# Overexpression of _RrGGP2_ and _RrDHAR_ Increases Ascorbic Acid Content in Tomato
## 4 Discussion
### Similarities and Differences for the Mechanism of AsA Accumulation in Tomato Fruit and Leaf  
The accumulation of AsA depends on the interplay between biosynthesis, catabolism, and cycling [73]. In this study, we comprehensively analyzed these three pathways to investigate AsA accumulation in the fruits and leaves of transgenic tomatoes. Our findings revealed both similarities and differences. GMP, GME, and GGP have been identified as good candidates for promoting AsA accumulation in tomato [63]. The overexpression of _SIGMP_, _SIGME_, or _SIGGP_ has been found to increase AsA content in tomato [35; 36; 63]. In our experiment, at least two of the three key genes were simultaneously upregulated in the fruits and leaves of transgenic tomatoes, which is likely why the AsA content was higher in transgenic tomatoes than in the control. Furthermore, through comparison and summarization, we suggest that there may be an additive model centered on GGP. From the perspective of increasing AsA content, GMP-GME-GGP co-expression yields higher results than GGP-GMP/GGP-GME, which is higher than GMP-GME and also higher than GMP/GME/GGP [15; 56; 74; 75]. This may explain why the AsA content in fruits of tomatoes overexpressing _RrGGP2_ is higher than that of _RrDHAR_ and why the AsA content in leaves of tomatoes overexpressing _RrDHAR_ is higher than that of _RrGGP2_. In transgenic _RrGGP2_ tomato fruits, the expression of _SIGME_ and _SIGGP_ was upregulated simultaneously, while in transgenic _RrDHAR_ tomato fruits, the expression of _SIGMP_ and _SIGME_ was upregulated and _SIGGP_ expression was downregulated. The expression of _SIGMP_, _SIGME_, and _SIGGP_ was simultaneously upregulated in transgenic _RrDHAR_ tomato leaves, but only _SIGMP_ and _SIGGP_ were upregulated in transgenic _RrGGP2_ tomato leaves, and _SIGME_ was downregulated. Additionally, the expression of _SIGGP_ in transgenic _RrDHAR_ tomato leaves was much higher than that in _RrGGP2_, and the correlation results further confirmed that the upregulation of key genes in the biosynthetic pathway, especially _SIGGP_, may be the main factor contributing to the increase in AsA. Recent studies have shown that GMP, GME, and GGP proteins are located in the cytoplasm [65] and that these enzyme complexes likely interact, demonstrating channelization [76]. This finding confirms the complex regulation of AsA pool size in tomato. In terms of catabolism and recycling pathways, _SIAPX_ was found to be significantly upregulated in the leaves of both transgenic tomatoes. It was observed that the high expression of _SIAPX_ could be detrimental to AsA accumulation, while almost no expression of _SIAO_ was very beneficial to AsA accumulation [77; 78]. Although the expression levels of the two genes were opposite, the final phenotypic results of tomatoes showed that AsA was still elevated. The correlation analysis results confirmed the positive effect of _SIAO_ on AsA accumulation. However, it was not immediately clear why the significant downregulation of _SIAPX_ expression did not result in AsA content remaining constant or decreasing. This may be related to the multiple functions of APX in plants; in addition to its oxidative function in AsA metabolism, APX also acts as an important antioxidant enzyme to scavenge ROS under various stress conditions. The overexpression of APX can improve tolerance to various stresses in tobacco [79], tomato [80], and _Arabidopsis_[81; 82]. Therefore, the upregulation of _SIAPX_ expression may not only affect the catabolism of AsA but also perform tasks related to stress resistance. Interestingly, the expression of _SIAO_ or _SIAPX_ in fruits was not significantly inhibited; this may be because fruits contain more abundant secondary metabolites and more hierarchical biological structures than leaves, thus having a potentially higher stress resistance effect.