## 4 Discussion  
To counteract the oxidative damage that continuously occurred due to abiotic stress, plant developed multiple enzymatic and non-enzymatic antioxidative defense systems to maintain the redox balance in the cell. The AsA-GSH pathway plays a vital role in detoxifying ROS and interacts with other defense systems to counteract various abiotic stresses. Both AsA and GSH are strong antioxidants which can donate one or two electrons to form oxidized form, DHA and GSSG, respectively. AsA is of particular importance among these, and is the most abundant water-soluble antioxidant in plants, e.g., AsA is present at concentrations of 20 mM or more in chloroplasts. In addition, AsA serves as an important co-factor in several pathways including the xanthophyll cycle, the synthesis of ethylene, and cell wall components. AsA is also involved in the regulation of cell division and cell expansion. Thus, the maintenance of proper AsA content is essential to the plant development and abiotic stress tolerance [6; 8; 21; 22; 23].  
Recycling of AsA from their oxidized forms is the necessity to keep the redox balance as well as the higher AsA content. The oxidized ascorbate, MDHA or DHA, can be recycled back to its reduced form by MDHAR or DHAR, respectively. Since MDHAR and DHAR are not rate-limiting steps in the recycling of AsA, loss-of-function mutations of their coding genes did not affect the pool size of total ascorbate [24; 25]. In this study, LT-tolerant inbred Jing72464 maintained similar AsA and GSH content as that of sensitive ones, whereas the oxidized substrates were significantly higher. This led to the increase of pool size in total ascorbate and glutathione, which contribute to the stress fitness of plant. Among the four antioxidative enzymes in AsA-GSH pathway, ZmAPX1 and ZmAPX2 showed significant transcriptional differences between the LT-tolerant and LT-sensitive inbred lines. Our results are consistent with previous study, in which _APX_ gene was more inducted by LT in LT-tolerant maize genotype Gurez local but not in LT-susceptible genotype G-M-6 [26]. This implied that APX might be the key enzyme to regulate low temperature tolerance of maize.  
Plant activates the AsA-GSH recycle pathway to ameliorate the oxidative damage, meanwhile requires the fine coordination of de novo antioxidant biosynthesis and recycling. There is compelling genetic evidence that the biosynthesis of AsA proceeds via the D-mannose/L-galactose pathway and is the most significant source of ascorbate in plants. This pathway consists of eight reaction processes catalyzed by constitutive enzymes, including GME, GGP, GPP, L-GalDH, L-GalLDH, and so on. GGP/VTC2 is the rate-limiting step in this pathway and is the only gene that causes a substantial increase in ascorbate contents by its overexpression [21; 27]. Among the biosynthesis genes analyzed in these studies, ZmVTC2 is the only gene that significantly induction by LT, which suggested its predominant role for abiotic stress fitness of maize. LT-tolerant inbred Jing72464 maintained significantly higher transcription level of ZmVTC2 at normal condition than that of LT-sensitive inbred JingX201, which might provide the plant with higher AsA pool to counteract the oxidative damage due to abiotic stress perturbation, e.g., occasional encountered low temperature during germination. In this context, ZmVTC2 would be a major target of genetic engineering for the improvement of AsA content and LT-stress fitness.