## 2 Results
### Pyramiding Biosynthetic Genes Increases AsA Transport Capacity  
It has been reported that AsA can be synthesized in both leaves and fruits and transported from leaves to fruits [45]. In order to investigate how pyramiding affects AsA transport capacity, we designed two experiments. First, AsA content was measured in leaf and fruit petioles of wild-type and pyramiding lines. The content of AsA showed no significant difference in leaf petioles between wild-type and pyramiding lines, but showed a significant increase in fruit petioles of _GMP_\(\times\)_GME_ and _GMP_\(\times\)_GME_\(\times\)_GGP_\(\times\)_GPP_ lines, respectively (Figure 3A), consistent with the AsA content in the red fruit (Figure 1B). The AsA content in the exudates of fruit and leaf petioles were also measured in AC and pyramiding lines. The results showed that the AsA content significantly increased in the secretion of both fruit and leaf petioles in pyramiding lines compared with AC (Figure 3B).  
Figure 2: Dynamic change of ascorbate accumulation in response to light in leaves of AC and pyramiding lines. Plants were grown in the greenhouse, and fully expanded leaves were harvested every 4 h, total ascorbate content was measured for 48 h (8 dark/16 light). Three replicate experiments were performed. Error bars represent standard error, means \(\pm\) SE. FW—fresh weight.  
Next, the petioles of mature green fruits were incubated with 5 mM AsA (water as a control) for 24 h at 16 light/8 dark, and the AsA content of fruit was measured. AsA concentration showed no change after feeding with AsA compared with water in the fruit of AC, but the content of AsA showed a significant increase in _GMP_\(\times\)_GME_, _GGP_\(\times\)_GPP_ and _GMP_\(\times\)_GME_\(\times\)_GGP_\(\times\)_GPP_ pyramiding lines, suggesting that pyramiding lines possessed stronger AsA transport capacity than AC (Figure 4B). To further detect the AsA content and its distribution in the fruits for which the petioles were incubated with 5 mM AsA, we investigated the localization of reduced AsA by AgNO\({}_{3}\) histochemical localization where the reduced AsA can react with AgNO\({}_{3}\) to form metallic Ag, and the content and distribution of \"black\" Ag ions represents the AsA. In AC fruit, Ag accumulation mainly locates in the inner wall of the pericarp, placental tissue, and seeds; but the distribution of Ag is broader, and its concentration is higher in the fruits of _GMP_\(\times\)_GME_, _GGP_\(\times\)_GPP_, and _GMP_\(\times\)_GME_\(\times\)_GGP_\(\times\)_GPP_ pyramiding lines (Figure 4A), consistent with the results shown in Figure 4B. All of these results indicated that gene pyramiding increases AsA transport capacity in both leaves and fruit of tomato.  
Figure 4: _Cont._  
Figure 3: Ascorbate concentration in leaf and fruit petioles and their exudates of wild-type and pyramiding tomato lines. (**A**) AsA levels in fruit petioles and leaf petioles of wild type and pyramiding lines. (**B**) AsA content in exudates of fruit and leaf petioles in AC and pyramiding lines. Three replicate experiments were performed. Error bars represent standard error, means \(\pm\) SE. FW—fresh weight. The asterisks represented significant differences from wild-type (AC), as indicated by the \(t\)-test (**\(p<0.01\)).