WRKY transcription factors play a nonnegligible role in plant growth and development, but little is known about the involvement of WRKY transcription factors in the regulation of fruit ripening. In this study, FaWRKY71 was identified to be closely related to fruit maturation in octoploid strawberry. FaWRKY71 protein localized in the nucleus and responded to cold, salt, low phosphate, ABA, and light quality in strawberry seedlings. The temporal and spatial pattern expression analysis indicated that FaWRKY71 was expressed in all the detected tissues, especially in the full red fruits. In addition, FaWRKY71 gave rise to the accumulation of anthocyanin content by promoting the expression of structural genes FaF3’H, FaLAR, FaANR, and transport factors FaTT19 and FaTT12 in the flavonoid pathway, and softening the texture of strawberry via up-regulating the abundance of FaPG19 and FaPG21. Furthermore, FaWRKY71 was a positive regulator that mediated resistance against reactive oxygen species by enhancing the enzyme activities of SOD, POD, and CAT, reducing the amount of MDA. Altogether, this study provides new and comprehensive insight into the regulatory mechanisms facilitating fruit ripening in strawberry.

B-box transcription factors (TFs) play a vital role in light-induced anthocyanin accumulation. Here, the FaBBX22 gene encoding 287 amino acids B-box TF was isolated from the cultivated strawberry variety ‘Benihoppe’ and characterized functionally. The expression analysis showed that FaBBX22 was expressed in the roots, stems, leaves, flowers and fruits, and its transcription level was upregulated under the red- or blue-light irradiation. FaBBX22 was localized in the nucleus and showed trans-acting activity in yeast cells. Ectopic overexpression of FaBBX22 in Arabidopsis enhanced the accumulation of anthocyanin. Additionally, we obtained transgenic strawberry calli that overexpressed the FaBBX22 gene, and strawberry calli coloration assays showed that FaBBX22 increased anthocyanin accumulation by upregulating the expression of anthocyanin biosynthetic genes (FaPAL, FaANS, FaF3′H, FaUFGT1) and transport gene FaRAP in a light-dependent manner. Yeast two-hybrid (Y2H) and bimolecular fluorescence complementation assays indicated that FaBBX22 interacted with FaHY5. Furthermore, mutation of the 70th Asp residue in FaBBX22 protein to an Ala residue disrupted the interaction between FaBBX22 and FaHY5. Further, a transient expression assay demonstrated that the co-expression of FaBBX22 and FaHY5 could strongly promote anthocyanin accumulation in strawberry fruits. Collectively, these results revealed the positive regulatory role of FaBBX22 in light-induced anthocyanin accumulation.

Anthocyanin content is important for both the external and internal fruit quality of cultivated strawberries, but the mechanism of its accumulation in pinkish-skinned and white-fleshed strawberries is puzzling. Here, we found that the factor determining variation in the flesh color was not the FaMYB10 but the FaC4H in the cultivated strawberry Benihoppe and its white-fleshed mutant Xiaobai. Compared with Benihoppe, there was no significant difference in the coding sequence and expression level of FaMYB10 in Xiaobai’s flesh. Instead, the transcription of FaC4H was dramatically inhibited. The combined analyses of transcriptomics and metabolomics showed that the differential genes and metabolites were significantly enriched in the phenylpropanoid biosynthesis pathway. Furthermore, the transient overexpression of FaC4H greatly restored anthocyanins’ accumulation in Xiaobai’s flesh and did not produce additional pigment species, as in Benihoppe. The transcriptional repression of FaC4H was not directly caused by promoter methylations, lncRNAs, or microRNAs. In addition, the unexpressed FaF3′H, which resulted in the loss of cyanidin 3-O-glucoside in the flesh, was not due to methylation in promoters. Our findings suggested that the repression of FaC4H was responsible for the natural formation of pinkish-skinned and white-fleshed strawberries.

C2H2-type zinc finger proteins (C2H2-ZFPs) play a key role in various plant biological processes and responses to environmental stresses. In Arabidopsisthaliana, C2H2-ZFP members with two zinc finger domains have been well-characterized in response to abiotic stresses. To date, the functions of these genes in strawberries are still uncharacterized. Here, 126 C2H2-ZFPs in cultivated strawberry were firstly identified using the recently sequenced Fragaria × ananassa genome. Among these C2H2-ZFPs, 46 members containing two zinc finger domains in cultivated strawberry were further identified as the C1-2i subclass. These genes were unevenly distributed on 21 chromosomes and classified into five groups according to the phylogenetic relationship, with similar physicochemical properties and motif compositions in the same group. Analyses of conserved domains and gene structures indicated the evolutionary conservation of the C1-2i subclass. A Ka/Ks analysis indicated that the C1-2i members were subjected to purifying selection during evolution. Furthermore, FaZAT10, a typical C2H2-ZFP, was isolated. FaZAT10 was expressed the highest in roots, and it was induced by drought, salt, low-temperature, ABA, and MeJA treatments. It was localized in the nucleus and showed no transactivation activity in yeast cells. Overall, these results provide useful information for enriching the analysis of the ZFPs gene family in strawberry, and they provide support for revealing the mechanism of FaZAT10 in the regulatory network of abiotic stress.

Anthocyanins widely accumulate in the vegetative and reproductive tissues of strawberries and play an important role in stress resistance and fruit quality. Compared with other fruits, little is known about the molecular mechanisms regulating anthocyanin accumulation in strawberry vegetative tissues. In this study, we revealed an R2R3–MYB transcription factor, FaMYB10-like (FaMYB10L), which positively regulated anthocyanin accumulation and was induced by light in the petiole and runner of cultivated strawberry. FaMYB10L is a homologue of FveMYB10-like and a nuclear localization protein. Transient overexpression of FaMYB10L in a white fruit strawberry variety (myb10 mutant) rescued fruit pigmentation, and further qR–PCR analysis revealed that FaMYB10L upregulated the expression levels of anthocyanin biosynthesis-related genes and transport gene. A dual luciferase assay showed that FaMYB10L could activate the anthocyanin transport gene FaRAP. Anthocyanin accumulation was observed in FaMYB10L-overexpressing strawberry calli, and light treatment enhanced anthocyanin accumulation. Furthermore, transcriptomic profiling indicated that the DEGs involved in the flavonoid biosynthesis pathway and induced by light were enriched in FaMYB10L-overexpressing strawberry calli. In addition, yeast two-hybrid assays and luciferase complementation assays indicated that FaMYB10L could interact with bHLH3. These findings enriched the light-involved regulatory network of anthocyanin metabolism in cultivated strawberries.

Cryptochrome 1 (CRY1), a main blue light receptor protein, plays a significant role in several biological processes. However, the expression patterns and function of CRY1 in strawberry have not been identified. Here, the expression profile of CRY1 in different tissues and developmental stages of strawberry fruit, and expression patterns response to abiotic stresses (low temperature, salt and drought) were analyzed. Its subcellular localization, interaction proteins and heterologous overexpression in tobacco were also investigated. The results showed that CRY1 was mainly expressed in leaves and fruits with an expression peak at the initial red stage in strawberry fruit. Abiotic stresses could significantly induce the expression of CRY1. The CRY1 protein was located in both nucleus and cytoplasm. Five proteins (CSN5a-like, JAZ5, eIF3G. NF-YC9, and NDUFB9) interacting with CRY1 were discovered. Genes related flowering times, such as HY5 and CO, in three overexpressed FaCRY1 tobacco lines, were significantly upregulated. Taken together, our results suggested CRY1 have a broad role in biological processes in strawberry.