CONTROL DE Salmonella spp y Campylobacter spp EN LA CADENA ALIMENTARIA MARCO

4. Salmonella spp y Campylobacter spp EN LA CADENA ALIMENTARIA

4.1. CONTROL DE Salmonella spp y Campylobacter spp EN LA CADENA ALIMENTARIA MARCO

To understand UV-B induction of flavonol biosynthesis in more detail, the activity of five VvFLS genes in Sauvignon blanc grape tissue from the vineyard experiment 2010 was measured by qPCR. Of the five genes, only two, VvFLS4 and VvFLS5, showed measureable expression in Sauvignon blanc berries (Figure 6.1). VvFLS4 transcripts exhibited peak accumulation at harvest and expression correlated with UV-B exposure at all stages of development measured. The LR and ACRYLIC treatments had significantly higher VvFLS4 expression when compared to the Control and UV/UV-B excluding treatments (POLYCARB and PETG). As shown in Figure 6.1a, VvFLS4 showed high transcript abundance in the UV-B exposure treatments (LR and ACRYLIC), which were 3 to 4 fold higher than the Control treatment. In contrast, the transcript abundance of VvFLS4 in the UV-B exclusion treatments (POLYCARB and PETG) completely removed these increases. In the POLYCARB and PETG treatments,

VvFLS4 had similar transcript abundance between each other, and the transcript abundance were

lower than the Control treatment at 3 and 6 weeks post-veraison. VvFLS5 showed a significant response to berry development, with higher transcript abundance at pre-veraison and lower transcript abundance at post-veraison (p<0.01; Figure 6.1b). No consistent leaf removal or UV response was seen with VvFLS5 gene expression at all developmental stages measured.

Figure 6.1 The effects of leaf removal and UV radiation on transcript abundance of VvFLS4 (a) and VvFLS5 (b) in Sauvignon blanc grape berries during berry development in 2010

Data shown are the average mean ± standard error of three replicates (n=3). Different letters indicate statistical significance (P<0.05) among different treatments at each developmental stage according to One-way ANOVA and a Fisher’s LSD test at the 5% level. The treatments are: vines with leaves maintained and no screen applied (Control); leaves removed and bunches exposed to sun until harvest, 6 weeks post-veraison (LR); leaves removed and bunches covered by an acrylic screen (ACRYLIC); leaves removed and bunches covered by a polycarbonate screen (POLYCARB); leaves removed and bunches covered by a glycol-modified polyethylene terephthalate screen (PETG). The leaf removal and screens were applied to vines at -5 weeks (pre-veraison). Samples were collected at four stages: 4 weeks pre-veraison (-4W PV); 1 week pre-veraison (-1W PV); 3 weeks post-veraison (3W PV) and harvest (6W PV). The transcript abundance in the Control treatment at -4W PV was set at 1.

The activity of three chalcone synthase (CHS) genes were analysed in berries at four stages of development. All of three VvCHS genes showed a significant response to berry development. These genes had a similar expression pattern throughout berry development, with significantly higher transcript abundance at pre-veraison and lower transcript abundance at 3 weeks post-veraison (p<0.05; Figure 6.2). VvCHS1 showed a significant UV-B response at all stages of development measured, even when the transcript abundance was very low at 3 weeks post-veraison (Figure 6.2a). The transcript abundance of VvCHS1 in berries was significantly higher in the UV-B exposure treatments (LR and ACRYLIC) than the Control and UV-B exclusion treatments (POLYCARB and PETG). When compared with VvCHS1 expression in the Control treatment, higher transcript abundances of

VvCHS1 were observed in the UV-B exclusion treatments (POLYCARB and PETG) at stages measured

pre-veraison. However, converse results were observed at 3 and 6 weeks post-veraison, with the UV- B exclusion treatments (POLYCARB and PETG) showing significantly lower transcript levels of VvCHS1

than the Control treatment. VvCHS2 showed higher expression at 4 weeks pre-veraison and harvest, when compared with other developmental stages measured (p<0.05; Figure 6.2b). A significant UV-B induction was also observed in the transcript abundance of VvCHS2 in the LR and ACRYLIC treatments,

butthis UV-B induction was only observed at 3 and 6 weeks post-veraison. No significant increase was induced by UV-B radiation at 4 weeks pre-veraison in these treatments. The transcript of VvCHS3

showed a developmental regulation, with significantly higher expressions at stages measured pre- veraison and lower expressions at post-veraison (p<0.05; Figure 6.2c). No consistent UV-B effect was observed in VvCHS3 expression at any stages of development measured.

Figure 6.2 The effects of leaf removal and UV radiation on transcript abundance of VvCHS1 (a), VvCHS2 (b)

and VvCHS3 (c) in Sauvignon blanc grape berries during berry development in 2010

Gene expression for transcription factors

It is well established that the temporal and spatial distribution and level of flavonols is determined by a MYB-bHLH-WD40 activation complex, with MYB being central to the regulation of the complex (Albert et al. 2013; Davies & Schwinn 2003). We tested members of this complex; VvMYB12, VvMYCA1,

VvWDR1 and VvWDR2 in relation to UV-B induced VvFLS activity in grapevines (Czemmel et al. 2009; Matus et al. 2009; Matus et al. 2010). VvMYB12 showed a significant response to berry development (Figure 6.3a). The transcript abundance of VvMYB12 had higher levels at both 4 weeks pre-veraison and 6 weeks post-veraison, when compared with other stages of development measured (p<0.01). A significant UV-B induction was also found in VvMYB12 expression. The LR and ACRYLIC treatments had significantly higher VvMYB12 expression than the Control treatment at most of developmental stages measured. In contrast, once the UV-B was excluded by screens in the POLYCARB and PETG treatments, the transcript of VvMYB12 declined to the low levels, similar or even lower than the Control treatment. Transcription factor VvMYCA1 also showed a developmental regulation throughout berry development, with significantly higher levels of transcript abundance at developmental stages pre- veraison and lower levels at post-veraison (p<0.01; Figure 6.3b). However, no significant UV-B response was observed at any stages of development measured. Two genes encoding the transcription factor VvWD40 were also analysed at different stages of berry development, VvWDR1 and VvWDR2

(Figure 6.3cd). VvWDR1 and VvWDR2 showed a similar transcript pattern throughout berry development, with the highest transcript abundance at 1 week pre-veraison. However, neither

VvWDR1 or VvWDR2 showed a significant UV-B response at any stages of development measured.

Figure 6.3 The effects of leaf removal and UV radiation on transcript abundance of VvMYB12 (a), VvMYCA1 (b), VvWDR1 (c) and VvWDR2 (d) in Sauvignon blanc grape berries during berry development in 2010

Gene expression for UV receptor UVR8 and its reaction partners

To investigate the signal transduction pathway from UV-B perception to flavonol biosynthesis in grapevine, gene activity for the UV-B photoreceptor (UVR8) and its reaction partners (HY5 and COP1) that are thought to be involved in the low fluence UV-B pathway in Arabidopsis thaliana (Cloix et al.

2012; Jenkins & Brown 2007; Stracke et al. 2010) were analysed. As shown in Figure 6.4a, VvUVR8

showed a significant response to berry development, with approximately 4 to 5 fold higher transcript abundance at developmental stages pre-veraison when compared with post-veraison (p<0.01), but at no stage of development was the VvUVR8 expression significantly influenced by UV-B radiation. In contrast, VvHY5 showed a significant UV-B response at all stages of development measured excepting 4 weeks pre-veraison (Figure 6.4b). The UV-B exposure treatments (LR and ACRYLIC) showed significantly higher VvHY5 expression from 1 week pre-veraion to harvest, when compared with the UV-B exclusion treatments (POLYCARB and PETG). Conversely, the Control treatment had low VvHY5

expression throughout all developmental stages measured, even lower than the POLYCARB and PETG treatments. VvCOP1 transcript abundance was present at all stages of development measured, but no significant UV-B response was observed at any developmental stages measured (Figure 6.4c).

Figure 6.4 The effects of leaf removal and UV radiation on transcript abundance of VvUVR8 (a), VvHY5 (b)

and VvCOP1(c) in Sauvignon blanc grape berries during berry development in 2010

Gene expression for pathogen-related proteins and MAPK pathway

A number of pathogen-related (PR) genes were also studied for gene activity in grape berries at different stages throughout berry development. These genes included three candidate genes for PR5 proteins (VvTL1, VvTL2 and VvTL3) and two candidate genes for PR3 proteins (VvChi4A and VvChi4B). As shown in Figure 6.5, five PR genes all showed a developmental regulation, with very low or no observable expression at stages measured pre-veraison and significantly higher transcript levels at 3 and 6 weeks post-veraison (p<0.01). There was, however, no evidence of a UV-B response at any stage of development measured in grape berries from vineyard trials.

In addition to PR proteins, MAPK3 that was thought to be involved in the UV-B signal regulation pathway was also determined in this study (Figure 6.5f). The transcript abundance of VvMAPK3 was present at all stages of development measured, but a significant UV-B response was only observed in berries at 3 weeks post-veraison.

Figure 6.5 The effects of leaf removal and UV radiation on transcript abundance of PR proteins and MAPK, VvTL1 (a), VvTL2 (b), VvTL3 (c), VvChi4A (d), VvChi4B (e) and VvMAPK3 (f) in Sauvignon blanc grape berries during berry development in 2010

In document Prevalencia de "Salmonella" spp y "Campylobacter" spp a lo largo de la cadena de sacrificio de porcino en mataderos de la Comunidad Valenciana (página 85-88)