Identification of candidate flavonoid pathway genes using transcriptome correlation network analysis in ripe strawberry (Fragaria × ananassa) fruits

Abstract

New modulators of the strawberry flavonoid pathway were identified through correlation network analysis. The transcriptomes of red, ripe fruit from two parental lines and 14 of their progeny were compared, and uncharacterized transcripts matching the expression patterns of known flavonoid-pathway genes were identified. Fifteen transcripts corresponded with putative transcription factors, and several of these were examined experimentally using transient expression in developing fruits. The results suggest that two of the newly-identified regulators likely contribute to discrete nodes of the flavonoid pathway. One increases only LEUCOANTHOCYANIDIN REDUCTASE (LAR) and FLAVONOL 3'-HYDROXYLASE (F3'H) transcript accumulation upon overexpression. Another affects LAR and FLAVONOL SYNTHASE (FLS) after overexpression. The third putative transcription factor appears to be a universal regulator of flavonoid-pathway genes, as many pathway transcripts decrease in abundance when this gene is silenced. This report demonstrates that such systems-level approaches may be especially powerful when connected to an effective transient expression system, helping to provide rapid and strong evidence of gene function in key fruit-ripening processes.

Keywords: Anthocyanin; coexpresssion; flavonoid; fruit ripening; strawberry; transcriptome..

Fig. 1.

Simplified flavonoid biosynthetic pathway. PAL, Phenylalanine ammonia lyase; C4H, cinnamate-4-hydroxylase; 4CL, 4-coumaroyl:CoA-ligase; CHS, Chalcone synthase; CHI, Chalcone isomerase; F3H, Flavanone 3 hydrolase; F3’H, Flavanone 3’ hydrolase; FLS, Flavonol synthase; DFR, Dihydroflavanol reductase; LAR, Leucoanthocyanidin reductase; ANR, Anthocyanidin reductase; ANS, Anthocyanidin synthase; UFGT, UDP-Glucose flavonoid glycosyl transferase.

Fig. 2.

Expression profile correlation analysis of the flavonoid biosynthetic pathway in the ‘Mara des Bois’ × ‘Elyana’ F1 population. Correlations are considered positive when Pearson’s r>0.65 (P<0.05). The thicker the line the more close to 1 the correlation is. PAL, phenylalanine ammonia lyase; C4H, cinnamate-4-hydroxylase; 4CL, 4-coumaroyl:CoA-ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3 hydrolase; F3’H, flavanone 3’ hydrolase; FLS, flavonol synthase; DFR, dihydroflavanol reductase; LAR, leucoanthocyanidin reductase; ANR, anthocyanidin reductase; ANS, anthocyanidin synthase; UFGT, UDP-glucose flavonoid glycosyl transferase.

Fig. 3.

Phylogenetic analysis using the Neighbor–Joining method and based on closest amino acid sequences of gene17673 from different plant species. Bootstrap values are based on 1000 replicates. Bars, 0.1 amino acid substitutions per site.

Fig. 4.

Expression patterns of FaTCP11, FaPCL1-like and FaSCL8 during fruit development. GR, green stage receptacle; WR, white stage receptacle; TR, turning stage receptacle; RR, red stage receptacle; GA, green stage achenes; WA, white stage achenes; TA, turning stage achenes; RA, red stage achenes. Significance was measured by a one-way ANOVA with P<0.001.

Fig. 5.

Expression profiles in agro-infiltrated fruits overexpressing FaTCP11. Significance was measured by taking into account the relative transcript accumulation in all of the independent fruits compared to all controls. **, P<0.01; ***, P<0.001. F3’H, flavanone 3’ hydrolase; LAR, leucoanthocyanidin reductase.

Fig. 6.

Expression profiles in agro-infiltrated fruits overexpressing PCL1-like. Significance was measured comparing expression levels in construct-containing, independent fruits compared to all controls. *, P<0.05; **, P<0.01. FLS, flavonol synthase; LAR, leucoanthocyanidin reductase.

Fig. 7.

Decreased transcript accumulation in agro-infiltrated fruits with FaSCL8 silencing. Significance was measured by taking in account all the independent fruits compared to all controls. * P<0.05; **, P<0.01. PAL, phenylalanine ammonia lyase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3 hydrolase; UFGT, UDP-glucose flavonoid glycosyl transferase.

Fig. 8.

Induced expression profiles in agro-infiltrated fruits with FaSCL8 suppression. Significance was assessed from the means and variation from the fruits treated with an experimental construct compared to controls. *, P<0.05; **, P<0.01. F3’H, flavanone 3’ hydrolase; ANR, anthocyanidin reductase.

Fig. 9.

Expression correlation analysis during fruit early development in Fragaria vesca. Correlations are considered positive when Pearson’s r>0.65 (P<0.05). The thicker the line the more close to 1 the correlation is. PAL, phenylalanine ammonia lyase; C4H, cinnamate-4-hydroxylase; 4CL, 4-coumaroyl:CoA-ligase; CHS, chalcone synthase; CHI, chalcone isomerase; F3H, flavanone 3 hydrolase; F3’H, flavanone 3’ hydrolase; FLS, flavonol synthase; DFR, dihydroflavanol reductase; LAR, leucoanthocyanidin reductase; ANR, anthocyanidin reductase; ANS, anthocyanidin synthase; UFGT, UDP-glucose flavonoid glycosyl transferase.