# Overexpression of _RrGGP2_ and _RrDHAR_ Increases Ascorbic Acid Content in Tomato
## 1 Introduction  
Ascorbic acid (AsA, Vitamin C) is an essential antioxidant in plants that scavenges reactive oxygen species (ROS) generated during exposure to biotic or abiotic stresses and those produced during normal growth and development [1, 2, 3]. Studies have also found that AsA regulates genes which control plant development [4, 5], as well as flowering time regulation, premature senescence, and programmed cell death [6]. Unlike plants, humans cannot synthesize AsA independently due to the absence of a crucial enzyme called L-gulonolactone oxidase, and insufficient dietary AsA intake can cause a deficiency and related diseases in humans [7]. The predominant source of AsA is plant products such as fresh fruits or vegetables [8]; therefore, genetic engineering techniques have been extensively studied to enhance the AsA content of horticultural plant products [9]. Several pathways for AsA biosynthesis have been identified, including the D-mannose/L-galactose pathway [10], the D-galacturonate pathway [11], the myoinositol pathway [12], and the L-glucose pathway [13]. Among them, the L-galactose pathway has been identified as the principal pathway for AsA biosynthesis in many higher plants, such as apple [14], kiwifruit [15], _R. raxburghii_[16], blueberry [17], and tomato [18], and is the only pathway present in _Arabidopsis_[19]. In this pathway, D-glucose-6-phosphate is used as a substrate, which sequentially passes through glucose-6-phosphate isomerase (GPI), phosphomannoseisomerase (PMI) [20], phosphomannose mutase (PMM) [21], GDP-mannose pyrophosphorylase (GMP) [22], GDP-mannose-3\({}^{\prime}\),5\({}^{\prime}\)-phenotypic isomerase (GME) [13], GDP-L-galactose phosphorylase (GGP) [19; 23], L-galactose-1-phosphatase (GPP) [24; 25], L-galactose dehydrogenase (GDH) [26], and L-galactose-1,4-lactone dehydrogenase (GLDH) [27]. As a vital antioxidant and electron donor, AsA is metabolized to monodehydroascorbic acid (MDHA) and dehydroacyclic acid (DHA) by the reactions of ascorbate oxidase (AO) and ascorbate peroxidase (APX) [28], and hydrogen peroxide (H\({}_{2}\)O\({}_{2}\)) is consumed to form malonyl H\({}_{2}\)O and O\({}_{2}\). MDHA and DHA are catalyzed by monodehydroascorbate reductase (MDHAR) and dehydroacorbate reductase (DHAR) [29; 30], respectively. All genes in this pathway have been cloned, with several identified as key genes for AsA accumulation in various plants. Interestingly, studies on the same plant have shown inconsistent findings regarding the key genes for AsA accumulation. For instance, in kiwifruit, GGP was originally believed to be the key gene [15; 31], but later, GPP was found to play this role [32]. In apple, initial evidence suggested that GDH, GPP, and GME expression were closely linked to AsA accumulation [14], but later studies showed that GGP was the key gene [33]. In tomato, GME was initially believed to be the key rate-limiting gene for AsA regulation [34; 35], but recent research indicates that GGP is the actual key gene [33; 36]. These variations in conclusions could be due to varietal differences or caused by experimental methods [33], and this controversy also exists in _R. roxburghii_. One research study has suggested that _RrDHAR_ plays a pivotal role in achieving the high levels of AsA accumulation seen in _R. roxburghii_ fruit [37], while another study has indicated that _RrGGP2_ plays a more significant regulatory role in _R. roxburghii_ AsA biosynthesis [38; 39]. In this study, we aim to explore the roles of _RrGGP2_ and _RrDHAR_ in AsA biosynthesis and determine which makes the greatest contribution to the accumulation of AsA in _R. roxburghii_. _R. roxburghii_ belongs to the Rosaceae family, which is cultivated as a functional fruit rich in a variety of nutrients and health-promoting compounds [40; 41; 42]. It is primarily found in Southwest China and referred to as the 'King of Vitamin C' because of its high vitamin C content [43; 44].