Metabolic and gene expression analysis of apple (Malus x domestica) carotenogenesis

Abstract

Carotenoid accumulation confers distinct colouration to plant tissues, with effects on plant response to light and as well as health benefits for consumers of plant products. The carotenoid pathway is controlled by flux of metabolites, rate-limiting enzyme steps, feed-back inhibition, and the strength of sink organelles, the plastids, in the cell. In apple (Malus × domestica Borkh), fruit carotenoid concentrations are low in comparison with those in other fruit species. The apple fruit flesh, in particular, begins development with high amounts of chlorophylls and carotenoids, but in all commercial cultivars a large proportion of this is lost by fruit maturity. To understand the control of carotenoid concentrations in apple fruit, metabolic and gene expression analysis of the carotenoid pathway were measured in genotypes with varying flesh and skin colour. Considerable variation in both carotenoid concentrations and compound profile was observed between tissues and genotypes, with carotenes and xanthophylls being found only in fruit accumulating high carotenoid concentrations. The study identified potential rate-limiting steps in carotenogenesis, which suggested that the expression of ZISO, CRTISO, and LCY-ε, in particular, were significant in predicting final carotenoid accumulation in mature apple fruit.

Fig. 1

. The apple carotenoid biosynthetic pathway with arrows showing enzymatic conversions of substrates from the isoprene biosynthesis pathway. CRH-β, β-carotene hydroxylase; CRH-ε, epsilon-carotene hydroxylase; CRTISO, carotene isomerase; DMAPP, dimethylallyl diphosphate; GGPPS, geranyl geranyl pyrophosphate synthase; IPP, isopentenyl pyrophosphate; IPPI, isopentenyl pyrophosphate isomerase; LCY-β, β-lycopene cyclase; LCY-ε, epsilon lycopene cyclase; NXS, neoxanthin synthase; PDS, phytoene desaturase; PSY, phytoene synthase; VDE, violaxanthin de-expoxidase; ZDS, φ-carotene desaturase; ZEP, zeaxanthin epoxidase; ZISO, φ-carotene isomerase.

Fig. 2

. Parents and progeny of a cross between apple ‘Aotea’ and ‘M. 9’. Apple fruit selected based on the pigmentation of their skin and flesh. (A) Ripe fruit from ‘Aotea’, ‘M. 9’ and two progeny from ‘Aotea’ × ‘M. 9’ cross, YAM9 and WAM9, had different fruit sizes. (B) Stained fruit sections of different cultivars showing plastids (arrows) in 20 days after full bloom (top and middle panels) and ripe tissues (bottom panel). Bars, 2 cm (A), 10 μm (B).

Fig. 3

. Total carotenoid concentration in ripe fruit skin and flesh of apple cultivars as measured by HPLC analysis. Fruits were harvested ripe from tree and separated into skin (peel) and flesh samples for carotenoid extraction and analysis. Error bars are standard errors of the mean from three technical replicates, with each tissue being the pooled sample of at least seven fruit, at P = 0.05 level. Bars with similar letters are not significantly different (P = 0.05), using one-way ANOVA analysis was followed by a multiple-comparisons T-test.

Fig. 4

. Fruit development series of apples used in carotenoid analysis. Fruit were harvested from ‘Aotea’, ‘M. 9’, YAM9, WAM9, ‘Granny Smith’, and ‘Royal Gala’ apple trees, growing under the same environmental conditions, at 20, 50 and 90 days after full bloom (dafb) and ripe fruit stages.

Fig. 5

. Expression of carotenoid biosynthetic pathway genes in ripe fruit skin of the different apple fruit cultivars, as determined by qRT-PCR relative to actin. Error bars are standard errors of the mean from four technical replicates.

Fig. 6

. Expression of carotenoid biosynthetic pathway genes in ripe fruit flesh of the different apple fruit genotypes, as determined by qRT-PCR relative to actin. Error bars are standard errors of the mean from four technical replicates.

Fig. 7

. Expression patterns of ZISO, CRTISO, LCY-ε and CRH-ε, which showed significant correlation with ripe fruit carotenoid concentrations, in fruit skin during fruit development. Gene expression was measured by qRT-PCR relative to actin. Error bars are standard errors of the mean from four technical replicates.

Fig. 8

. Expression patterns of ZISO, CRTISO, and LCY-ε, which showed significant correlation with ripe fruit carotenoid concentrations, in fruit flesh during fruit development. Gene expression was measured by qRT-PCR relative to actin. Error bars are standard errors of the mean from four technical replicates.