## 3 Discussion  
In the past decades, many studies on genetic engineering of plant disease resistance, drought resistance, and salt tolerance have been reported [42; 44; 46; 47]. Also, using genetic engineering methods to develop food crops with increased contents of specific micronutrients and phytonutrients such as high-zinc rice, provitamin A-enriched maize, and anthocyanin-enriched \"Purple Tomatoes\", have been well developed [36; 41]. However, the genetic engineering of AsA pathway has rarely been reported. In this study, effective biofortification for improving AsA content in tomato has been conducted by pyramiding \(GMP\times\)\(GME\), \(GGP\times\)\(GPP\), and tetravalent pyramiding \(GMP\times\)\(GME\times\)\(GGP\times\)\(GPP\). \(GMP\)[22], \(GME\)[26], \(GGP\)[48], and \(GPP\)[49] are the major genes in the d-Man/l-Gal pathway of ascorbate biosynthesis and engineering of these genes can improve the AsA content to an extended degree. Our results showed that the increase in AsA content of pyramiding lines was comparable to the single-gene overexpression lines, e.g., \(GMP\) overexpressing lines (Figure 1), suggesting that AsA accumulation will not increase without limit by gene pyramiding in tomato. We hypothesize a rate-limiting step for AsA synthesis and self-regulating mechanism for AsA homeostasis. Figure 6: The fruit weight (**A**), transverse diameter (**B**), longitudinal diameter (**C**), and brix degree (**D**) in AC and pyramiding lines. Three replicate experiments were performed. Error bars represent standard error, means \(\pm\) SE. \(G\times G\times G\times G\) means \(GMP\times GME\times\)\(GGP\times GPP\). The asterisks represented significant differences from wild type (AC), as indicated by the \(t\)-test (* \(p\) < 0.05; ** \(p\) < 0.01). In this study, a series of experiments were conducted to reveal the molecular mechanism for the increase in AsA content in plants of pyramiding AsA-related genes. The possible reasons for the increase of AsA content in pyramiding lines were assumed to be multifactorial. First, compared with AC, the transcript level of most AsA biosynthesis-related genes were upregulated in the leaves and red ripened fruits of pyramiding lines (Figure 1A,C). Therefore, the activated AsA pathway may be one of the reasons for the increase of AsA content in pyramiding lines. Second, pyramiding did not completely break the basic rule of the circadian rhythm of AsA but increased the amplitude of fluctuation during the 48 h period (Figure 2). We assumed that the pyramiding enhances plant sensitivity to light and increases the product of photosynthesis substrate, which is involved in the main biosynthetic pathway of AsA [50], and thus increases the accumulation of AsA. Third, a precursor feeding experiment showed that the d-Man/L-Gal pathway especially contributes to AsA synthesis in the leaves of AC and pyramiding tomato lines. However, besides contribution of d-Man/L-Gal pathway and myo-inositol pathway to AsA biosynthesis in both AC and pyramiding lines, d-galacturonate pathway also plays a vital role in the synthesis of AsA in the fruits of the _GMP_\(\times\)_GME_ and _GMP_\(\times\)_GME_\(\times\)_GGP_\(\times\)_GPP_ pyramiding plants (Table 1 and Figure 4B). This is the reason why AC and _GGP_\(\times\)_GPP_ plants showed lower AsA content than _GMP_\(\times\)_GME_ and _GMP_\(\times\)_GME_\(\times\)_GGP_\(\times\)_GPP_ plants (Figure 1B). Besides, pyramiding biosynthetic genes also increased the transport capacity of AsA. Taken together, key structural gene pyramiding enhances synthesis and transport capacity of AsA in tomato. 200 mg powdered fruits were used for RNA extraction. Complementary DNAs were synthesized from total RNA using the HiScript@II Reverse Transcriptase (Vazyme, Miramar Beach, FL, USA) following the manufacturer's protocol.