## 4 Discussion  
### Effect of VI on Proline Content of Spinach Leaves  
Our results clearly show that impregnation of GABA, sucrose and SA into spinach tissue have resulted in the increment of proline by 240%, 153% and 103% respectively on day 1 of storage in chilling temperature (Figure 1a). Amino acid proline is known as one of the major organic osmolytes that accumulate in a variety of plants in response to environmental stresses such as temperatures, drought, salinity, UV radiation and heavy metals [30]. [PERSON] et al. [31] stated that proline defends plants by functioning as a cellular osmotic regulator between cytoplasm and vacuole and by detoxifying of reactive oxygen species (ROS), thus protecting membrane integrity and stabilizing antioxidant enzymes. Therefore, higher proline accumulation can help in reducing the damages resulted from low temperature abuse during cold storage of spinach leaves.  
In plants, the precursors for proline biosynthesis are L-glutamic acid and ornithine. Two enzymes involved in the proline biosynthetic pathway (Figure 5) through L-glutamic acid are _pyrroline-5-carboxylate synthetase_ (P5 CS) and _pyrroline-5-carboxylate reductase_ (P5 CR). Meanwhile, _ornithine \(\delta\)-amino-transferase_ (OAT) takes part through the ornithine pathway [32].  
Based on our results, the highest proline content was observed in GABA impregnated leaves, followed by sucrose and SA impregnated leaves. In contrast, the proline content in non-impregnated leaves was decreasing by as much as 94% throughout 7 days of storage. The results were supported by previous research of [PERSON] et al. [32] regarding the effect of exogenous GABA treatment on chilling injury in peach after long term cold storage. It has been reported that immersion of peaches in GABA for 10 min could reduce chilling injury by enhancing the accumulation of proline and also endogenous GABA. The study also mentioned that proline accumulation depends on its degradation, which catalyzed by _proline dehydrogenase_ (PDH). [PERSON] et al. [32] also revealed that treatment with GABA increased the activities of P5 CS and OAT but decreased PDH activity in peaches under chilling stress. The increase of P5 CS and OAT activities could enhance the proline biosynthesis, while the decrease of PDH activity would contribute to the lower degradation of proline in the GABA-treated peaches.  
The increment of proline content in sucrose impregnated leaves was supported by previous research of [PERSON] et al. [33]. In this study, it was found that the effect of exogenous sucrose feeding to cucumber seedlings on chilling tolerance and proline content has resulted in higher proline. [PERSON] and [PERSON] [30] explained that proline helps in chilling injury by providing sufficient reducing agents upon relief of stress, which later supports mitochondrial oxidative phosphorylation and generation of ATP for recovery from stress and repairing of stress-induced damages. In addition to proline's role in chilling tolerance, endogenous sucrose also contributes to the mitigation of chilling injury by activating the antioxidant enzymes [33].  
Previous researches [9; 10; 11] reported that the application of exogenous SA could improve proline accumulation in fruits and vegetables, thus alleviating damages from chilling temperature storage. This was also found in our results where vacuum impregnation  
Figure 5: Proline biosynthesis from glutamate [30].  
of SA in spinach tissue can increase proline accumulation by 103% as compared to untreated leaves. It is believed that exogenous SA can activate P5 CS which triggers proline accumulation [34].