## 3 Results
### FaAKR23 Regulates Anthocyanin and AsA Accumulation in Strawberry Fruits  
To investigate the potential role of _FaAKR23_ in strawberry fruit ripening and quality formation, we transiently expressed _FaAKR23_ in octoploid strawberry fruits via agroinfiltration. The visual phenotype suggested that _FaAKR23_ was involved in anthocyanin accumulation (Figure 3A). Using qRT-PCR analysis, we confirmed the lower relative _FaAKR23_ mRNA level in _FaAKR23_-RNAi transiently transformed strawberry fruits (Figure 3B). Furthermore, the AKR family is widely reported to regulate cell redox levels in both animals and plants [28] because AsA and anthocyanin are important antioxidants in strawberry fruits. Hence, we further determined the AsA and anthocyanin contents in EV-RNAi and _FaAKR23_-RNAi transformed fruits, and it was found that the contents of both AsA and anthocyanin were obviously reduced in the _FaAKR23_-RNAi fruits compared to control; the AsA level decreased by approximately 30%, and the anthocyanin content was reduced by nearly 50% (Figure 3C,D).  
Figure 2: The phenotypes of different tissues in strawberry, expression patterns, protein structure, and subcellular localization of FaAKR23. (**A**) Phenotypes of strawberry leaf, flower, and fruits in different development stages. (**B**) The transcript levels of _FaAKR23_ in different strawberry tissues. L, leaf, F, flower, SG, small green fruit stage, BG, big green fruit stage, W, white fruit stage, T, turning stage, R, fully red stage. (**C**) Protein structure of FaAKR23. (**D**) Subcellular localization of FaAKR23. Scale bars: 50 μm.  
Expression Patterns of AsA and Anthocyanin Metabolism-Related Genes in EV-RNAi and FaAKR23-RNAi Fruits  
To decipher the molecular mechanisms underlying _FaAKR23_-mediated AsA and anthocyanin accumulation in strawberry fruits, we performed transcriptome deep sequencing (RNA-seq) using EV-RNAi and _FaAKR23_-RNAi fruits. FDR < 0.01 and fold change \(\geq\) 2 were set as the threshold for significantly differential expression (Figures 4 and 5). We also validated the expression of several genes of interest by qRT-PCR (Figure 6). These results showed that the AsA biosynthetic genes, such as _FaGal_UR, _FaGME_, and _FaGGP_ (Figure 4), and anthocyanin-synthesis-related genes, such as _FaCHS, FaCHI, Fa3H, FaDR_, and _FaANS_, as well as _FaMYB10_ and _FaRAP_, were significantly downregulated (Figure 5).  
Figure 3: Transient silenced expression of _FaAKR23_ in strawberry fruits. (**A**) Phenotypes of EV-RNAi and _FaAKR23_-RNAi fruits; EV, empty vector; scale Bars: 1 cm. (**B**) The expression levels of _FaAKR23_ in EV-RNAi and _FaAKR23_-RNAi fruits. Expression values are means \(\pm\) SEMs of three biological replicates and are normalized using _FaCHP1_ as an internal control. (**C**) AsA content in EV-RNAi and _FaAKR23_-RNAi fruits. (**D**) Anthocyanin content in EV-RNAi and _FaAKR23_-RNAi fruits. Statistically significant differences from control were determined by Student’s \(t\)-test: **_p_ < 0.01. Values are means \(\pm\) SEMs of three biological replicates.  
Figure 4: Expression profiles of AsA metabolism-related genes differently expressed between EV-RNAi and _FaAKR_23-RNAi fruits.  
Figure 5: Expression profiles of anthocyanin biosynthesis genes differently expressed between EV-RNAi and _FaAKR_23-RNAi fruits.