Ectopic expression of VpSTS29, a stilbene synthase gene from Vitis pseudoreticulata, indicates STS presence in cytosolic oil bodies
Stilbene synthase (STS) and its metabolic products are accumulated in senescing grapevine leaves. Ectopic expression of VpSTS29 in Arabidopsis shows the presence of VpSTS29 in oil bodies and increases trans-piceid in developing leaves.
Stilbenes are the natural antimicrobial phytoalexins that are synthesised via the phenylpropanoid pathway. STS is the key enzyme catalysing the production of stilbenes. We have previously reported that the VpSTS29 gene plays an important role in powdery mildew resistance in Vitis pseudoreticulata. However, the synthesis and accumulation of these stilbene products in plant cells remain unclear. Here, we demonstrate that VpSTS29 is present in cytosolic oil bodies and can be transported into the vacuole at particular plant-developmental stages. Western blot and high-performance liquid chromatography showed that STS and trans-piceid accumulated in senescent grape leaves and in pVpSTS29::VpSTS29-expressing Arabidopsis during age-dependent leaf senescence. Subcellular localisation analyses indicated VpSTS29-GFP was present in the cytoplasm and in STS-containing bodies in Arabidopsis. Nile red staining, co-localisation and immunohistochemistry analyses of leaves confirmed that the STS-containing bodies were oil bodies and that these moved randomly in the cytoplasm and vacuole. Detection of protein profiles revealed that no free GFP was detected in the pVpSTS29::VpSTS29-GFP-expressing protoplasts or in Arabidopsis during the dark–light cycle, demonstrating that GFP fluorescence distributed in the STS-containing bodies and vacuole was the VpSTS29-GFP fusion protein. Intriguingly, in comparison to the controls, over-expression of VpSTS29 in Arabidopsis resulted in relatively high levels of trans-piceid, chlorophyll content and of photochemical efficiency accompanied by delayed leaf senescence. These results provide exciting new insights into the subcellular localisation of STS in plant cells and information about stilbene synthesis and storage.
KeywordsChinese wild grape Stilbene synthase gene Expression profile Subcellular localisation Oil body Leaf senescence
Green fluorescent protein
High-performance liquid chromatography
This work was supported by the Grants from the National Science Foundation of China (Grant No. 31672129). The authors specifically thank Dr Alexander (Sandy) Lang from RESCRIPT Co. (New Zealand) for useful comments and language editing which have greatly improved the manuscript.
- Cheng S, Xie X, Xu Y, Zhang C, Wang X, Zhang J, Wang Y (2016) Genetic transformation of a fruit-specific, highly expressed stilbene synthase gene from Chinese wild Vitis quinquangularis. Planta 243(4):1–13Google Scholar
- David S, Brenda WS (2001) Localization of flavonoid enzymes in Arabidopsis roots. Plant J 27(27):37–48Google Scholar
- Dercks W, Creasy LL (1989) The significance of stilbene phytoalexins in the Plasmopara viticola-grapevine interaction. Physiol Mol Plant Pathol 13(4):351–371Google Scholar
- Krokene P (2015) Conifer defense and resistance to bark beetles. Bark beetles: biology and ecology of native and invasive species. Elsevier, Oxford, pp 177–207Google Scholar
- Parage C, Tavares R, Réty S, Baltenweckguyot R, Poutaraud A, Renault L, Heintz D, Lugan R, Marais GA, Aubourg S (2012) Structural, functional, and evolutionary analysis of the unusually large stilbene synthase gene family in grapevine. Plant Physiol 160(3):1407–1419CrossRefPubMedPubMedCentralGoogle Scholar
- Richter A, Jacobsen HJ, Kathen AD, Lorenzo GD, Briviba K, Hain R, Ramsay G, Kiesecker H (2006) Transgenic peas (Pisum sativum) expressing polygalacturonase inhibiting protein from raspberry (Rubus idaeus) and stilbene synthase from grape (Vitis vinifera). Plant Cell Rep 25(11):1166–1173CrossRefPubMedGoogle Scholar
- Wang Y, Liu Y, He P, Chen J, Lamikanra O, Lu J (1995) Evaluation of foliar resistance to Uncinula necator in Chinese wild Vitis species. Vitis 34(3):159–164Google Scholar
- Yin X, Singer SD, Qiao H, Liu Y, Jiao C, Wang H, Li Z, Fei Z, Wang Y, Fan C (2016) Insights into the mechanisms underlying ultraviolet-C induced resveratrol metabolism in grapevine (V. amurensis Rupr.) cv. “Tonghua-3”. Front. Plant Sci 7:503Google Scholar