## 2 Results
### Light-Dependent Fluctuation of AsA Content in Wild Type and Pyramiding Lines  
It has been reported that light intensity is one of the critical environmental factors affecting AsA accumulation [29], and the feeding experiment with its precursors in leaves and fruits indicated that glucose, as a product of photosynthesis, had a significant influence on the AsA content in leaves. To further study the effect of pyramiding on light-dependent ascorbate metabolism, we measured the AsA content in leaves under different light treatments for 48 h.  
\begin{table}
\begin{tabular}{l l l l l l} \hline \hline \multirow{2}{*}{**Lines**} & \multicolumn{2}{c}{Feeding-leaf- total ascorbate content (ug g\({}^{-1}\) FW)} & \multirow{2}{*}{**GLA**} & \multirow{2}{*}{**AsA**} \\  & **H\({}_{2}\)O** & **MI** & & & \\ \hline AC & 496.2 \(\pm\) 6.3 & 422.5 \(\pm\) 15.9 ** & 591.2 \(\pm\) 7.6 ** & 215.6 \(\pm\) 8.5 ** & 2139.9 \(\pm\) 59.9 ** \\ _GMP_\(\times\)_GME_ & 554.6 \(\pm\) 14.2 & 452.4 \(\pm\) 14.2 ** & 742.4 \(\pm\) 10.5 ** & 162.5 \(\pm\) 9.9 ** & 2733.7 \(\pm\) 291.6 ** \\ _GGP_\(\times\)_GPP_ & 579.8 \(\pm\) 15.9 & 427.8 \(\pm\) 3.2 ** & 623.8 \(\pm\) 6.5 ** & 158.0 \(\pm\) 9.8 ** & 2220.3 \(\pm\) 71.9 ** \\ _GMP_\(\times\)_GME_\(\times\)_GGP_\(\times\)_GPP_ & 626.1 \(\pm\) 10.2 & 508.1 \(\pm\) 17.8 ** & 790.9 \(\pm\) 6.4 ** & 283.1 \(\pm\) 13.3 ** & 2584.4 \(\pm\) 72.2 ** \\ \hline \multirow{2}{*}{**Lines**} & \multicolumn{2}{c}{Feeding-fruit- total ascorbate content (ug g\({}^{-1}\) FW)} & \multirow{2}{*}{**GLA**} & \multirow{2}{*}{**AsA**} \\  & **H\({}_{2}\)O** & **MI** & & & \\ \hline AC & 254.3 \(\pm\) 3.6 & 276.1 \(\pm\) 1.4 * & 287.9 \(\pm\) 3.0 ** & 259.9 \(\pm\) 5.2 & 296.3 \(\pm\) 1.2 ** \\ _GMP_\(\times\)_GME_ & 287.5 \(\pm\) 4.0 & 384.6 \(\pm\) 8.0 & 396.2 \(\pm\) 2.5 ** & 325.8 \(\pm\) 4.9 ** & 368.4 \(\pm\) 11.3 ** \\ _GGP_\(\times\)_GPP_ & 251.0 \(\pm\) 2.8 & 265.9 \(\pm\) 4.7 * & 350.1 \(\pm\) 6.9 ** & 261.9 \(\pm\) 6.1 & 351.5 \(\pm\) 34.8 ** \\ _GMP_\(\times\)_GME_\(\times\)_GGP_\(\times\)_GPP_ & 301.9 \(\pm\) 8.3 & 373.7 \(\pm\) 5.9 ** & 390.4 \(\pm\) 7.4 ** & 331.5 \(\pm\) 5.4 ** & 354.0 \(\pm\) 7.0 ** \\ \hline \end{tabular}  
* The leaves (upper panel) and fruits (lower panel) were fed with H\({}_{2}\)O, inositol (MI), glucose (GLc), galacturonic acid (GLA), and ascorbate (AsA) and incubated under light for 24 h. Three replicate experiments were performed. The data presented are means \(\pm\) SE. FW—fresh weight. The asterisks represented significant differences from wild type (AC), as indicated by the \(t\)-test (*_p_ < 0.05; **_p_ < 0.01).  
\end{table}
Table 1: Effect of feeding AsA biosynthesis precursors on the content of ascorbate in tomato.  
The total AsA content exhibited similar dynamic changes, as a model of \"down-up-down-up-down\", in both wild-type and pyramiding lines under different light treatments for 48 h. Therefore, when we compared the AsA content in AC and _GGP_\(\times\)_GPP_ line, the AsA content in _GMP_\(\times\)_GME_, and _GMP_\(\times\)_GME_\(\times\)_GGP_\(\times\)_GPP_ lines were higher at most of the time points and had larger fluctuations (Figure 2). These findings suggested that the accumulation of AsA in tomato showed a specific response to photoperiod, and light is indeed an important environmental factor affecting AsA levels. The AsA content in leaves increased at the first stage of light treatment but decreased during further illumination, suggesting that the accumulation of AsA maximizes within a certain light frame (8 h, 6:00 to 14:00), but will not further increase with the extension of light. Taken together, gene pyramiding enhanced the light-induced AsA accumulation in tomato leaves and the fluctuation amplitude.