Flavonoid 3'-hydroxylase of Camellia Nitidissima Chi. Can Promote the Synthesis of Polyphenols Better Than Flavonoids


 Camellia nitidissima Chi is an ornamental plant of the genus Camellia L. Its flowers contain a lot of flavonoids and polyphenols. Flavonoid 3'-hydroxylase (F3'H) plays an important role in the synthesis of flavonoids, polyphenols and anthocyanins. We cloned CnF3'H from the petal of C. nitidissima (GenBank code: HQ290518.1). The full length of CnF3'H was 1859 bp, with an open reading frame of 1577 bp, and encoded 518 amino acid. A phylogenetic tree analysis showed that CnF3'H was closely related to Camellia sinensis L. and C. sinensis cultivar Zhonghuang. CnF3'H was expressed in flowers, leaves, fruits, sepals, petals and stamens of C. nitidissima, and during the flowering process the expression level in flower decreased initially and then increased. CnF3'H expression was significantly positive correlated with polyphenol contents in C. nitidissima. A CnF3'H-EGFP expression vector was constructed to do the subcellular localization, we found that CnF3'H was obviously localized in the nuclear envelope and cytomembrane. In transgenic tobacco flowers, the total polyphenol content and various polyphenol constituents were significantly increased with high CnF3'H expression level, while total flavonoid contents and some flavonol constituents were increased slightly. These findings suggest that CnF3'H can promote the synthesis of polyphenols better than flavonoids.


Introduction
Camellia nitidissima Chi. was rst discovered in Guangxi Province, China. It is an evergreen shrub or small tree belonging to Sect. Chrysantha Chang in the genus Camellia L. of Theaceae (Zhang and Ren 1998). The owers of C. nitidissima have a particular golden yellow color, making it desirable as an ornamental plant that used as a genetic resource for yellow Camellia breeding. Studies have showen that C. nitidissima, like Camellia sinensis L., containes a large number of avonoids and polyphenols in its leaves and owers (Jiang et (Brugliera et al. 1999) were the rst to isolate the F3'H gene from Petunia hybrida and identi ed it as belonging to the CYP75B2 gene family (Schoenbohm et al. 2000;Sirim et al. 2009). Several F3'H genes have since been identi ed, such as in Arabidopsis thaliana (Han et al. 2010), Ginkgo biloba (Li et al. 2015), C. sinensis (Zhou et al. 2016) and Canarium album (Huang et al. 2017 In this study, we cloned the full-length sequence of the CnF3'H gene from the petals of C. nitidissima and studied its expression pattern and subcellular localization. We also overexpressed CnF3'H in tobacco to verify its function. The results showed a functional diversi cation of the avonoid 3'-hydroxylase from C. nitidissima, which is different from other plants slightly. This nding is important for the study of ower color formation in C. nitidissima.
The materials were frozen in liquid nitrogen and stored at -80℃ for later use.  (Figure 1a). There was 78-97% amino acid sequence similarity between CnF3'H and other F3'H proteins and in the N-terminal of the amino acid sequence there were possible anchoring residues (LPPGP), while in the C-terminal contained residues of a heme-binding region (PFGAGRRICAG), which indicated that CnF3'H belongs to the P450 super family (Chapple 1998;He et al. 2008) (Figure 1b).
Phylogenetic tree analysis (Figure 1c)   Using EXclone technology, we contained CnF3'H fused to green uorescent protein (EGFP). After transforming the epidermal cells of Nicotiana benthamiana leaves, a laser confocal microscope was used to identify the subcellular localization of CnF3'H. When excitated at 488 nm, green uorescence signal of the empty 35s-EGFP vector appeared in the cell nucleus, cytomembrane and cytoplasm of the cells, and the signal was dispersed throughout the whole cell (Figure 4a), indicating that the free EGFP could be successfully expressed. In the epidermal cells containing CnF3'H-EGFP vector, green uorescence signal appeared in the nuclear envelope and cytomembrane, and the contours were clear (Figure 4b), indicating that CnF3'H protein was probably located on the nuclear envelope and cytomembrane.

Functional Analyses of CnF3'H in Tobacco
After overexpressing CnF3'H in tobacco, the gene was positively identi ed by PCR. No signi cant changes in leaf or ower color were found in positive tobacco lines. We randomly selected 6 positive lines and measured the expression of CnF3'H in owers. Compared with wild-type tobacco (WT), the CnF3'H expression levels increased signi cantly in owers of positive lines, and the expression levels of CnF3'H were signi cantly different among the positive lines. The highest expression level (F3'H-4) was 3.9 times greater than the lowest (F3'H-6) (Figure 5a). We also determined the total avonoid content, total polyphenol content and total anthocyanin content in owers of the 6 positive lines. We found the total polyphenol and total avonoid contents in the positive lines were signi cantly higher than that in wildtype tobacco, and the total polyphenol content was obviously higher than the total avonoid content. The total anthocyanin content could not be detected in either wild-type or positive lines (Figure 5b).
We detected six avonoid constituents and six polyphenol constituents in owers of the CnF3'H positive lines by HPLC. Except for F3'H-6, with low expression level, the 6 polyphenol constituents (EGC, EGCG, GC, GCG, ECG and CG) in the other 5 lines were all signi cantly higher than those in wild-type tobacco ( Figure  5c, d). Among the avonoids, Qu3R and Ka were not signi cantly increased in F3'H-17 but were signi cantly higher in the other 5 lines compared to wild-type. DHQ was signi cantly higher in F3'H-3 than wild-type tobacco and the other lines. Ru, Qu7G and Qu3G were signi cantly higher in the 6 CnF3'H positive lines (Figure 5e, f). This indicated that CnF3'H promoted the synthesis of avonols and polyphenols, and the promotion of polyphenol synthesis was greater than that of avonoid synthesis. These ndings show that CnF3'H preferentially promote the synthesis of polyphenols.

Discussion
In this study, we obtained a CnF3'H gene encoded 518 amino acids. The P450 family is an important and large gene family in plants, and is mainly involved in biosynthetic and biodetoxi cation processes (He et al. 2008). Seitz et al. (Seitz et al. 2007) constructed chimeric genes and found that the area near the Nterminals of F3'H and F3'5'H determined their action characteristics, while the C-terminal determined functional differences between the enzymes. The "PPGP" sequence was determined to be a conserved sequence, and "FGAGRRICAG" was identi ed as the C-terminal heme-binding region necessary for cytochrome enzymes (Murakami et al. 1994;Yamazaki et al. 1993). We founed that CnF3'H had typical F3'H characteristics (Figure 1b). Multiple sequence alignment showed that CnF3'H was highly similar (98%) to C. sinensis F3'H (Figure 1b), The phylogenetic tree analysis also indicated that CnF3'H was closely related to C. sinensisF3'H and C. sinensis cultivar Zhonghuang F3'H ( Figure 1c). In the protein sequence of CsF3'H, there were four conserved cytochrome P450-featured motifs and three F3'H-speci c conserved motifs (Zhou et al. 2016), these structures were also found in CnF3'H.  (Toda et al. 2002) found that F3'H in soybean was located in vacuoles in the seed coat umbilicus, and Li et al. (Li et al. 2015) suggested that GbG3'H protein might be located in the endoplasmic reticulum as a complex, according to the analysis of signal peptide (MHLFLPPLFFFHINSVCNPE) (Stafford 1974). However, our result was not the case with the instantaneous conversion of Nicotiana benthamiana differed from the previous ndings. In the current study, CnF3'H-EGFP protein was identi ed from the strong uorescence signal on the nuclear envelope and cytomembrane (Figure 3b). Thus, CnF3'H probably locates to the nuclear envelope and cytomembrane.
In this study, the expression of CnF3'H showed signi cant tissue speci city, which was similar to the results of previous studies. In addition, F3'H gene expression was found to be primarily related to avonoid metabolic processes. The higher expression level of F3'H in Ginkgo biloba leaves may be related to the accumulation of secondary metabolites such as avonoids (Li et al. 2015). The expression of CaF3'H in Canarium album was detected during fruits development and it was highest 50 days after owering (Huang et al. 2017). CsF3'H1 gene in tea plants regulated the avonoid metabolism pathway, the gene was highly expressed in the young leaves in which polyphenols were concentrated (Jiang et al. 2013), and it also showed the importance in the biosynthesis of catechins and avanols in tea leaves (Zhou et al. 2016). In C. nitidissima, CnF3'H expression was proportional to polyphenol contents and not signi cant with avonoids contents. It can be infered that F3'H expression changes among plants and is mostly related to the metabolism of avonoids and polyphenols.
After transferring CnF3'H into tobacco, we found signi cative increases in polyphenol and avonol contents in positive lines, indicating that CnF3'H could indeed promote the synthesis of avonols and polyphenols. Previous researches have also shown that F3'H gene can interfere with the metabolic process of plant avonoids. The total avonoid content in transgenic tobacco containing the CoF3'H gene of Chromolaena odorata was signi cantly increased, indicating CoF3'H promoted the synthesis of avonoids and was one of the key genes in avonoids synthesis process (He et al. 2015b). The F3'H gene of Eupatorium adenophorum Sprengel was highly homologous with the endogenous F3'H tobacco gene, the endogenous F3'H gene of tobacco was inhibited after the transformation, leading to decreased F3'H expression and decreased anthocyanin accumulation in tobacco (Zhang et al. 2009). Although there was no signi cant change in the color of positive transgenic lines in our study, CnF3'H was found to preferentially convert dihydroquercetin into polyphenols, which suppressed the formation of a yellow ower color. The co-regulatory effect of CnF3'H and CnFLS on the ower color requires further study.

Conclusions
We identi ed one F3'H homolog from C. nitidissima (CnF3'H), and phylogenetic analysis showed that F3'Hs of Camellia species formed a clade that was close to Rhododendron x pulchrum. Gene expression analysis revealed that the expression of CnF3'H was positively correlated with polyphenols but negatively with yellow coloration. Subcellular localization of CnF3'H showed a likely dual localization in the nuclear envelope and cytomembrane. Furthermore, in the transgenic tobaccos, it was found that the content of polyphenols increased signi cantly, while the content of avonols increased a bit. These ndings show that CnF3'H promote the synthesis of polyphenols better than avonoids.