There have already been many reports on

There have already been many reports on the AFS gene (Beuning et al., 2010, Green et al., 2007, Li et al., 2006, Pechous and Whitaker, 2004, Pechous et al., 2005, Yuan et al., 2007, Yuan et al., 2010) and HMGR gene (Pechous and Whitaker, 2002, Rupasinghe et al., 2001). The recent advances concerning AFS gene, HMGR gene and FPPS gene have been summarized in a review paper (Lurie and Watkins, 2012). The farnesyl diphosphate synthase is the key enzyme in the synthesis of alpha-farnesene. It catalyzes the conversion of IPP (isopentenyl diphosphate synthase) and DMAPP (dimethylallyl diphosphate synthase) to FPP (farnesyl diphosphate, the substrate of alpha-farnesene synthesis) and this reaction is considered to be a rate-limiting step in isoprenoid biosynthesis (Cunillera et al., 1996). So far, more than two polyprenyl synthetase genes found be up-regulated by just a moment (Schaffer et al., 2007), the cDNA encoding for farnesyl diphosphate synthase in apples has already been cloned (GenBank accession no. AY083165), a promotor sequence of the FPPS gene in apples has already been reported (Yuan et al., 2009), the genomic organization and sequence polymorphism of FPPS1 gene in apples was analyzed (Yuan et al., 2011). When doing research on FPPS1 gene, some sequences of another FPPS2 gene were found; it once interferes our FPPS1 gene sequence splice analysis, as its sequences in many gene regions were the same as those of FPPS1 gene. To avoid similar problem in the future and to understand whether it involved in the synthesis of alpha-farnesene in apple fruit, it is necessary to make clear its genomic sequence and organization and to conduct gene expression research.
Materials and methods
Results
Discussion Although farnesyl diphosphate synthase (FPPS) is a key enzyme in the pathway of alpha-farnesene synthesis, there is not much work concerning the FPPS gene in the apples. So far, only one cDNA sequence was deposited in the GenBank (accession no. AY083165); we reported a promotor sequence of the FPPS gene in apples, as well as the genomic organization and coding sequence polymorphism of FPPS1 gene in apples (Yuan et al., 2009, Yuan et al., 2011). Here, our further research identified another FPPS2 gene which expressed in fruit and leaf, and revealed that FPPS1 gene expressed in fruit and leaf. We know that the FPPS gene involved in the synthesis of alpha-farnesene in apple fruit. Now we wonder what function(s) both FPPS1 and FPPS2 genes had in fruit; did one or both of them involved in the synthesis of alpha-farnesene in apple fruit? Some research reported that two polyprenyl synthetases (PPS7, PPS8) and alpha-farnesene synthase 1 (AFS1) showed increase in gene expression due to the application of ethylene (Schaffer et al., 2007), but it just mentioned that polyprenyl synthetases (PPS7, PPS8) are among the geranyl diphosphate, farnesyl diphosphate and geranylgeranyl diphosphate synthases; it is not clear whether PPS7 and PPS8 are farnesyl diphosphate synthase. Here, we demonstrated at first that both FPPS1 and FPPS2 genes expressed in apple fruit. The genomic sequence and intron–exon organization of FPPS2 gene in apples were obtained, with 11 introns and 12 exons. As each intron size of FPPS2 gene was different from that of FPPS1 gene we reported before (Yuan et al., 2011), the genomic organization of FPPS2 gene is obviously different from that of FPPS1 gene, but its various exon sizes were the same as those of FPPS1 gene. In this study, exon sequence comparison results indicated that there were 30 nucleotide differences between the coding region sequences of FPPS1 gene and FPPS2 gene; this result is very useful for our research on FPPS1 gene and FPPS2 gene. In this paper, some nucleotide differences have been utilized to design specific primers and compare expression levels of FPPS1 gene and FPPS2 gene. The experiment of specific primers in the C-terminal of both genes revealed that the expression level of FPPS2 gene was obviously lower than that of FPPS1 gene when fruit was stored at 4°C for 5weeks, and this result was confirmed by the experiment of specific primers in the N-terminal of both genes, so this result is reliable. In fact, using early-picked fruit of another available apple cultivar “Ralls” as experiment material, we also found that the expression level of FPPS2 gene was slightly higher than that of FPPS1 gene after 10weeks of storage at 4°C (unpublished data). Using expression level of FPPS1 gene as a reference, the expression level of FPPS2 gene was lower in “White Winter Pearmain” fruit after 5weeks of storage and higher in early-picked “Ralls” fruit after 10weeks of storage. This result is interesting; it might imply that the expression level of FPPS2 gene changed relative to that of FPPS1 gene during fruit storage, or its expression was influenced by fruit maturity. As apple and pear fruit begin to accumulate alpha-farnesene in the skin tissues shortly after they are placed in cold storage, generally reaching a peak during the first 8–12weeks of storage (Lurie and Watkins, 2012), and the alpha-farnesene in both early-picked and normal harvest “Ralls” fruits reached a peak after the first two months of storage (Ju et al., 1992), we guess that FPPS2 gene may be related to the synthesis of alpha-farnesene in fruits. Certainly, further analyses of FPPS1 and FPPS2 expressions in relation to alpha-farnesene changes during fruit storage are needed to confirm this guess. Related work is planned to be done in the future research.