FaMYB5 Interacts with FaBBX24 to Regulate Anthocyanin and Proanthocyanidin Biosynthesis in Strawberry (Fragaria × ananassa).

MYB and BBX transcription factors play important roles in flavonoid biosynthesis. Here, we obtained transgenic woodland strawberry with stable overexpression of FaMYB5, demonstrating that FaMYB5 can increase anthocyanin and proanthocyanidin content in roots, stems and leaves of woodland strawberry. In addition, bimolecular fluorescence complementation assays and yeast two-hybridization demonstrated that the N-terminal (1-99aa) of FaBBX24 interacts with FaMYB5. Transient co-expression of FaBBX24 and FaMYB5 in cultivated strawberry 'Xiaobai' showed that co-expression strongly promoted the expression of F3'H, 4CL-2, TT12, AHA10 and ANR and then increased the content of anthocyanin and proanthocyanidin in strawberry fruits. We also determined that FaBBX24 is also a positive regulator of anthocyanin and proanthocyanidin biosynthesis in strawberry. The results reveal a novel mechanism by which the FaMYB5-FaBBX24 module collaboratively regulates anthocyanin and proanthocyanidin in strawberry fruit.

Regulation of flavonoids in strawberry fruits by FaMYB5/FaMYB10 dominated MYB-bHLH-WD40 ternary complexes.

Anthocyanins endowing strawberry fruit red color are regulated by the MYB-bHLH-WD40 complex. By analyzing the MYBs involved in the flavonoid biosynthesis in strawberry, we found that R2R3-FaMYB5 promoted the content of anthocyanin and proanthocyanidins in strawberry fruits. Yeast two-hybrid and BiFC assays confirmed that MBW complexes connected with flavonoid metabolism were FaMYB5/FaMYB10-FaEGL3 (bHLH)-FaLWD1/FaLWD1-like (WD40). Transient overexpression and qRT-PCR analysis revealed that disparate MBW models hold different patterns in the regulation of flavonoid biosynthesis in strawberry fruits. Compared with FaMYB10, FaMYB5 and its dominant complexes showed a more specific regulatory range on strawberry flavonoid biosynthetic pathway, while FaMYB10 was more extensive. In addition, the complexes involved in FaMYB5 facilitated PAs accumulation primarily through the LAR tributary while FaMYB10 mainly by the ANR branch. FaMYB9 and FaMYB11 tremendously elicited the accumulation of proanthocyanidins by up-regulating the expression levels of both LAR and ANR, and also affected anthocyanin metabolism by changing the ratio of Cy3G and Pg3G which were constituted as two major anthocyanin monomers in strawberries. Our study also illustrated that FaMYB5-FaEGL3-FaLWD1-like directly targeted the promoters of F3'H, LAR, and AHA10 thus committing to flavonoid accumulation. These results allow the specific members involved in the MBW complex to be deciphered and provide new insights into the regulatory mechanisms of anthocyanins and proanthocyanidins regulated by the MBW complex.

A novel R2R3-MYB transcription factor FaMYB5 positively regulates anthocyanin and proanthocyanidin biosynthesis in cultivated strawberries (Fragaria × ananassa).

Flavonoids have a major contribution to the fruit quality in cultivated strawberries and are regulated by MYB, bHLH and WD40 transcriptional factors. We reported here the identification of the FaMYB5, an R2R3-MYB transcription factor, which positively regulated the accumulation of anthocyanins and proanthocyanidins through the trans-activation of the F3'H and LAR. The strawberry FaEGL3 and FaLWD1/FaLWD1-like interact with the R2R3-FaMYB5 to form an MYB-bHLH-WD40 complex (MBW), enhancing the regulatory efficiency. The R2R3-FaMYB5 was constitutively expressed in various tissues and in fruits of different developmental stages, which was strikingly contrasting to the fruit-specific expression patterns of FaMYB10. Meanwhile, R2R3-FaMYB5 failed to promote a stable accumulation of anthocyanin glycosides in the mature fruits of the myb10 mutant, mainly due to the suppressed expression of TT19. The R2R3-FaMYB5 was regulated by an antisense long noncoding RNA lncRNA-myb5. Additionally, the R2R3-FaMYB5 protein could interact with FaBT2 and was degraded through the ubiquitin/26 S proteasome pathway. Transcriptome and metabolome data showed that R2R3-FaMYB5 enhanced the gene expression and the metabolite accumulation involved in the flavonoid, phenylpropanoid and lignin biosynthesis pathways. Collectively, we conclude that the FaMYB5 is an R2R3-MYB activator involved in the composition of MBW, which positively regulates the biosynthesis of anthocyanin and proanthocyanidin. These findings provided new insights into the molecular mechanisms that regulate flavonoids in strawberry fruits.

Genome-Wide Identification of Strawberry C2H2-ZFP C1-2i Subclass and the Potential Function of FaZAT10 in Abiotic Stress.

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.

Importance of FaWRKY71 in Strawberry (Fragaria × ananassa) Fruit Ripening.

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.

Alterations of Phenylpropanoid Biosynthesis Lead to the Natural Formation of Pinkish-Skinned and White-Fleshed Strawberry (Fragaria × ananassa).

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.

B-Box Transcription Factor FaBBX22 Promotes Light-Induced Anthocyanin Accumulation in Strawberry (Fragaria × ananassa).

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.

Transcriptomic insights into citrus segment membrane's cell wall components relating to fruit sensory texture.

BACKGROUND: During fresh fruit consumption, sensory texture is one factor that affects the organoleptic qualities. Chemical components of plant cell walls, including pectin, cellulose, hemicellulose and lignin, play central roles in determining the textural qualities. To explore the genes and regulatory pathways involved in fresh citrus' perceived sensory texture, we performed mRNA-seq analyses of the segment membranes of two citrus cultivars, Shiranui and Kiyomi, with different organoleptic textures. RESULTS: Segment membranes were sampled at two developmental stages of citrus fruit, the beginning and end of the expansion period. More than 3000 differentially expressed genes were identified. The gene ontology analysis revealed that more categories were significantly enriched in 'Shiranui' than in 'Kiyomi' at both developmental stages. In total, 108 significantly enriched pathways were obtained, with most belonging to metabolism. A detailed transcriptomic analysis revealed potential critical genes involved in the metabolism of cell wall structures, for example, GAUT4 in pectin synthesis, CESA1, 3 and 6, and SUS4 in cellulose synthesis, CSLC5, XXT1 and XXT2 in hemicellulose synthesis, and CSE in lignin synthesis. Low levels, or no expression, of genes involved in cellulose and hemicellulose, such as CESA4, CESA7, CESA8, IRX9 and IRX14, confirmed that secondary cell walls were negligible or absent in citrus segment membranes. A chemical component analysis of the segment membranes from mature fruit revealed that the pectin, cellulose and lignin contents, and the segment membrane's weight (% of segment) were greater in 'Kiyomi'. CONCLUSION: Organoleptic quality of citrus is easily overlooked. It is mainly determined by sensory texture perceived in citrus segment membrane properties. We performed mRNA-seq analyses of citrus segment membranes to explore the genes and regulatory pathways involved in fresh citrus' perceived sensory texture. Transcriptomic data showed high repeatability between two independent biological replicates. The expression levels of genes involved in cell wall structure metabolism, including pectin, cellulose, hemicellulose and lignin, were investigated. Meanwhile, chemical component contents of the segment membranes from mature fruit were analyzed. This study provided detailed transcriptional regulatory profiles of different organoleptic citrus qualities and integrated insights into the mechanisms affecting citrus' sensory texture.