Introduction of apple ANR genes into tobacco inhibits expression of both CHI and DFR genes in flowers, leading to loss of anthocyanin

Abstract

Three genes encoding anthocyanidin reductase (ANR) in apple (Malus×domestica Borkh.), designated MdANR1, MdANR2a, and MdANR2b, have been cloned and characterized. MdANR1 shows 91% identity in coding DNA sequences with MdANR2a and MdANR2b, while MdANR2a and MdANR2b are allelic and share 99% nucleotide sequence identity in the coding region. MdANR1 and MdANR2 genes are located on linkage groups 10 and 5, respectively. Expression levels of both MdANR1 and MdANR2 genes are generally higher in yellow-skinned cv. Golden Delicious than in red-skinned cv. Red Delicious. Transcript accumulation of MdANR1 and MdANR2 genes in fruits gradually decreased throughout fruit development. Ectopic expression of apple MdANR genes in tobacco positively and negatively regulates the biosynthesis of proanthocyanidins (PAs) and anthocyanin, respectively, resulting in white, pale pink-coloured, and white/red variegated flowers. The accumulation of anthocyanin is significantly reduced in all tobacco transgenic flowers, while catechin and epicatechin contents in transgenic flowers are significantly higher than those in flowers of wild-type plants. The inhibition of anthocyanin synthesis in tobacco transgenic flowers overexpressing MdANR genes is probably attributed to down-regulation of CHALCONE ISOMERASE (CHI) and DIHYDROFLAVONOL-4-REDUCTASE (DFR) genes involved in the anthocyanin pathway. Interestingly, several transgenic lines show no detectable transcripts of the gene encoding leucoanthocyanidin reductase (LAR) in flowers, but accumulate higher levels of catechin in flowers of transgenic plants than those of wild-type plants. This finding suggests that the ANR gene may be capable of generating catechin via an alternative route, although this mechanism is yet to be further elucidated.

Fig. 1.

A general schematic diagram of the flavonoid biosynthetic pathway. CHI, chalcone isomerase; CHS, chalcone synthase; F3H, flavonoid 3-hydroxylase; F3′H, flavonoid 3′-hydroxylase; FLS, flavonol synthase; DFR or BAN, dihydroflavonol reductase; LAR, leucoanthocyanidin reductase; ANS, anthocyanidin synthase; ANR, anthocyanidin reductase; UFGT, glucose transferase.

Fig. 2.

Southern blot of BAC clones containing MdANR genes. Genomic DNA (gDNA) from apple ‘GoldRush’ was used to determine the number of gene copies of MdANR genes in the apple genome. The five positive BAC clones were designated as B1, B5, B6, B7, and B8. The digoxigenin (DIG)-labelled DNA probes correspond to cDNA fragments of the last two exons.

Fig. 3.

Phylogenetic tree derived from nucleotide sequences of genes encoding ANR and DFR in plants. The scale bar represents 0.05 substitutions per site, and the numbers next to the nodes are bootstrap values from 1000 replicates. The GenBank accession numbers are as follows: PcANR (DQ251189), FaANR (DQ664192), VvANR (XM_002271336), PtANR (XM_002317234), TcANR (GU324348), GhANR (EF187443), DkANR (AB195284), CsANR (AY641729), LuANR (EF197823), RcANR (XM_002518532), PcANR (BN000164), MsANR (HM754630), AtANR (NM_104854), HvANR1 (AK373696), HvANR2 (AK365124), OsANR (NM_001060512), SbANR (XM_002447113), ZmANR (BT064433), PcDFR (AY227730), IhDFR (GQ180935), IbDFR (EU402466), CaDFR (EU189076), and GhDFR (FJ713480).

Fig. 4.

Mapping of MdANR1 and MdANR2 genes onto apple genetic linkage groups 10 and 5, respectively.

Fig. 5.

Real-time PCR analysis of MdANR1 and MdANR2a/b gene expression in various floral and fruit tissues of ‘Red Delicious’ and ‘Golden Delicious’. Differential expression of MdANR1 and MdANR2 between the two apple cultivars Red Delicious and Golden Delicious was compared. Abbreviations for stages of development are as follows: Fw1, flower buds at the pink stage; Fw2, flower buds at the balloon stage; Fw3, flowers at full bloom; Ft1, fruitlets 9 DAP; Ft2, fruitlets 16 DAP; Ft3, fruitlets 44 DAP; Ft4, fruits at 104 DAP; Ft5, fruits at 145 DAP; RD, ‘Red Delicious’; GD, ‘Golden Delicious’. All expression data were normalized to the apple actin gene, and values are means of three technical replicates.

Fig. 6.

Ectopic expression of MdANR1 and MdANR2 genes in tobacco. (A) Tobacco flowers of wild-type (WT) and T₂ transgenic lines. Transgenic lines containing MdANR1, MdANR2a, and MdANR2b genes were pre-fixed with MdANR1, MdANR2a, and MdANR2b, respectively. (B) Expression profiles of MdANR genes in flowers of transgenic tobacco lines.

Fig. 7.

Expression profiles of flavonoid-related structural biosynthetic genes in flowers of transgenic tobacco lines carrying MdANR genes. All mRNA transcripts expressed in transgenic flowers were quantified relative to those expressed in wild-type tobacco flowers.