Synthesis of flavour-related linalool is regulated by PpbHLH1 and associated with changes in DNA methylation during peach fruit ripening

Abstract

Linalool is one of the common flavour-related volatiles across the plant kingdom and plays an essential role in determining consumer liking of plant foods. Although great process has been made in identifying terpene synthase (TPS) genes associated with linalool synthesis, much less is known about regulation of this pathway. We initiated study by identifying PpTPS3 encoding protein catalysing enantiomer (S)-(+)-linalool synthesis, which is a major linalool component (˜70%) observed in ripe peach fruit. Overexpression of PpTPS3 led to linalool accumulation, while virus-induced gene silencing of PpTPS3 led to a 66.5% reduction in linalool content in peach fruit. We next identified transcription factor (TF) PpbHLH1 directly binds to E-box (CACATG) in the PpTPS3 promoter and activates its expression based on yeast one-hybrid assay and EMSA analysis. Significantly positive correlation was also observed between PpbHLH1 expression and linalool production across peach cultivars. Peach fruit accumulated more linalool after overexpressing PpbHLH1 in peach fruit and reduced approximately 54.4% linalool production after silencing this TF. DNA methylation analysis showed increased PpTPS3 expression was associated with decreased 5 mC level in its promoter during peach fruit ripening, but no reverse pattern was observed for PpbHLH1. Arabidopsis and tomato fruits transgenic for peach PpbHLH1 synthesize and accumulate higher levels of linalool compared with wild-type controls. Taken together, these results would greatly facilitate efforts to enhance linalool production and thus improve flavour of fruits.

Keywords: epigenetics; fruit aroma; linalool; transcription factor.

Figure 1

Changes in volatiles during peach fruit development and ripening. (a) Photo of peach fruit cv Hujingmilu at different stages: S1 (34 days after bloom, DAB), S2 (71 DAB), S3 (94 DAB), S4 (108 DAB) and S5 (111 DAB). Bars = 1 cm. (b) Volatile content of peach fruit. Averaged data from three biological replicates are shown. Standard errors are presented in Table S1.

Figure 2

Characterization of PpTPS3 related to linalool production. (a) Content of linalool during peach fruit development and ripening. The linalool enantiomers were distinguished using chiral GC–MS analysis. (b) Expression pattern of peach TPS genes during peach development and ripening. (c) Phylogenetic analysis of plant TPSs based on deduced amino acid sequence. Accession numbers for these TPSs are given in Table S2.

Figure 3

PpTPS3 catalyses linalool synthesis both in vitro and in vivo. (a) Enzymatic activity assay of PpTPS3 protein towards geranyl pyrophosphate (GPP) as substrate in vitro. (b) Schematic diagram for gene transient expression in peach fruits. (c) Transient overexpression of PpTPS3 increases linalool content in peach fruits. Empty SK vector was used as a control. (d) Silencing PpTPS3 by VIGS decreases linalool content in peach fruits. Empty pTRV1 + pTRV2 vector was used as a control. Phytoene desaturase (PDS) gene was used as a reporter gene for VIGS (Figure S3). (e) Overexpressing PpTPS3 induces linalool synthesis in Arabidopsis. tps10 mutant was used as a control. (f) Increased linalool content in tomato fruits after overexpressing PpTPS3. Wild‐type (WT) fruits were used as controls. (g) Subcellular localization of PpTPS3. Bars = 20 µm. Data are presented as mean ± standard error from three independent biological replicates. Significant differences are indicated with asterisks above the bars (*, P < 0.05; **, P < 0.01; and ***, P < 0.001). N.D., not detected.

Figure 4

PpbHLH1 activates PpTPS3 and binds to its promoter. (a) Regulatory effects of transcription factors on the promoter of PpTPS3. Means and standard errors were calculated from six replicates. (b) Yeast one‐hybrid analysis of PpbHLH1 binding to the PpTPS3 promoter. Autoactivation was tested on SD‐Ura in the presence of AbA. AD‐empty and pAbAi‐PpTPS3 were used as negative controls. (c) G‐box (CACGTG) and E‐boxes (CACATG) of bHLH protein‐binding sites in the PpTPS3 promoter. (d) EMSA of 3’ boitin‐labelled dsDNA probes with the PpbHLH1‐binding protein. Presence or absence of specific probes is marked by symbol + or ‐. The mutated nucleotides in probe are indicated in red lowercase letters. (e) Subcellular localization of PpbHLH1 in Nicotiana benthamiana leaves. GFP, GFP channel; nucleus–RFP, transgenic tobacco plants with red fluorescence in the nucleus; merge, merged image of the GFP and nucleus–RFP channels; bright‐field, light microscopy image; bars = 50 µm.

Figure 5

Correlation between transcript levels of PpbHLH1, PpTPS3 and contents of linalool in peach fruit. (a) Content of linalool and expression levels of PpTPS3 and PpbHLH1 across peach cultivars. (b) Correlation analysis between content of linalool and expression levels of PpTPS3 and PpbHLH1 across peach cultivars. (c) Analysis of cis‐acting elements of PpbHLH1 2kb promoter sequence based on the PlantCARE database. (d) Effect of UV‐B light treatment on content of linalool and expression levels of PpTPS3 and PpbHLH1 in peach fruit. Relative expression levels were determined using RT‐qPCR. Data are presented as mean ± standard error from three independent biological replicates. Significant differences are indicated with asterisks above the bars (**, P < 0.01).

Figure 6

Regulatory effects of PpbHLH1 on linalool synthesis in peach fruit. (a) Overexpressing PpbHLH1 induces expression of PpTPS3 and linalool accumulation in peach fruits. Empty SK vector was used as a control. Significant differences are indicated with asterisks above the bars (*, P < 0.05; **, P < 0.01; and ***, P < 0.001). (b) Chiral GC‐MS analysis of linalool enantiomers in transiently overexpressed peach fruits. (c) Silencing PpbHLH1 by VIGS decreases PpTPS3 expression and linalool content in peach fruit. Empty pTRV1 + pTRV2 vector was used as a control. (d) Changes in transcript levels and DNA methylation levels of PpTPS3 and PpbHLH1 during peach fruit ripening. (e) Effects of UV‐B irradiation on transcript levels and DNA methylation levels of PpTPS3 and PpbHLH1 in peach fruit. (f) A proposed regulatory model of linalool synthesis in peach fruit.

Figure 7

Overexpression of PpbHLH1 increases content of linalool in planta. (a) Transient overexpressing PpbHLH1 induces linalool accumulation in tobacco (Nicotiana benthamiana). (b) Transgenic overexpressing PpbHLH1 causes linalool accumulation in Arabidopsis. Wild‐type Arabidopsis Col‐0 was used as control. (c) Transgenic tomato fruits with overexpressing PpTPS3 produces higher content of linalool than wild‐type controls. Relative expression levels were determined using RT‐qPCR. Data are presented as mean ± standard error from three independent biological replicates. Significant differences are indicated with asterisks above the bars (*, P < 0.05; **, P < 0.01; and ***, P < 0.001). N.D., not detected.