# Overexpression of _RrGGP2_ and _RrDHAR_ Increases Ascorbic Acid Content in Tomato
## 4 Discussion  
Overexpression _RrGGP2_ and _RrDHAR_ in Tomato Indicates That RrGGP2 Is the Key Control Point of AsA Biosynthesis and Metabolism  
Over the years, genes relating to AsA biosynthesis and metabolism in _R. roxburghii_ have been identified and cloned, and their functions have been verified by heterologous overexpression. For instance, the overexpression of _RrGDH_ and _RrGGP2_ in tobacco increased the leaf AsA content by an average of 1.1-fold and 12-fold [16; 39], while increasing the leaf AsA content by 3.02-fold and 2.11-fold was achieved by overexpressing _RrDHAR_ and _RrGME_ in _Arabidopsis_[37]. Indeed, _RrGGP2_ and _RrDHAR_ are particularly good candidates for increasing AsA biosynthesis in _R. roxburghii_[49]; however, further validation of these two genes was required in a model plant that has edible fruit, such as tomato, to assess their effectiveness in a plant with commercial applications. Our results reveal that overexpressing _RrGGP2_ increases AsA content in tomato fruits to a greater extent than _RrDHAR_, which suggests that _RrGGP2_ may have greater potential to enhance fruit AsA content in other fruit crops via molecular breeding. GGP catalyzes the conversion of GDP-L-galactose into L-galactose-1-phosphate, making it the first specific enzyme in the L-galactose pathway. Numerous previous studies on a variety of crops have demonstrated that overexpressing GGP can significantly augment AsA content; these crops include _Arabidopsis_[50; 51; 52; 15], tobacco [53; 54; 39; 55; 23], tomato [56; 57; 31], rice [58; 59; 2], kiwifruit [60], strawberry, and potato [31]. Although other genes in this pathway may also amplify AsA content, such as PMM in Acerola and GME in alfalfa [50; 61], not all genes have the same effect on recipient plants. For instance, GMP and GME in peach [62] were found to not increase AsA content. After summarizing experimental data from previous studies and conducting a comparative analysis, we have determined that overexpressing native GGP has a greater impact on fruit AsA content compared to overexpressing other genes in the AsA pathway. For instance, overexpressing _SIGGP_ in tomatoes resulted in a 3-fold increase in fruit AsA content [36], which was higher than the increases observed with _SIGMP_ (1.22 to 1.60-fold) [63], _SIGME_ (1.22 to 1.42-fold) [35], _SIDHAR_ (1.4 to 1.5-fold), and _SIMDHAR_[64]. Furthermore, overexpression _Arabidopsis_ AsA biosynthesis-related GMP, GME, GGP, GPP, GDH, and GLDH in _Arabidopsis_ leaves resulted in 1.3, 1.4, 2.9, 1.5, 1.2, and 1.8-fold increases in the leaves' AsA content, respectively [51]. Although not all the differences were significant, it offers initial evidence that indicates that the single transformation of GGP is more effective than other \begin{table}
\begin{tabular}{c c c c c c c c c c c c c c c} \hline \hline  & & \multicolumn{6}{c}{**Correlation Coefficient**} \\ \hline  & **GPI** & **PMI** & **PMM** & **GMP** & **GME** & **GGP** & **GPP** & **GDH** & **CLDH** & **AO** & **APX** & **SIDHAR** & **MDHAR** \\ \hline _RrGGP2_ & AsA & 0.844 & \(-0.869\) & \(-0.435\) & 0.684 & \(-0.882^{*}\) & 0.885* & \(-0.9429*\) & \(-0.521\) & 0.002 & \(-0.950*\) & 0.463 & 0.626* & \(-0.952*\) \\ _RrGGP2_ & DHA & 0.866 & \(-0.891\) & \(-0.030\) & 0.482 & \(-0.807\) & 0.485* & \(-0.239\) & 0.007 & 0.856 & \(-0.423\) & 0.986* & \(-0.962*\) & \(-0.623\) \\ \hline _RrDHAR_ & AsA & 0.457 & \(-0.658\) & \(-0.501\) & 0.468 & 0.663 & \(0.956*\) & \(-0.214\) & \(-0.275\) & 0.629 & \(-0.904*\) & 0.872 & 0.718 & \(-0.265\) \\ \hline _RrDHAR_ & DHA & 0.279 & \(-0.180\) & \(-0.106\) & 0.326 & 0.203 & 0.816 & 0.147 & \(-0.366\) & 0.274 & \(-0.697\) & 0.761 & 0.765 & \(-0.066\) \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Correlation analysis between gene expression and AsA content in transgenic tomato leaves. genes in increasing AsA content, while other genes have additive effects compared to GGP alone.