In this study, a rapid, efficient, and stable Agrobacterium-mediated genetic transformation system was successfully developed using stem slices of fig cultivar ‘117D’ as explants. In the Murashige and Skoog (MS) basal medium supplemented with 2 mg·L^-1 thidiazuron (TDZ) and 0.05 mg·L^-1 1-naphthaleneacetic acid (NAA), the highest shoot-induction rate was 53.33%, while the medium with a hormone ratio of 3 mg·L^-1 TDZ and 0.05 mg·L^-1 NAA achieved a maximum callus-induction rate of 78.89%. The optimal infection parameters for fig callus and stem slices were obtained by infecting thin cell layers (TCLs) with Agrobacterium tumefaciens K599 (OD₆₀₀ = 0.6) under vacuum for 10 min, and shaking at 120 r · min⁻¹, 28 ℃ for 30 min. Finally, transgenic callus and plants were identified through green fluorescent protein (GFP) screening, β-glucuronidase (GUS) staining, PCR analysis, and Western blot analysis, successfully obtaining positive FcMYB114-overexpressing (FcMYB114-OE) callus and three transgenic plants. Utilizing these FcMYB114 transgenic callus and plants allows us to gain deeper insights into their roles in the growth, development, and metabolism of fig. In the future, we aim to identify new regulatory factors and leverage this efficient transgenic technology to investigate the regulatory mechanisms of key molecules in fig. The advancement of fig regeneration and transgenic systems provides a valuable tool for validating genetic functions in fruit trees and enhancing the agronomic traits of fig.

Dry fig is a traditional healthy snack and has important economic value in a number of Mediterranean and Middle Eastern countries. Cultivars with no anthocyanin accumulation in the fruit peel are preferred for dry fig production. R2R3-MYB transcription factors have promotive or repressive regulatory roles in plant anthocyanin biosynthesis. In this study, 113 R2R3-MYB genes were identified in Ficus carica, 3 of which were assigned to the S4 subfamily of flavonoid-biosynthesis repressors. FcMYB57 was further recruited as a candidate anthocyanin-biosynthesis repressor based on its sequence features and expression, which was significantly negatively correlated with that of anthocyanin-biosynthesis structural genes. Transient overexpression of FcMYB57 in strawberry totally inhibited fruit pigmentation and significantly increased fruit firmness. The metabolomic analysis confirmed a significant reduction in the contents of cyanidin-3-O-glucoside and pelargonidin-3-O-glucoside, as well as other flavonoids, and transmission electron microscopy revealed an increment in cell-wall thickness. Transcriptome analysis showed downregulation of anthocyanin-biosynthesis structural genes and upregulation of genes related to xylan synthesis. Yeast one-hybrid and dual luciferase assays demonstrated a negative regulatory effect of FcMYB57 on the promoter of FcUFGT3 (UDP glucose-flavonoid 3-O-glcosyl-transferase). Yeast two-hybrid assay showed that FcMYB57 does not interact with FcbHLH42, 3, 14, MYC2, or FcTTG1, all of which have a previously identified or predicted role in flavonoid biosynthesis, however, interaction was detected with FcTPL (Topless), suggesting that FcMYB57 serves as an active repressor of anthocyanin biosynthesis. This is the first identification of an anthocyanin-biosynthesis repressor in fig, with a possible role in fig fruit quality.

Fig fruit firmness decreases rapidly during ripening and after harvest, resulting in poor storability and transportation loss, which severely restricts development of the fresh fig industry. APETALA2/ethylene-responsive factor (AP2/ERF) transcription factors are downstream components of the ethylene-signaling pathway that play crucial roles in quality formation during fruit ripening. In this study, Ficus carica (Fc) ERF12 was clustered in repressor subfamily Ⅷ of ERFs through phylogenetic analysis, and further recruited by its two EAR motifs and expression pattern during fig ripening. DNA affinity purification sequencing analysis indicated that FcERF12 binds to the promoter or gene body regions of multiple ripening-related genes, including cell wall-modification genes FcPG, FcXTH and FcPME, and ethylene-biosynthesis genes FcACS and FcACO. Yeast two-hybrid assay demonstrated that FcERF12 interacts with TOPLESS (TPL) co-repressors FcTPL1, FcTPL4 and FcTPL5, and histone deacetylases FcHDA6 and FcHDA19; interaction with FcTPL4 and FcTPL5 relied on the C-terminal EAR motif. Overexpressing FcERF12 in tomato did not change fruit size or yield, but resulted in an 18.37% increment in fruit firmness and a 49.62% reduction in ethylene-release rate at fruit ripening, accompanied by a significant decrease in seed number per fruit. Transcriptomic analysis revealed downregulation of tomato cell wall-modification genes SlPL, SlEXP and SlPG, and ethylene-synthesis genes SlACO and SlACS. Metabolomic profiling identified 82 differentially accumulated flavonoid metabolites, 61 of them showing significantly decreased contents. Taken together, our results exhibit the negative regulatory role of FcERF12 in fig ethylene-signal transduction, providing new information on precise control of fruit firmness and other quality traits at ripening.