Search for transcriptional and metabolic markers of grape pre-ripening and ripening and insights into specific aroma development in three Portuguese cultivars

Abstract

Background: Grapes (Vitis species) are economically the most important fruit crop worldwide. However, the complexity of molecular and biochemical events that lead to ripening of berries as well as how aroma is developed are not fully understood.

Methodology/principal findings: In an attempt to identify the common mechanisms associated with the onset of ripening independently of the cultivar, grapes of Portuguese elite cultivars, Trincadeira, Aragonês, and Touriga Nacional, were studied. The mRNA expression profiles corresponding to veraison (EL35) and mature berries (EL36) were compared. Across the three varieties, 9,8% (2255) probesets corresponding to 1915 unigenes were robustly differentially expressed at EL 36 compared to EL 35. Eleven functional categories were represented in this differential gene set. Information on gene expression related to primary and secondary metabolism was verified by RT-qPCR analysis of selected candidate genes at four developmental stages (EL32, EL35, EL36 and EL 38). Gene expression data were integrated with metabolic profiling data from GC-EI-TOF/MS and headspace GC-EI-MS platforms.

Conclusions/significance: Putative molecular and metabolic markers of grape pre-ripening and ripening related to primary and secondary metabolism were established and revealed a substantial developmental reprogramming of cellular metabolism. Altogether the results provide valuable new information on the main metabolic events leading to grape ripening. Furthermore, we provide first hints about how the development of a cultivar specific aroma is controlled at transcriptional level.

Figure 1. Fresh berry weight (g) and total anthocyanin content (absorbance at 530 nm per g of freeze dried material) of berries at developmental stages of EL 32, EL 35, EL 36 and EL 38.

Bars represent standard deviation.

Figure 2. Relative quantification of malic acid, tartaric acid, fructose and glucose based on ion peak response.

Malate and tartrate contents are higher at pre-véraison stages peaking at EL 32 whereas contents in fructose and glucose increase at post-véraison stages reaching maximal levels at EL 38.

Figure 3. Score plot of ICA showing metabolic discrimination of developmental stages and cultivars.

A Polar metabolites B Volatiles.

Figure 4. Heat map showing metabolite levels analyzed during berry ripening.

Each square represents the average of the biological replicates.

Figure 5. PCA plot showing transcriptional discrimination of developmental stages and cultivars.

The first (PC1) and the second (PC2) principal components are represented.

Figure 6. Functional categories distribution in the common gene set of the 2255 modulated probesets and in the entire GrapeGen Chip®.

Figure 7. Real time qPCR validation of the expression profiles of ten genes in the three varieties under analysis.

Data are reported as means ± SE of three technical and two-three biological replicates. Transcript levels were calculated using the standard curve method and normalized against grapevine actin gene (VVTU17999_s_at) used as reference control. Flavonol synthase (VVTU16103_at); Palmitoyl-monogalactosyldiacylglycerol delta-7 desaturase, chloroplast (VVTU3709_at); Succinate semialdehyde dehydrogenase (VVTU35625_s_at); Tocophenol cyclase (VVTU22626_s_at); L-Galactono-1,4-lactone dehydrogenase (VVTU8069_at); Limonoid UPD-glycosyltransferase (VVTU17111_s_at); Terpene synthase (VvTPS10); Terpene synthase (VvTPS34); Cytochrome 450 monooxygenase CYP89H3 (VVTU21329_at) and Caffeic acid O-methyltransferase (VVTU36927_x_at).

Figure 8. Phenylpropanoids-related transcripts and metabolites showing significant differences between véraison and ripening (EL 36 vs EL 35) in Touriga Nacional cultivar; pathways are represented in VitisNet networks with the Cytoscape software.

Parallelograms represent non-redundant transcripts differentially expressed. Ovals and hexagons represent metabolites and enzymes, respectively. Pink lines show metabolic reactions and blue lines catalytic reactions; bold arrows highlight the reactions starting from phenylalanine leading to anthocyanidin biosynthesis. Green represents transcripts down-regulated or decrease in metabolites values, whereas purple represents transcripts up-regulated or increase in metabolites values.

Figure 9. Candidate molecular and metabolic markers of pre-ripe and ripe berries established in three Portuguese varieties.