nuclei were stained with DAPI (b, e) or TMR-red for TUNEL assay (c, f), and observed under UV light, with low (b, c) and high (e, f) magnification (Panel a). TUNEL assay was performed in GSC and RSC grown under light or dark conditions at day 0 and 6, to evaluate the percentage of cells undergoing PCD (Panel b), counting the cells with red fluorescent-stained nuclei as apoptotic-like dead cells. Bars are means ± S.D. of at least three different experiments. Different letters indicate significant difference (P ≤ 0.05), evaluated by ANOVA test

Fig. 4

Cytochrome c release in cytosolic fractions isolated from V. vinifera (cv. Limberger) suspension cell cultures. GSC and RSC were grown either under light or dark conditions for 6 days. Samples were obtained at day 0 and 6, respectively, for the analysis of cytochrome c release. The densitometric analysis of cross-reactivity signals were detected after Western blot of cytosolic proteins isolated from cell cultures, incubated with anti-cytochrome c primary antibody. Bars are means ± S.D. of at least three independent experiments. Different letters indicate significant difference (P ≤ 0.05), evaluated by ANOVA test

Fig. 5

ATP content in V. vinifera (cv. Limberger) suspension cell cultures. GSC and RSC were grown under light or dark conditions for 6 days, and sampled at day 0, 3 and 6, respectively (Panel a). Necrotic- and apoptotic-like samples, used as positive controls, were obtained on GSC at day 0, after incubation at 80 °C for 10 min or with 10 % (v/v) ethanol for 24 h, respectively (Panel b). Bars are means ± S.D. of at least three independent experiments. Different letters indicate significant difference (P ≤ 0.05), evaluated by ANOVA test

Fig. 6

Reactive oxygen species (ROS) formation in V. vinifera (cv. Limberger) suspension cell cultures. ROS generation was estimated as fluorescence intensity generated by H₂DFCA in GSC and RSC at day 0. Cell cultures were incubated in the absence (Panel a) or presence (Panel b) of 1 mM ABAP. Insets represent the total amount of dead cells, evaluated by FDA staining at either 0 and 105 min. Bars are means ± S.D. of at least three independent experiments