Transcriptional analysis of apple fruit proanthocyanidin biosynthesis

Abstract

Proanthocyanidins (PAs) are products of the flavonoid pathway, which also leads to the production of anthocyanins and flavonols. Many flavonoids have antioxidant properties and may have beneficial effects for human health. PAs are found in the seeds and fruits of many plants. In apple fruit (Malus × domestica Borkh.), the flavonoid biosynthetic pathway is most active in the skin, with the flavan-3-ols, catechin, and epicatechin acting as the initiating units for the synthesis of PA polymers. This study examined the genes involved in the production of PAs in three apple cultivars: two heritage apple cultivars, Hetlina and Devonshire Quarrenden, and a commercial cultivar, Royal Gala. HPLC analysis shows that tree-ripe fruit from Hetlina and Devonshire Quarrenden had a higher phenolic content than Royal Gala. Epicatechin and catechin biosynthesis is under the control of the biosynthetic enzymes anthocyanidin reductase (ANR) and leucoanthocyanidin reductase (LAR1), respectively. Counter-intuitively, real-time quantitative PCR analysis showed that the expression levels of Royal Gala LAR1 and ANR were significantly higher than those of both Devonshire Quarrenden and Hetlina. This suggests that a compensatory feedback mechanism may be active, whereby low concentrations of PAs may induce higher expression of gene transcripts. Further investigation is required into the regulation of these key enzymes in apple.

Fig. 1.

Scheme of the apple polyphenolic pathway, showing structural genes involved in flavonoid biosynthesis in Arabidopsis thalianaseed and Malus × domestica fruit, annotated in bold with the TAIR locus and GenBank accession numbers, and their predicted linkage group in the apple reference genome, with approximate position (to the nearest 100kb). PAL, phenylalanine ammonia lyase;C4H, cinnamate-4-hydroxymate; 4CL, 4-coumarate:coenzyme A ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3-hydroxylase, F3'H, flavanone 3'-hydroxylase; DFR, dihydroflavonol 4-reductase; ANS, anthocyanidin synthase; UFGT, UDP-glucose flavonoid 3-O-glucosyl transferase; FLS, flavonol synthase; LAR1/2, leucoanthocyandin reductase; ANR, anthocyanidin reductase; GT1/2, glycosyltransferases; HQT/HCT, quinate hydroxycinnamoyl/hydrxycinnamoyl CoA shikimate; C3H, p-coumarate 3-hydroxylase.

Fig. 2.

(A) Apple fruit developmental series. Developing Hetlina, Devonshire Quarrenden, and Royal Gala apple fruit were collected at five time points throughout the 2007/2008 growing season. (B) HPLC data of apple skin and cortex for total polyphenolic composition for Hetlina, Devonshire Quarrenden, and Royal Gala over a developmental series. (C) HPLC data of apple skin and cortex for total flavan-3-ols (catechin and epicatechin) for Hetlina, Devonshire Quarrenden, and Royal Gala over a developmental series. Error bars show SE. Data points with the same letter are not significantly different at P = 0.05, using one-way ANOVA analysis, followed by a multiple-comparison t-test.

Fig. 3.

HPLC analysis for identification of the polyphenolic compounds in tree-ripe (stage 5) apple skin and cortex from Hetlina, Devonshire Quarrenden, and Royal Gala cultivars. Error bars show SE.

Fig. 4.

Real-time qPCR expression analysis for all major steps of the polyphenolic biosynthetic pathway in apple skin from Hetlina, Devonshire Quarrenden, and Royal Gala cultivars. MdActin used as the reference gene. Error bars show SD.

Fig. 5.

Real-time qPCR expression analysis for all major steps of the polyphenolic biosynthetic pathway in apple cortex from three apple cultivars, Hetlina, Devonshire Quarrenden, and Royal Gala. MdActin used as the reference gene. Error bars show SD.

Fig. 6.

Phylogenetic relationships for the deduced amino acid sequences of the ANR, LAR1, LAR2, and DFR biosynthetic genes from Malus × domestica and other species. All ANR protein sequences clustered together, as did LAR1, LAR2, and DFR.

Fig. 7.

Protein sequence alignment of various apple cultivars for (A) ANR protein and (B) LAR1 and LAR2 protein. The conserved reductase Rossmann dinucleotide-binding domain (Bottoms et al., 2002) is shown for ANR, LAR1, and LAR2. An additional motif at the C-terminal end differing between LAR1 and LAR2 is marked I (Takos et al., 2006c).