## 2 Results and Discussion
### Subcellular Localization of VvRiLinNer Protein  
Differential subcellular compartmentalization for the biosynthesis of FPP and GPP (the _in vitro_ substrates of VvRiLinNerin plastids and the cytosol, respectively) was found in most plants including grape [8,9,21,27]. To determine _in vivo_ localization of this enzyme, we examined the subcellular localization of the VvRiLinNer through transient expression of _VvRiLinNer_ fused with a _GFP_ reporter gene in _Arabidopsis_ prototoplasts. GFP fluorescence was clearly restricted in the chloroplasts, and no fluorescence signal was observed  
Figure 3: Analysis of SDS polyacrylamide gels of the purified recombinant VvRiLinNer proteins (**A**) and GC-MS analysis of the products of recombinant VvRiLinNer reaction system (**B**,**C**). In (**A**), M: Protein Molecular Weight Marker; 1: the purified protein VvRiLinNer. The apparent molecular weight of expressed recombinant proteins VvRiLinNer was estimated approximately 82 kDa, which is bigger than the predicted proteins from cDNA sequences because of the presence of the His-Tag (approximately 16 kDa); In (**B**) and (**C**), the reaction system contained the prokaryoticly-expressed VvRiLinNer and geranyl diphosphate (**B**) or farnesyl diphosphate (**C**). The reaction product (35)-linalool or (_E_)-neroidol was authentically identified. Compounds marked in numbers of (**B**) and (**C**) are listed as follows: 1 \(\beta\)-myrcene, 2 (_Z_)-\(\beta\)-ocimene, 3 (_E_)-\(\beta\)-ocimene, 4 allocimene, 5 \(\beta\)-farnesene, 6 \(\alpha\)-bisabolol, 7 \(\alpha\)-farnesene. Among them, the compounds one & five were authentically identified, the others were tentatively identified according to MS spectra comparison with NIST 05 library. in the cytoplasm (Figure 4), which means that the present VvRiLinNer is mainly responsible for the synthesis of 3_S_-linalool _in vivo_. This inference was made mainly from two aspects of evidence. Firstly, using intact plastids separated from cell suspensions of _Vitis vinifera_ Muscat de Frontignan, the authors have found that this organelle exhibits the activity of VvGPPS responsible for the synthesis of monoterpene precursor GPP [27]. Secondly, although FPPS and trace FPP pool in wild tomato also have been found to be chloroplast-localized [21, 28], the feeding experiment in grape berries with the deuterium labeled precursors [5,5-H-2(2)]-1-deoxy-d-xylulose and [5,5-H-2(2)]-mevalonic acid lactone, a cytosolic MVA pathway-specific precursor, has recently demonstrated that adding IPP concentration would promote more production of sesquiterpenes, but hardly alter level of monoterpenes [22]. IPP is produced from the cytosolic MVA pathway. Up to date, there is still no evidence on presence of FPPS or FPP pool in the plastid of grape berry. Moreover, VvFPPS lacks a plastid signal peptide at _N_-terminal [29]. Combined the present subcellular localization with previous reports, we inferred that the VvRiLinNer acquired in this study should not participate in the synthesis of sesquiterpenes in grape berry. Correspondence Relationship between VvRiLinNer Expression and Linalool Accumulation in Three Grape Cultivars  
To further determine the role of the VvRiLinNer in linalool accumulation during grape berry development, we examined free and glycosidically-bound forms of linalool and its structure-linked oxides in three grape cultivars containing Cabernet Sauvignon (CS, a neutral cultivar), Gewurztraminer (Gw, a muscat cultivar) and Riesling (a non-muscat aromatic cultivar) (Figure 5). Figure 4: Confocal laser scanning microscopy of transiently expressed VvRiLinNer-GFP fusions in _Arabidopsis_ protoplasts (**A**) Green fluorescent protein fluorescence of VvRiLinNer-GFP; (**B**) Chlorophyll autofluorescence; (**C**) Light-microscopy image of intact protoplast. Bar: 5 \(\upmu\)m; (**D**) Merged images. All images were taken with the 40\(\times\) water-immersion objectives on the Zeiss LSM 510 (Zeiss, Germany).