Olive phenolic compounds: metabolic and transcriptional profiling during fruit development

Abstract

Background: Olive (Olea europaea L.) fruits contain numerous secondary metabolites, primarily phenolics, terpenes and sterols, some of which are particularly interesting for their nutraceutical properties. This study will attempt to provide further insight into the profile of olive phenolic compounds during fruit development and to identify the major genetic determinants of phenolic metabolism.

Results: The concentration of the major phenolic compounds, such as oleuropein, demethyloleuropein, 3-4 DHPEA-EDA, ligstroside, tyrosol, hydroxytyrosol, verbascoside and lignans, were measured in the developing fruits of 12 olive cultivars. The content of these compounds varied significantly among the cultivars and decreased during fruit development and maturation, with some compounds showing specificity for certain cultivars. Thirty-five olive transcripts homologous to genes involved in the pathways of the main secondary metabolites were identified from the massive sequencing data of the olive fruit transcriptome or from cDNA-AFLP analysis. Their mRNA levels were determined using RT-qPCR analysis on fruits of high- and low-phenolic varieties (Coratina and Dolce d'Andria, respectively) during three different fruit developmental stages. A strong correlation was observed between phenolic compound concentrations and transcripts putatively involved in their biosynthesis, suggesting a transcriptional regulation of the corresponding pathways. OeDXS, OeGES, OeGE10H and OeADH, encoding putative 1-deoxy-D-xylulose-5-P synthase, geraniol synthase, geraniol 10-hydroxylase and arogenate dehydrogenase, respectively, were almost exclusively present at 45 days after flowering (DAF), suggesting that these compounds might play a key role in regulating secoiridoid accumulation during fruit development.

Conclusions: Metabolic and transcriptional profiling led to the identification of some major players putatively involved in biosynthesis of secondary compounds in the olive tree. Our data represent the first step towards the functional characterisation of important genes for the determination of olive fruit quality.

Figure 1

Concentration of total phenols and secoiridoids compounds in olive fruits. Phenolic compounds of 12 cultivars during fruit development (45, 60, 75, 90, 105, 120, 135, 150 and 165 DAF) were considered. A) Total biophenols, B) Oleuropein, C) Demethyloleuropein, D) 3,4-DHPEA-EDA, and E) Ligstroside. Demethyloleuropein was not detected in cvs. Dolce d’Andria, Nocellara del Belice and Nocellara Etnea. Red and Blue lines represent high (HP) and low phenolic (LP) cultivars, respectively. The standard errors are not shown in the graphs because the values were lower than 5%.

Figure 2

Concentration of phenolic compounds in olive fruits. Phenolic compounds of 12 cultivars during fruit growth (45, 60, 75, 90, 105, 120, 135, 150 and 165 DAF) were considered. A) Tyrosol, B) Hydroxytyrosol, C) Verbascoside, and D) Lignans. Red and Blue lines represent high (HP) and low phenolic (LP) cultivars, respectively. The standard errors are not shown in the graphs because the values were lower than 5%.

Figure 3

Schematic representation showing the putative biosynthetic pathways of main secondary compounds of olive fruits. G3P: glyceraldehyde 3-phosphate; DMAPP: Dimethylallyl diphosphate; IPP: Isopentenyl diphosphate; AC: Acetyl-CoA; MVAPP: Mevalonate diphosphate; GPP: Geranyl diphosphate; FPP: Farnesyl diphosphate; and GGPP: Geranyl geranyl pyrophosphate. Dotted arrows indicate uncertain biosynthetic steps.

Figure 4

The expression of genes putatively involved in the MEP and MVA pathways. The mRNA expression of genes putatively involved in 2-C-methyl-d-erythritol 4-phosphate (MEP) and mevalonic acid (MVA) pathways, as determined using RT-qPCR and a schematic representation of these pathways. G3P: glyceraldehyde 3-phosphate; DXP: 1-deoxy-D-xylulose-5-P; DXS: DXP synthase; DXR: DXP reductoisomerase; MEP: 2-C-methyl-D-erythritol-4-P; CDPME: 4-(CDP)-2-C-methyl-D-erythritol; CDPMES: CDPME synthase; CDPMEK: CDPME kinase; CDPME2P: 4-(CDP)-2-C-methyl-D-erythritol-2-P; MECP: 2-C-methyl-D-erythritol 2,4-cyclo-PP; MECPS: MECP synthase; HMBPP: 1-hydroxy-2-methyl-2-(E)-butenyl-4-PP; HMBPPS: HMBPP synthase; HMBPPR: HMBPP reductase; DMAPP: Dimethylallyl diphosphate; IPP: Isopentenyl diphosphate; IPPI: IPP delta isomerase; AC: Acetyl-CoA; ACC: Acetoacetyl-CoA; HMG: 3-hydroxy-3-methylglutaryl-CoA; HMGR: HMGC reductase; MVAK: MVA kinase; MVAP: mevalonate phosphate; MVAPK: MVAP kinase; MVAPP: Mevalonate diphosphate; MVAPPD: MVAPP decarboxylase; and DAPP: Dimethylallyl diphosphate. The relative mRNA levels are expressed as ΔΔCt. Bars = ±SE, n = 3. Different letters indicate significant differences between samples, as determined using analysis of variance (Bonferroni’s post hoc tests, P < 0.05).

Figure 5

The expression of genes putatively involved in the metabolism of secoiridoids and other phenolics compounds. The mRNA expression of genes putatively involved in the biosynthesis of terpenic (A) and phenolic moieties (B) of secoiridoids, in the biosynthesis of phenylpropanoids (C), in the degradation of phenolic compounds, and a schematic representation of their metabolism. The mRNA level was determined using RT-qPCR. GES: geraniol synthase; G10H: Geraniol 10-hydroxylase; NDHI: NADH dehydrogenase I; GT: SLS: Secologanin synthase; LAMT: Loganic acid methyltransferase; ADH: Arogenate dehydrogenase; CuAO: Copper amine oxidase; p-HPPA: p-hydroxyphenylpyruvic acid; p-HPAA: p-hydroxyphenylacetic acid; TYRD: Tyrosine/dopa decarboxylase; ALDH: Alcohol dehydrogenase; PPO: Polyphenol oxidase; PAL: Phenylalanine ammonia-lyase; and 4CL: 4-coumarate coenzyme A ligase. The relative mRNA levels are expressed as ΔΔCt. Bars = ±SE, n = 3. Different letters indicate significant differences between samples as determined using analysis of variance (Bonferroni’s post hoc tests, P < 0.05). Grey dotted box includes the secoiridoids compounds. Dotted arrows indicates uncertain biosynthetic steps.

Figure 6

The expression of transcripts putatively involved in the biosynthesis of terpenes and sterols. The mRNA expression of genes putatively involved in the biosynthesis of volatile monoterpenes (A), tri-terpenoids and sterols (B), di- and tetra-terpenoids (C), as determined using RT-qPCR, and a schematic representation of this pathway. IPP: Isopentenyl diphosphate; GPP: Geranyl diphosphate; GES: geraniol synthase; LS: Limonene synthase; FPP: Farnesyl diphosphate; FPPS: FPP synthase; GGPP: Geranyl geranyl pyrophosphate; GGPS: GGPP synthase; SQS: Squalene synthase; LUPS: Lupeol synthase. The relative mRNA levels are expressed as ΔΔCt. Bars = ±SE, n = 3. Different letters indicate significant differences between samples as determined using analysis of variance (Bonferroni’s post hoc tests, P < 0.05).