Light conditions can cause quantitative and qualitative changes in anthocyanin. However, little is known about the underlying mechanism of light quality-regulated anthocyanin accumulation in fruits. In this study, light-emitting diodes (LEDs) were applied to explore the effect of red and blue light on strawberry coloration. The results showed contents of total anthocyanins (TA), pelargonidin 3-glucoside (Pg3G) and pelargonidin 3-malonylglucoside (Pg3MG) significantly increased after blue and red light treatment. Pg3G was the major anthocyanin component in strawberry fruits, accounting for more than 80% of TA, whereas Pg3MG accounted for a smaller proportion. Comparative transcriptome analysis was conducted using libraries from the treated strawberries. A total of 1402, 5034, and 3764 differentially-expressed genes (DEGs) were identified in three pairwise comparisons (red light versus white light, RL-VS-WL; blue light versus white light, BL-VS-WL; blue light versus red light, BL-VS-RL), respectively. Photoreceptors and light transduction components remained dynamic to up-regulate the expression of regulatory factors and structural genes related to anthocyanin biosynthesis under red and white light, whereas most genes had low expression levels that were not consistent with the highest total anthocyanin content under blue light. Therefore, the results indicated that light was an essential environmental factor for anthocyanin biosynthesis before the anthocyanin concentration reached saturation in strawberry fruits, and blue light could quickly stimulate the accumulation of anthocyanin in the fruit. In addition, red light might contribute to the synthesis of proanthocyanidins by inducing LAR and ANR.

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.

Flowering connects vegetative and generative developmental phases and plays a significant role in strawberry production. The mechanisms that regulate strawberry flowering time are unclear. B-box transcription factors (BBXs) play important roles in the flowering time regulation of plants. Nevertheless, BBXs in octoploid cultivated strawberry (Fragaria ananassa) and their functions in flowering time regulation have not been identified. Here, we identified 51 FaBBXs from cultivated strawberry and 16 FvBBXs from diploid wild strawberry (Fragaria vesca), which were classified into five groups according to phylogenetic analysis. Further evolutionary analysis showed that whole-genome duplication or segmental duplication is a crucial factor that leads to the expansion of the BBX gene family in two strawberry species. Moreover, some loss and acquisition events of FaBBX genes were identified in the genome of cultivated strawberry that could have affected traits of agronomic interest, such as fruit quality. The promoters of FaBBX genes showed an enrichment in light-responsive, cis-regulatory elements, with 16 of these genes showing changes in their transcriptional activity in response to blue light treatment. On the other hand, FaBBX28c1, whose transcriptional activity is reduced in response to blue light, showed a delay in flowering time in Arabidopsis transgenic lines, suggesting its role in the regulation of flowering time in cultivated strawberry. Our results provide new evolutionary insight into the BBX gene family in cultivated strawberry and clues regarding their function in flowering time regulation in plants.

Selecting an appropriate reference gene is of crucial importance for improving the accuracy of qRT-PCR analyses. In this study, strawberry (Fragaria ananassa) seedlings were subjected to different environmental conditions including heat, cold, drought, salt, white-light, blue-light, and red-light treatments. The expression levels of seven candidate reference genes, including Fa18S, FaGAPDH, FaPIRUV, FaDBP, FaHISTH4, FaACTIN1, and FaACTIN2, in the strawberry leaves were measured by qRT-PCR. Then, four programs (geNorm, NormFinder, BestKeeper, and RefFinder) were employed as tools to evaluate the expression stability of the candidate reference genes. The results showed that the expression stability of the reference genes varied under different conditions. For the cold stress and white-light treatments, FaACTIN2 was evaluated to be the most stable reference gene. FaGAPDH should be used as the reference gene under salt-stress condition and red-light treatment. For the data normalization under drought-stress treatment, FaDBP is the recommended reference gene with the highest expression stability. FaHISTH4 was observed to be the best reference gene for data normalization under heat stress and blue-light treatment. This work provides information on selecting reference genes for accurate gene expression analyses of target genes in strawberry leaves under various abiotic stress and light-quality conditions.

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.