Functional Diversification of the Dihydroflavonol 4-Reductase from Camellia nitidissima Chi. in the Control of Polyphenol Biosynthesis

Abstract

Plant secondary metabolism is complex in its diverse chemical composition and dynamic regulation of biosynthesis. How the functional diversification of enzymes contributes to the diversity is largely unknown. In the flavonoids pathway, dihydroflavonol 4-reductase (DFR) is a key enzyme mediating dihydroflavanol into anthocyanins biosynthesis. Here, the DFR homolog was identified from Camellia nitidissima Chi. (CnDFR) which is a unique species of the genus Camellia with golden yellow petals. Sequence analysis showed that CnDFR possessed not only conserved catalytic domains, but also some amino acids peculiar to Camellia species. Gene expression analysis revealed that CnDFR was expressed in all tissues and the expression of CnDFR was positively correlated with polyphenols but negatively with yellow coloration. The subcellular localization of CnDFR by the tobacco infiltration assay showed a likely dual localization in the nucleus and cell membrane. Furthermore, overexpression transgenic lines were generated in tobacco to understand the molecular function of CnDFR. The analyses of metabolites suggested that ectopic expression of CnDFR enhanced the biosynthesis of polyphenols, while no accumulation of anthocyanins was detected. These results indicate a functional diversification of the reductase activities in Camellia plants and provide molecular insights into the regulation of floral color.

Keywords: Camellia nitidissima Chi.; anthocyanins; dihydroflavonol 4-reduetase; floral pigmentation; polyphenols.

Figure 1

Amino acid alignment and phylogenetic analysis of the homolog plant DFRs of Camellia nitidissima Chi. (CnDFR). (A) Alignment of DFR-like protein sequences. CnDFR, Camellia nitidissima DFR; CsDFR, Camellia sinensis DFR; CcDFR, Camellia chekiangoleosa DFR; RsDFR, Rhododendron simsii DFR; AcDFR, Actinidia chinensis DFR; GhDFR, Gerbera hybrida DFR; PhDFR, Petunia hybrida DFR. The red boxed region is a putative NADPH-binding region. The region underlined is predicted to be the substrate specificity-determining region. At the 134 th amino acid, there is an asparagine (N) different from Petunia but identical to Gerbera and so on. The red triangle is the different amino acids of Camellia DFRs from other plants. (B) Phylogenetic tree of CnDFR constructed with MEGA 5.0, using the neighbor-joining (NJ) method and 1000 bootstrap replicates. CnDFR sequence was found to be 75% to 99% similar to homological DFR genes.

Figure 2

Relative expression level of CnDFR in C.nitidissima Chi. (A) Tissues of C.nitidissima Chi.: root, leaf, fruit, flower, speal, petal, stamen, pistil. (B) Flowers at different developmental stages of C.nitidissima Chi.: bud in 10 mm, bud in 20 mm, bud in 30 mm, half-open flower, blooming flower, withered flower. (C) Relative expression level of CnDFR in different tissues of C.nitidissima Chi. CnDFR expressed in all of the tissues, while the expression was the highest in the flower and the lowest in the sepal. (D) Relative expression level of CnDFR in flowers at different developmental stages. The expression of CnDFR showed a trend of first decreasing then increasing and then decreasing, and the expression was the highest in the half-open flower.

Figure 3

The relationship between DFR relative expression level and the chemical components content in petals of C. nitidissima Chi. (A) Petals in 5 stages of C. nitidissima Chi.: petals of young bud, petals of big bud, petals of half-open flower, petals of blooming flower, petals of withered flower. (B) CnDFR relative expression level and flavonoids contents and b* in petals of C. nitidissima Chi. DFR-RQ (the relative expression level of DFR) in 5 stages had an M-shaped trend. TF (the content of total flavonoids), Qu7 G (quercetin-7-O-β-D- glucopyranoside) and b* (yellow color index of petals) were negatively correlated with the expression level of CnDFR. (C) CnDFR expression level and polyphenols components. TP (the content of total polyphenols), EC (the content of epicatechin) and C (the content of catechin) were positively correlated with the expression level of CnDFR.

Figure 4

The subcellular localization of CnDFR. (A) Observation by LSM510 Meta of the EGFP empty vector. White scale: 20 µm. The green fluorescence signals appeared in the nucleus, cell membrane and cytoplasm under the excitation of the wavelength of 488 nm. (B) Observation of the lower epidermal cells of Nicotiana benthamiana leaves with the CnDFR-EGFP vector. White scale: 20 µm. The nucleus and cell membrane expressed a strong green fluorescence signal.

Figure 5

Relative expression of CnDFR and contents of total flavonoids, total polyphenols and total anthocyanins in flowers of transgenic tobaccos. (A) The expression of CnDFR in tobacco flowers. CnDFR expression of transgenic lines was significantly higher than the wild type. (B) Flowers of wild type and transgenic tobaccos. The flowers of wild type and transgenic strains were white with no significant difference. (C) The contents of total polyphenols and total flavonoids. Total polyphenols in most of transgenic strains were about twice the wild type. Total flavonoids were very low overall, and the contents in transgenic strains were 1.5 times the wild type. The letters represent the level of difference.

Figure 6

The content of flavonoids and polyphenols in flowers of transgenic tobaccos. (A) The content of EGC (epigallocatechin), GC (gallocatechin) and GCG (gallocatechin gallate) in flowers of tobaccos. (B) The content of EGCG (epigallocatechin gallate), CG CG (catechin gallate) and ECG (epicatechin gallate) in flowers of tobaccos. The contents of EGC, GC, GCG (except DFR-17), EGCG (except DFR-50), CG (except DFR-18) and ECG (except DFR-50) in the positive lines were significantly higher than those in the wild type tobacco except one line. (C) The content of Ka (kaempferol), Qu3 G (quercetin-3-O-glucopyranoside) and Qu(quercetin) in flowers of tobaccos. (D) The content of DHQ (dihydroquercetin), Qu7 G (quercetin-7-O-β-D-glucopyranoside) and Ka3 G (kaempferol-3-glucopyranoside) in flowers of tobaccos. DHQ (except DFR-33) and Ka in the positive lines were significantly higher than those in the wild type tobacco. The contents of flavonols (Qu3 G, Qu7 G and Ka3 G) have no significant change between the positive lines and the wild type. The letters represent the level of difference.