This work characterized the role of the R2R3-MYB10 transcription factor (TF) in strawberry fruit ripening. The expression of this TF takes place mainly in the fruit receptacle and is repressed by auxins and activated by abscisic acid (ABA), in parallel to the ripening process. Anthocyanin was not produced when FaMYB10 expression was transiently silenced in fruit receptacles. An increase in FaMYB10 expression was observed in water-stressed fruits, which was accompanied by an increase in both ABA and anthocyanin content. High-throughput transcriptomic analyses performed in fruits with downregulated FaMYB10 expression indicated that this TF regulates the expression of most of the Early-regulated Biosynthesis Genes (EBGs) and the Late-regulated Biosynthesis Genes (LBGs) genes involved in anthocyanin production in ripened fruit receptacles. Besides, the expression of FaMYB10 was not regulated by FaMYB1 and vice versa. Taken together, all these data clearly indicate that the Fragaria × ananassa MYB10 TF plays a general regulatory role in the flavonoid/phenylpropanoid pathway during the ripening of strawberry.

Plant phenolics have drawn increasing attention due to their potential nutritional benefits. Although the basic reactions of the phenolics biosynthetic pathways in plants have been intensively analyzed, the regulation of their accumulation and flux through the pathway is not that well established. The aim of this study was to use a strawberry (Fragaria × ananassa) microarray to investigate gene expression patterns associated with the accumulation of phenylpropanoids, flavonoids, and anthocyanins in strawberry fruit. An examination of the transcriptome, coupled with metabolite profiling data from different commercial varieties, was undertaken to identify genes whose expression correlated with altered phenolics composition. Seventeen comparative microarray analyses revealed 15 genes that were differentially (more than 200-fold) expressed in phenolics-rich versus phenolics-poor varieties. The results were validated by heterologous expression of the peroxidase FaPRX27 gene, which showed the highest altered expression level (more than 900-fold). The encoded protein was functionally characterized and is assumed to be involved in lignin formation during strawberry fruit ripening. Quantitative trait locus analysis indicated that the genomic region of FaPRX27 is associated with the fruit color trait. Down-regulation of the CHALCONE SYNTHASE gene and concomitant induction of FaPRX27 expression diverted the flux from anthocyanins to lignin. The results highlight the competition of the different phenolics pathways for their common precursors. The list of the 15 candidates provides new genes that are likely to impact polyphenol accumulation in strawberry fruit and could be used to develop molecular markers to select phenolics-rich germplasm.

Short-chain esters contribute to the blend of volatiles that define the strawberry aroma. The last step in their biosynthesis involves an alcohol acyltransferase that catalyses the esterification of an acyl moiety of acyl-CoA with an alcohol. This study identified a novel strawberry alcohol acyltransferase gene (FaAAT2) whose expression pattern during fruit receptacle growth and ripening is in accordance with the production of esters throughout strawberry fruit ripening. The full-length FaAAT2 cDNA was cloned and expressed in Escherichia coli and its activity was analysed with acyl-CoA and alcohol substrates. The semi-purified FaAAT2 enzyme had activity with C1-C8 straight-chain alcohols and aromatic alcohols in the presence of acetyl-CoA. Cinnamyl alcohol was the most efficient acyl acceptor. When FaAAT2 expression was transiently downregulated in the fruit receptacle by agroinfiltration, the volatile ester production was significantly reduced in strawberry fruit. The results suggest that FaAAT2 plays a significant role in the production of esters that contribute to the final strawberry fruit flavour.

Summary Strawberry (Fragaria × ananassa) is a fruit crop with a distinct biphasic flavonoid biosynthesis. Whereas, in the immature receptacle, high levels of proanthocyanidins accumulate, which are associated with herbivore deterrence and pathogen defense, the prominent color‐giving anthocyanins are primarily produced in ripe ‘fruits’ helping to attract herbivores for seed dispersal. Here, constitutive experimental down‐regulation of one branch of proanthocyanidin biosynthesis was performed. As a result, the proportion of epicatechin monomeric units within the proanthocyanidin polymer chains was reduced, but this was not the case for the epicatechin starter unit. Shortened chain lengths of proanthocyanidins were also observed. All enzymatic activities for the production of color‐giving anthocyanins were already present in unripe fruits at levels allowing a striking red anthocyanin phenotype in unripe fruits of the RNAi silencing lines. An immediately recognizable phenotype was also observed for the stigmata of flowers, which is another epicatechin‐forming tissue. Thus, the down‐regulation of anthocyanidin reductase (ANR) induced a redirection of the proanthocyanidin pathway, leading to premature and ectopic anthocyanin biosynthesis via enzymatic glycosylation as the alternative pathway. This redirection is also seen in flavonol biosynthesis, which is paralleled by higher pollen viability in silencing lines. ANRi transgenic lines of strawberry provide a versatile tool for the study of the biological functions of proanthocyanidins.

Flavonoids are products from specialized metabolism that contribute to fruit sensorial (color) and nutritional (antioxidant properties) quality. Here, using a pseudo full-sibling F1 progeny previously studied for fruit sensorial quality of cultivated strawberry (Fragaria × ananassa), we explored over two successive years the genetic architecture of flavonoid-related traits using liquid chromatography electrospray ionization tandem mass spectrometry (13 compounds including anthocyanins, flavonols, and flavan-3-ols) and colorimetric assays (anthocyanins, flavonoids, phenolics, and total antioxidant capacity (ferric reducing antioxidant power and Trolox equivalent antioxidant capacity)). Network correlation analysis highlighted the high connectivity of flavonoid compounds within each chemical class and low correlation with colorimetric traits except for anthocyanins. Mapping onto the female and male linkage maps of 152 flavonoid metabolic quantitative trait loci (mQTLs) and of 26 colorimetric QTLs indicated colocalization on few linkage groups of major flavonoid- and taste-related QTLs previously uncovered. These results pave the way for the discovery of genetic variations underlying flavonoid mQTLs and for marker-assisted selection of strawberry varieties with improved sensorial and nutritional quality.