Abstract
The analyses of the grapevine (Vitis vinifera L.) genome have revealed an unusually large and closely related stilbene synthase (VvSTS) gene family. Interestingly, despite the high sequence similarity among those genes, several studies have observed clear differences between their expression patterns. Here, we studied the transcriptional responses to different elicitors of several VvSTSs in cellular suspension cultures. Primarily, we performed the in silico analysis of the VvSTS regulatory sequences and found the presence of several putative cis-regulatory elements. Then, we evaluated the effect of three treatments—naphtalene acetic acid, methyl jasmonate (MeJA), and ethylene—over the gene expression and found that the genes follow expression patterns probably specific to their sequences. According to this, we focused our study on their regulatory regions and adopted a novel and efficient transient expression assay to determine the activity of these promoters. The results demonstrated that variation in gene expression could be assessed through the analysis of VvSTS regulatory sequences under the effect of different stimuli such as MeJA and cyclodextrins. Furthermore, taking advantage of the lower sequence identity at the promoter level, this strategy accomplished a more accurate alternative to differentiate the members of a large multi-gene family such as STS.
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Abbreviations
- BAP:
-
6-Benzylaminopurine
- CDs:
-
Cyclodextrins
- C4H:
-
Cinnamate-4-hydroxylase
- CHS:
-
Chalcone synthase
- 4CL:
-
Coumaroyl-CoA ligase
- CREs:
-
Cis-regulatory elements
- HREs:
-
Hormone response elements
- IBA:
-
Indole-3-butyric acid
- Ja:
-
Jasmonate
- MeJA:
-
Methyl jasmonate
- NAA:
-
Naphthalene acetic acid
- PAL:
-
Phenylalanine ammonia lyase
- STS:
-
Stilbene synthase
- Un:
-
Untreated
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Acknowledgments
The authors thank to Dr. Sebastián Gómez Talquenca for his technical support (INTA, EEA Mendoza, Luján de Cuyo, Mendoza, Argentina) and Diana Segura and Martín López Appiolaza for the assistance with plant material at IBAM-CONICET (Chacras de Coria, Mendoza, Argentina).
Funding
This work was supported by Agencia Nacional de Promoción Científica y Tecnológica [PAE-PICT-2007-02360 and PICT-2008-00270]; SeCTyP-Universidad Nacional de Cuyo [A504A/11 and 06//A587]; and Ministerio de Ciencia, Tecnología e Innovación Productiva [CH/14/02]. C.C. PhD studies were supported by CONICET and partially by BEC.AR.
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C.C., H.P., and D.L. conceived and designed the experiments. C.C. performed the in silico, in vitro, and transcriptomic experiments. C.M. provided the technical support on the transcritomic analysis. L.C. helped with the general data analysis and graphical presentations. H.P. and M.M. designed and tested the transient transformation assay. M.M and E.E. provided the technical support on the in vitro experiments. C.C. and D.L. wrote the paper. All the authors critically revised the manuscript and approved its final version.
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Fig. A1
Heatmap created from the expression changes of a set of STS genes in response to the signaling molecule MeJA in grapevine cellular suspension cultures of cv. Cabernet Sauvignon. Gene expression was evaluated through RT-qPCR at different time points. The differences are represented with color according to the scale shown (GIF 174 kb) (JPEG 16 kb)
Fig. A2
Heatmap created from the expression changes of a set of STS genes in response to the phytohormone ethylene in grapevine cellular suspension cultures of cv. Cabernet Sauvignon. Gene expression was evaluated through RT-qPCR at different time points. The differences are represented with color according to the scale shown (JPEG 17 kb)
Fig. A3
Heatmap created from the expression changes of a set of VvSTSs in response to the phytohormone NAA in grapevine cellular suspension cultures of cv. Cabernet Sauvignon. Gene expression was evaluated through RT-qPCR at six time points. The expression differences are represented with colors according to the scale shown (JPEG 16 kb)
Fig. A4
Grapevine plant agro-infiltration assay using beta-glucuronidase (GUS) in different grapevine cultivars. Whole grapevine plantlets from in vitro populations kept as indicated by Materials and Methods were infiltrated with a 35S:GUS vector to determine the transient transformation extent in leaves. Insets correspond to microscopy images (total magnification 200 X) from leaves from infiltration experiments using Agrobacterium transformed with the 35S:GUS construct (right) and non-transformed (left). GI and TS, “Thompson Seedless”; CH, “Chardonnay”; PM, “Pinot Meunier”; Car, “Carménere”; H, Harmony; SC, Salt Creek. (GIF 174 kb)
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Chialva, C., Muñoz, C., Miccono, M. et al. Differential Expression Patterns Within the Grapevine Stilbene Synthase Gene Family Revealed Through Their Regulatory Regions. Plant Mol Biol Rep 36, 225–238 (2018). https://doi.org/10.1007/s11105-018-1073-3
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DOI: https://doi.org/10.1007/s11105-018-1073-3