Ectopic expression of apple F3'H genes contributes to anthocyanin accumulation in the Arabidopsis tt7 mutant grown under nitrogen stress

Abstract

Three genes encoding flavonoid 3'-hydroxylase (F3'H) in apple (Malus x domestica), designated MdF3'HI, MdF3'HIIa, and MdF3'HIIb, have been identified. MdF3'HIIa and MdF3'HIIb are almost identical in amino acid sequences, and they are allelic, whereas MdF3'HI has 91% nucleotide sequence identity in the coding region to both MdF3'HIIa and MdF3'HIIb. MdF3'HI and MdF3'HII genes are mapped onto linkage groups 14 and 6, respectively, of the apple genome. Throughout the development of apple fruit, transcriptional levels of MdF3'H genes along with other anthocyanin biosynthesis genes are higher in the red-skinned cv Red Delicious than that in the yellow-skinned cv Golden Delicious. Moreover, patterns of MdF3'H gene expression correspond to accumulation patterns of flavonoids in apple fruit. These findings suggest that MdF3'H genes are coordinately expressed with other genes in the anthocyanin biosynthetic pathway in apple. The functionality of these apple F3'H genes has been demonstrated via their ectopic expression in both the Arabidopsis (Arabidopsis thaliana) transparent testa7-1 (tt7) mutant and tobacco (Nicotiana tabacum). When grown under nitrogen-deficient conditions, transgenic Arabidopsis tt7 seedlings expressing apple F3'H regained red color pigmentation and significantly accumulated both 4'-hydrylated pelargonidin and 3',4'-hydrylated cyanidin. When compared with wild-type plants, flowers of transgenic tobacco lines overexpressing apple F3'H genes exhibited enhanced red color pigmentation. This suggests that the F3'H enzyme may coordinately interact with other flavonoid enzymes in the anthocyanin biosynthesis pathway.

Figure 1.

The biosynthesis pathways of the most abundant anthocyanin pigments. The proposed pathway in apple is marked with dotted lines.

Figure 2.

Southern-blot analysis of apple genomic DNA and BAC DNAs. The cDNA probe corresponds to a partial region of the last exon of MdF3′HI. M, Standard DNA marker; G, apple genomic DNA. B1 to B6 correspond to six positive BAC clones. Each BAC DNA yields a strong band corresponding to one of the three bands from the genomic DNA.

Figure 3.

Comparisons of the deduced amino acid sequences of the three apple F3′H genes. Alignment was carried out using ClustalX. Sequence mismatches are noted with lowercase letters.

Figure 4.

Phylogenetic tree derived from amino acid sequences of genes encoding flavonoid hydroxylase in plants. The phylogenetic analysis was performed using the maximum parsimony method. Numbers on branches correspond to bootstrap estimates for 100 replicate analyses using 500× stepwise addition of taxa; values less than 50% are not indicated.

Figure 5.

Genetic mapping of F3′H genes in apple. Markers linked to F3′H genes are marked in boldface. The MdF3′HI gene is tagged with F3′HI-SSR, while the MdF3′HII gene is anchored by F3′HII-SSR or F3′HII-Indel. LG6 and LG14 represent linkage groups 6 and 14, respectively.

Figure 6.

Analysis of expression profiles of anthocyanin genes in apple cv Red Delicious (red skin) and cv Golden Delicious (yellow skin) using real-time PCR. The cDNA templates are listed as follows: L, young leaves; FWI, flower buds at the pink stage; FWII, flower buds at the balloon stage; FWIII, flowers at full bloom; FTI, 2 weeks after pollination (WAP); FTII, 6 WAP; FTIII, 15 WAP; FTIV, 20 WAP; FTV, 24 WAP. Transcriptional levels were normalized to expression of an apple actin gene. All data correspond to mean values of three biological replicates. [See online article for color version of this figure.]

Figure 7.

Complementation of the pigmentation of Arabidopsis tt7 mutant seedlings of the ecotype Landsberg erecta with apple F3′H genes. A, Phenotypes of wild-type, mutant, and transgenic Arabidopsis seedlings grown in nitrogen-deficient MS medium. B, Phenotypes of wild-type, mutant, and transgenic Arabidopsis seeds. C, Contents of anthocyanidins and flavonols in Arabidopsis seedlings grown in nitrogen-deficient MS medium. Data correspond to means of three biological replicates. Means with different letters within the same column are significantly different at the 0.01 level of probability. Two additional transgenic lines each of MdF3′HI and MdF3′HII were analyzed, and these exhibited similar phenotypes and HPLC profiles as shown. N/D, Not determined.

Figure 8.

Functional characterization of MdF3′H genes following their overexpression in transgenic tobacco lines. A, Differences in color between transgenic and wild-type tobacco flowers. B, Contents of anthocyanidins and flavonols in transgenic and wild-type tobacco flowers. All data correspond to mean values of three biological replicates. Values with different letters within the same column are significantly different at the 0.01 level of probability. Two transgenic lines each of MdF3′HI and MdF3′HII were analyzed and showed similar phenotypes and HPLC profiles as shown. N/D, Not determined.

Figure 9.

Analysis of expression profiles of anthocyanin genes in Arabidopsis seedlings grown in both MS medium and nitrogen-free MS medium using real-time PCR. The cDNA templates are listed as follows: 1, the wild type; 2, tt7 mutant; 3, MdF3′HI transgenic line; 4, MdF3′HIIb transgenic line. Transcriptional levels were normalized to expression of an Arabidopsis actin gene. All data correspond to mean values of three biological replicates. [See online article for color version of this figure.]