## 2 Results  
Gene Pyramiding Altered Expression of AsA-Related Genes and AsA Concentration in Both Leaves and Fruits  
The _GMP_\(\times\)_GME_ and _GGP_\(\times\)_GPP_ pyramiding lines were generated by conventional crossing of _GMP_ with _GME_ and _GGP_ with _GPP_, respectively. Then, in the same way, four gene pyramiding lines (_GMP_\(\times\)_GME_\(\times\)_GGP_\(\times\)_GPP_) were generated. The expression of AsA-related genes and content of AsA were measured in the wild-type and three pyramiding lines (Figure 1). In leaves, the total AsA and reduced AsA levels in _GGP_ overexpression line and _GMP_\(\times\)_GME_, _GMP_\(\times\)_GME_\(\times\)_GGP_\(\times\)_GPP_ pyramiding lines showed a 2-fold increase compared with wild-type, and significant increase was also observed in _GMP_, _GME_, _GPP_, _GGP_\(\times\)_GPP_ lines (Figure 1A). The considerable amount of ascorbate in _GME_, _GGP_, _GPP_\(\times\)_GME_, _GGP_\(\times\)_GPP_, and _GMP_\(\times\)_GME_\(\times\)_GGP_\(\times\)_GPP_ indicated that there is a limit in the increase in AsA content in tomato leaves and that the biosynthesis of AsA may be subjected to feedback inhibition. The ascorbate content in fruits in _GME_, _GMP_ overexpression lines, and _GMP_\(\times\)_GME_, _GMP_\(\times\)_GME_\(\times\)_GGP_\(\times\)_GPP_ pyramiding lines increased significantly compared with the wild-type, while no significant ascorbate content changes were observed in _GGP_, _GPP_ and _GGP_\(\times\)_GPP_ lines (Figure 1B). In addition to the increased expression of the pyramided genes themselves in the three pyramiding lines, such as the increased expression of _GME2_ and _GMP_ in _GMP_\(\times\)_GME_ pyramiding line and increased expression of _GGP_ and _GPP1_ in _GGP_\(\times\)_GPP_ pyramiding lines, the transcript level of other AsA biosynthetic and recycling genes showed slight or significant decline, especially in leaves of the _GMP_\(\times\)_GME_\(\times\)_GGP_\(\times\)_GPP_ pyramiding line (Figure 1C). In fruits, however, except _PML_, _GLDH_, and _MIOX_, the expression of most AsA-related structural genes, such as the _GPI_, _GMP_, _GME1_, _GME2_, _GPP1_, and _DHARI_, showed a significant increase in three pyramiding lines (Figure 1D). Taken together, we speculated that in the d-Man/l-Gal pathway, all _GMP_, _GME_, _GGP_, and _GPP_ contribute to the biosynthesis of AsA in leaves, and _GGP_ and _GPP_ function in AsA accumulation in tomato leaf more efficiently than in fruits. The biosynthesis of AsA may be subjected to feedback inhibition, that is, overexpression of the target gene in the pyramiding lines increases the content of AsA, but downregulates the expression of other structural genes by feedback inhibition. Figure 1: Relative expression of AsA biosynthesis, recycling-related genes, and ascorbate concentration in leaf and red ripe fruit of transgenic and pyramiding tomato lines. (**A**) Reduced and total ascorbate content in young leaf. (**B**) Reduced and total ascorbate contents in red ripe fruit. (**C**) Relative expression of AsA biosynthesis and recycling-related genes in leaf. (**D**) Relative expression of AsA biosynthesis and recycling-related genes of red ripe fruit.