## 4 Discussion  
The AKR superfamily is a large enzyme group of oxidoreductases that mainly use NADP(H) as a cofactor; this group is widely distributed in animals, plants, yeast, and bacteria and regulates cell oxidation reduction under multiple stress conditions. Although AKRs play numerous roles in the metabolism of steroids, sugars, and other carbonyls, many AKR superfamily members remain uncharacterized [39]. In octopoloid strawberry, a total of 80 _FaAKR_ genes were identified from the genome and divided into 20 subgroups [33]. Among the members, _FaGalUR_ (_FaAKR24_) has been characterized and proven to play an important role in AsA biosynthesis [6]. Overexpression of _FaGalUR_ in _Arabidopsis_, potato, and tomato leads to a 2-3-fold increase in AsA content in the transgenic plant leaves and fruits compared with the wild-type [6; 7; 8]. However, whether other AKR genes in strawberry also contribute to AsA accumulation or fruit ripening is not known. Recently, we performed a genome-wide analysis of the AKR superfamily in cultivated strawberry and found that _FaAKR23_ was the most extremely upregulated _FaAKR_ gene during fruit development and ripening [33] (Figure 2B). Moreover, _FaAKR23_ and _FaGalUR_ (_FaAKR24_) were in a close phylogenetic relationship, and _AtA6 PR1_ and _AtA6 PR2_ were also located in the same cluster (Figure 1A). Previously, _AtA6 PR1_ and _AtA6 PR2_ were proposed to be related to the aldose 6-phosphate reductase pathway, which is involved in sorbitol synthesis. Sorbitol is the main form of photosynthetically fixed carbon, translocated via the phloem from source to sink organs in plants [40]; consequently, we speculated that _FaAKR23_ might be involved in strawberry fruit ripening and quality formation. To confirm this deduction, we manipulated _FaAKR23_ by transient expression in strawberry fruits and conducted metabolic and molecular analyses. The results demonstrated that _FaAKR23_ is indeed involved in AsA and anthocyanin accumulation through the regulation of the transcription level of downstream structural genes as well as some regulator factors of these two metabolic pathways. AsA content obviously decreased in the _FaAKR23_-RNAAi fruits (Figure 3C). The RNA-seq and qPCR analyses showed that the expression of multiple AsA biosynthetic genes were significantly suppressed, especially _FaGalUR_, _FaGGP_, and _FaGME_ (Figures 4 and 6A). Interestingly, we found that the FaAKR23 protein was located in both cytoplasm and nuclear, which suggests that _FaAKR23_ might work as a transcription factor (Figure 2D). To date, a number of TFs have been reported to be involved in AsA biosynthesis. These include the AP2 ethylene response factors _ERF98_[42]; _AB14_[43], the basic helix-loop helix _bHLH59_[44], the HD-Zip family TF _SIHZ24_[45], and SINFYA10 [46] bind to _VTC1_, _GGP_, and _GME_ to promote or reduce AsA synthesis. More recently, in kiwrift, ethylene response factor _AcERF91_ has been found to increase the expression of _AcGGP3_[47], and transcription factors _AccMYSB1_ and _AccGBF3_ influence AsA biosynthesis by activating the transcription of _AccGGP3_[48]. Consistent with this knowledge, it is necessary that the specific regulatory mechanism of _FaAKR23_ in AsA accumulation be further clarified. In addition, anthocyanin was also dramatically decreased in _FaAKR23_-RNAi fruits (Figure 3D), along with lower expression of anthocyanin biosynthetic genes and regulatory factors (Figures 5 and 6B). In _FaAKR23_-RNAi transcriptome data and qRT-PCR analysis, the transcript levels of anthocyanin biosynthetic genes _FaCHS_, _FaCHI_, _FaSHA_, _FaDFR_, and _FaANS_ were significantly repressed. Moreover, the expression of _FaMYB10_ was decreased in _FaAKR23_-RNAi fruits; it is a powerful and conserved regulator of anthocyanin biosynthesis in diploid and octopoloid strawberry fruits [12; 13; 14; 21] and also regulates the expression of _CHS1_, _CHI_, _DFR_, _F3H_, and _DFR_ individuals by forming an MYB-bHLH-WD complex, thereby regulating anthocyanin accumulation [13].