Chlorophyll, carotenoid and vitamin C metabolism regulation in Actinidia chinensis 'Hongyang' outer pericarp during fruit development

Abstract

Ascorbic acid (AsA), chlorophyll and carotenoid contents and their associated gene expression patterns were analysed in Actinidia chinensis 'Hongyang' outer pericarp. The results showed chlorophyll degradation during fruit development and softening, exposed the yellow carotenoid pigments. LHCB1 and CLS1 gene expressions were decreased, while PPH2 and PPH3 gene expressions were increased, indicating that downregulation of chlorophyll biosynthesis and upregulation of its degradation, caused chlorophyll degradation. A decrease in the expression of the late carotenoid biosynthesis and maintenance genes (LCYB1, LCYE1, CYP1, CYP2, ZEP1, VDE1, VDE2, and NCED2) and degradation gene (CCD1), showed biosynthesis and degradation of carotenoid could be regulatory factors involved in fruit development. Most genes expression data of L-galactose and recycling pathway were agreement with the AsA concentrations in the fruit, suggesting these are the predominant pathways of AsA biosynthesis. GMP1, GME1 and GGP1 were identified as the key genes controlling AsA biosynthesis in 'Hongyang' outer pericarp.

Fig 1

Development of A. chinensis var. chinensis ‘Hongyang’ from the open flower (0 days after anthesis, DAA) to ripened fruit including fruit weight (A), size (B), dry matter (C), °Brix (D), soluble sugar content (E), titratable acid (F), ascorbic acid (G), firmness (H) and phenotype (I, Bars indicated 1 cm). Each value is presented as the mean ± standard deviation (n = 10).

Fig 2. Change in chlorophyll a, chlorophyll b, total chlorophyll, and total carotenoid contents during A. chinensis var. chinensis ‘Hongyang’ fruit development.

Each value is presented as the mean ± standard deviation (n = 10).DAA, days after anthesis.

Fig 3. Expression of chlorophyll biosynthetic pathway associated genes in A. chinensis var. chinensis Hongyang’ outer pericarp during fruit development.

CAO1, chlorophyll a oxygenase 1; GluTR1, glutamyl-tRNA synthase 1; RBCS1, ribulose-1,5-bisphosphate carboxylase/oxygenase small subunit 1; LHCB1, light-harvesting chlorophyll a/b binding complex 1; LHCB2, light-harvesting chlorophyll a/b binding complex 2; CLS1, chlorophyll synthase 1. Error bars indicate standard error (n = 3). The asterisk (*) represents that the sample was used as reference in relative comparison. The different small letters for number in a same gene represent significant difference at 0.05 level. Heatmap were performed using the software of MEV (Multi Experiment Viewer). Color scale represents log2^−ΔΔCt counts where blue indicates low level and red indicate high level.

Fig 4. Gene expression of chlorophyll degradation pathway in A. chinensis var. chinensis ‘Hongyang’ outer pericarp during fruit development.

CBR1, chlorophyll b reductase; PAO1, pheophorbide a oxygenase 1; PAO2, pheophorbide a oxygenase 2; PPH1, pheophytin pheophorbide hydrolase 1; PPH2, pheophytin pheophorbide hydrolase 2; PPH3, pheophytin pheophorbide hydrolase 3; SGR1, stay-green 1; CLH1, chlorophyllase 1; CLH2, chlorophyllase 2. Error bars indicate standard error (n = 3). The asterisk (*) represents that the sample was used as reference in relative comparison. The different small letters for number in a same gene represent significant difference at 0.05 level. Heatmap were performed using the software of MEV. Color scale represents log2^−ΔΔCt counts where blue indicates low level and red indicate high level.

Fig 5. The expression of carotenoid biosynthesis and degradation genes in A. chinensis var. chinensis ‘Hongyang’ outer pericarp during fruit development.

CCD, carotenoid cleavage dioxygenases; CHY, non-heme hydroxylases; CRTISO, 7,9,7',9'-tetra-cis-lycopene isomerase; CYP, P450 hydroxylase; LCYB, lycopene β-cyclase; LCYE, lycopene γ-cyclase; NCED, 9-cis-epoxycarotenoid dioxygenase; PDS, phytoene desaturase; PSY, phytoene synthase; PTOX, alternative oxidase; VDE, violaxanthin de-epoxidase; ZDS, ζ-carotene desaturase; ZEP, zeaxanthin epoxidase; ZISO, 9,15,9’-tri-cis-ζ-carotene isomerase. Error bars indicate standard error (n = 3). The asterisk (*) represents that the sample was used as reference in relative comparison. The different small letters for number in a same gene represent significant difference at 0.05 level. Heatmap was performed using the software of MEV. Color scale represents log2^−ΔΔCt counts where blue indicates low level and red indicate high level.

Fig 6. The expression of ascorbic acid biosynthesis and recycling pathway genes in A. chinensis var. chinensis ‘Hongyang’ outer pericarp during fruit development.

AO, L-ascorbate oxidase; APX, L-ascorbate peroxidase; DHAR, dehydroascorbate reductase; GalLDH, L-galactono-1,4-lactone dehydrogenase; GDH, L-galactose dehydrogenase; GGP, GDP-L-galactose phosphorylase; GME, GDP-D-mannose-3,5-epimerase; GMP, GDP-D-mannose pyrophosphorylase; GPP, L-galactose-1-phosphate phosphatase; MDHAR, monodehydroascorbate reductase; PGI, glucose-6-phosphate isomerase; PME, pectinesterase; PMI, mannose-6-phosphate isomerase; PMM, phosphomannomutase. Error bars indicate standard error (n = 3).The asterisk (*) represents that the sample was used as reference in relative comparison. The different small letters for number in a same gene represent significant difference at 0.05 level. Heatmap was performed using the software of MEV. Color scale represents log2^−ΔΔCt counts where blue indicates low level and red indicate high level.