Controlled environment experiments to study the effects of UV-B on gene activity

Chapter 6 The effects of UV-B radiation on gene expression in Sauvignon blanc grape berries

6.2.3 Controlled environment experiments to study the effects of UV-B on gene activity

To contrast the vineyard trial, experiments were carried out in a controlled environment (CE) cabinet using potted vines in response to a relatively high fluence UV-B radiation of UVI 12. The transcript abundance of candidate genes were analysed in potted berries at 10 and 17 weeks post bud burst.

Gene expression for key genes in the flavonoid biosynthetic pathway

The transcript abundance of VvFLS4 in potted berries at both developmental stages showed a significant UV-B response. Figure 6.10a showed VvFLS4 transcript levels in berries at 10 weeks post bud burst from both the Control and UV-B treatments. After 2 and 3 days of PAR+UV-B exposure, the transcript abundance of VvFLS4 in the UV-B treatment was over 1.5 fold higher than the Control treatment. When compared to berries at 10 weeks post bud burst, berries at the 17 weeks post bud burst had lower level of VvFLS4 expression but more significant UV-B response (Figure 6.10b). VvFLS4

expression showed a significant UV-B induction in the UV-B treatment, and this induction increased significantly from day 1 to day 3. After 1 day of PAR+UV-B exposure, the transcript abundance of

VvFLS4 in the UV-B treatment was 9.8 fold higher than the Control treatment. The transcript

abundance of VvFLS4 in berries after 2 days of PAR+UV-B exposure was 17 fold higher compared with berries after 2 days of PAR exposure, and after 3 days of PAR+UV-B exposure, this increase reached as high as 23.5 folds. In addition to UV-B response, a developmental regulation was also observed in

VvFLS4 expression in potted berries, with higher expression at 10 weeks post bud burst and lower

expression at 17 weeks post bud burst (P<0.01).

The transcript abundance of VvFLS5 also showed a significant response to berry development (Figure 6.10cd). The control berries at 10 weeks post bud burst had over 10 fold higher of transcript abundance than berries at 17 weeks post bud burst (P<0.01). However, no UV-B effect was observed with VvFLS5

expression under CE environments. VvFLS5 expression showed no significant difference between the Control and UV-B treatments in potted berries at 10 weeks post bud burst, and at 17 weeks post bud burst no consistent UV-B response was observed.

Figure 6.10 The effects of UV-B on transcript abundance of VvFLS4 (a b) and VvFLS5 (c d)in Sauvignon blanc grape berries after 1-3 days of PAR or PAR+UV-B exposure

Data shown are the average mean ± standard error of three replicates (n=3). *Significant difference comparing the Control treatment at each time point according to One-way ANOVA test (*P<0.05, **P<0.01). Different letters indicate statistical significance (P<0.05) among different time points for the Control treatment (a, b, c) and UV-B treatment (d, e, f) according to One-way ANOVA and a Fisher’s LSD test at the 5% level. The treatments are: vines exposed to PAR (Control); vines exposed to PAR+UV-B radiation (UV-B). Berries used for experiments are at 10 (Blue) and 17 (Green) weeks post bud burst, respectively. The transcript abundance in the Control treatment after 1 day of PAR exposure at 10 weeks post bud burst was set at 1.

The transcript abundance of three VvCHS genes in potted berries showed a significant response to berry development, with higher transcript abundance at 10 weeks post bud burst and lower transcript abundance at 17 weeks post bud burst (p<0.05; Figure 6.11). VvCHS1 expression also showed a significant UV-B induction in the UV-B treatment, and this UV-B induction was observed in potted berries at both developmental stages measured (Figure 6.11ab). The transcript abundance of VvCHS1

showed a significant increase after UV-B exposure in the UV-B treatment, when compared to the Control treatment. This increase could be detected after 2 days of PAR+UV-B exposure at 10 weeks post bud burst, and throughout the whole period from day 1 to 3 at 17 weeks post bud burst. A significant UV-B response was also observed in VvCHS2 expression in potted berries at 17 weeks post bud burst, with the transcript abundance significantly increasing in berries after UV-B exposure in the UV-B treatment (Figure 6.11d). However, VvCHS2 expression showed no consistent UV-B response in berries at 10 weeks post bud burst (Figure 6.11c). VvCHS3 expression showed no significant difference between the Control and UV-B treatments in potted berries at 10 weeks post bud burst, and at 17 weeks post bud burst no consistent UV-B response was observed (Figure 6.11ef).

Figure 6.11 The effects of UV-B on transcript abundance of VvCHS1 (a b), VvCHS2 (c d)and VvCHS3 (e f)in Sauvignon blanc grape berries after 1-3 days of PAR or PAR+UV-B exposure

Gene expression for transcription factors

The gene activity of transcription factors were also analyzed in berries from controlled environment experiments, including VvMYB12, VvMYCA1, VvWDR1 and VvWDR2. Consistent with vineyard trials, the transcript of VvMYB12 also showed a significant response to berry development under the CE environments (Figure 6.12ab). VvMYB12 had high transcript abundance in berries at 10 weeks post bud burst and low transcript abundance in berries at 17 weeks post bud burst (p<0.05). A significant UV-B response was also observed in VvMYB12 expression in potted berries, regardless of developmental stages. VvMYB12 showed significant increases in transcript abundance after UV-B exposure in the UV-B treatment compared with the Control treatment, and these increases were clearly detected in berries at both 10 and 17 weeks post bud burst. When compared with berries at 10 weeks post bud burst, berries at 17 weeks post bud burst showed lower VvMYB12 expression but a significant UV-B induction. However, no significant UV-B response was observed for VvMYCA1,

VvWDR1 and VvWDR2 at both developmental stages (Figure 6.12c-h). At 10 weeks post bud burst, the

transcript abundance of VvMYCA1, VvWDR1 and VvWDR2 showed an UV-B induction in the UV-B treatment at day 1 and 2, but not at day 3. At 17 weeks post bud burst, no significant UV-B response was observed for these genes.

Figure 6.12 The effects of UV-B on transcript abundance of VvMYB12 (a b), VvMYCA1 (c d), VvWDR1 (e f)and

VvWDR2 (g h)in Sauvignon blanc grape berries after 1-3 days of PAR or PAR+UV-B exposure

Gene expression for UV receptor UVR8 and its reaction partners

Gene activity of UV photoreceptor VvUVR8 and its reaction partners (VvCOP1 and VvHY5) were also analyzed in response to relatively high fluence UV-B in berries from the CE experiments. Neither

VvUVR8 nor VvCOP1 showed a significant UV-B response in gene expression (Figure 6.13a-d). VvUVR8

showed a developmental regulation, with higher transcript abundance in berries at 10 weeks post bud burst and lower transcript abundance in berries at 17 weeks post bud burst (p<0.05). But no stage of development was found with significant expression difference between the Control and UV-B treatments. Similarly, no significant change was found in VvCOP1 expression in response to UV-B exposure at 10 and 17 weeks post bud burst, and the transcripts of VvCOP1 showed similar levels in berries at both stages of development. However, VvHY5 showed a significant UV-B response in gene expression at both developmental stages under the same CE environments (Figure 6.13ef). Berries exposed to PAR+UV-B in the UV-B treatment had about 2 fold higher VvHY5 expression, when compared with berries exposed to PAR in the Control treatment.

Figure 6.13 The effects of UV-B on transcript abundance of VvUVR8 (a b), VvCOP1 (c d)and VvHY5 (e f)in Sauvignon blanc grape berries after 1-3 days of PAR or PAR+UV-B exposure

Gene expression for pathogen-related proteins and MAPK pathway

For genes that are thought to be involved in the high fluence UV-B response, five PR genes were studied in the berries from CE experiments. All PR genes showed a developmental regulation, with significantly higher expressions at 17 weeks post bud burst and lower or no detectable expression at 10 weeks post bud burst (p<0.05; Figure 6.14). UV-B response in transcript abundance was varied between genes and developmental stages. VvTL1 expression was significantly increased by UV-B exposure in berries at 10 weeks post bud burst, but not in berries at 17 weeks post bud burst (Figure 6.14ab). VvTL2 had no detectable expression in berries at 10 weeks post bud burst, and at 17 weeks post bud burst no significant UV-B induction (Figure 6.14cd). No significant UV-B response was found in VvTL3 expression at both developmental stages (Figure 6.14ef). However, both VvChi4A and VvChi4B showed a significant UV-B induction in the UV-B treatment at both developmental stages, with higher gene expression in berries exposed to PAR+UV-B radiation in the UV-B treatment and lower expression in berries exposed to PAR radiation in the Control treatment (Figure 6.14g-j).

The gene activity of VvMAPK3 was also determined in response to UV-B exposure under CE conditions.

VvMAPK3 showed no significant response to either berry development or UV-B exposure. VvMAPK3

had similar transcript abundance in berries at both 10 and 17 weeks post bud burst, and no stage was a UV-B response observed in berries (Figure 6.15).

Figure 6.14 The effects of UV-B on transcript abundance of VvTL1 (a b), VvTL2 (c d), VvTL3 (e f) VvChi4A (g h) and VvChi4B (i j)in Sauvignon blanc grape berries after 1-3 days of PAR or PAR+UV-B exposure

Figure 6.15 The effects of UV-B on transcript abundance of VvMAPK3 in Sauvignon blanc grape berries after 1-3 days of PAR or PAR+UV-B exposure

6.3 Discussion

In document The effects of UV radiation on flavonoid accumulation and gene expression in Sauvignon blanc grape berries (Page 133-144)