Effects of exogenous trehalose on sugar metabolism and energy sensing in response to chilling injury of ‘Kim Ju’ guava fruit during low temperature storage

Abstract

Chilling injury (CI) is a devastating postharvest disorder of fruit that can occur under low temperature stress in cold storage and is closely related to intracellular energy deficiency and loss of membrane stability. Trehalose, a non-reducing sugar generated by sugar metabolism, plays a protective role against different physical stresses. Maintenance of energy level by postharvest trehalose treatment has contributed to alleviated CI and improved chilling tolerance in many harvested crops. However, ability of trehalose on homeostatic regulation of cellular energy metabolism in response to CI has not been well studied. The aim of this study was to investigate the effects of exogenous trehalose on sugar metabolism in relation to the regulation of cellular energy balance and energy sensing in reducing CI of ‘Kim Ju’ guava fruit under exposure to low temperature storage. The whole experiment process was divided into four parts. In the first experiment, the effects of low temperature storage on CI and postharvest quality of ‘Kim Ju’ guava fruit was investigated. The fruit were stored at 6, 8, 10, 12 and 14 °C and transferred to room temperature at 25 °C. The fruit from each group was randomly taken every 2 d during cold storage and every day at room temperature to determine the fruit quality and CI symptoms. The results showed that the temperature at 6, 8 and 10 °C induced CI symptoms within 6, 10 and 10 d, respectively and CI became more aggravated after being transferred to room temperature. The CI symptoms at 6 ºC was more severe than that at 8 and 10 °C, while at 12 °C showed slight CI and at 14 °C did not show CI symptoms. The maximum storage life after cold storage at 6, 8, 10, 12 and 14 °C was 10, 12, 10, 8 and 8 d, respectively. The eating quality of fruit stored at 6 and 8 °C was higher than that stored at 10, 12 and 14 °C indicating that 8 °C was the optimal storage temperature for providing high overall quality and minimal CI symptoms of ‘Kim Ju’ guava fruit. In the second experiment, the relationship among energy status, membrane stability and CI of ‘Kim Ju’ guava fruit during low temperature storage was investigated. The fruit were immersed in distilled water (control); 0.5, 1 and 2 mM of adenosine triphosphate (ATP); 0.5, 1 and 2 mM of 2,4-dinitrophenol (DNP; ATP production inhibitor) for 30 min and stored at 8 °C for 14 d followed by 4 d of shelf storage at 25 °C. The fruit from each group was randomly taken as previously described to investigate energy level and membrane stability. It was shown that the intracellular energy (ATP content and energy charge) of untreated fruit decreased gradually during low temperature storage. This reduction coincided with the increases in membrane damage and CI index as well as a decrease in membrane transport enzyme (H+/Ca2+-ATPase) activities. The fruit treated with ATP had higher levels of cellular energy and ATPase activity but had lower membrane damage and CI index than the controls. In contrast, treatment with DNP exhibited opposite results by reducing cellular energy and ATPase activity and increasing membrane damage and CI index comparing with ATP treatment. These results suggest that CI development of ‘Kim Ju’ guava fruit was associated with a decrease in energy level and loss of membrane stability. In the third experiment, the impacts of exogenous trehalose on soluble sugar and energy metabolisms in relation to membrane stability and CI of ‘Kim Ju’ guava fruit during low temperature storage were investigated. The fruit were immersed in 0, 50, 100, 200 and 400 mM trehalose for 30 min and stored at 8 °C for 14 d followed by 4 d of shelf storage at 25 °C. The fruit from each group was randomly taken as previously described to determine the energy status, enzyme activities in sugar metabolism, soluble sugar contents, membrane stability and CI symptoms. It was shown that trehalose at 200 mM was the most effective concentration in lowering CI. The trehalose-treated fruit exhibited a higher index of membrane stability and energy level accompanied by a rise in endogenous trehalose, sucrose, glucose 6 phosphate and fructose 6 phosphate levels. Trehalose treatment promoted the activities of sucrose synthetic enzyme including, sucrose phosphate synthase and sucrose synthase (synthesis) as well as trehalose hydrolytic enzyme such as trehalase and glycolytic enzymes including, hexokinase, fructokinase, phosphoglucoisomerase and pyruvate kinase. On the other hand, it inhibited activities of sucrose hydrolytic enzymes such as sucrose synthase (cleavage) and invertase. These results suggest that trehalose treatment could maintain membrane integrity under regulation of adequate energy balance by enhancing the activities of energy and sugar metabolism‐related enzymes, thus contributing to elevated energy status and reduced CI in cold‐stored ‘Kim Ju’ guava fruit. In the fourth experiment, the effects of exogenous trehalose on sugar signaling-mediated energy sensing and energy homeostasis of ‘Kim Ju’ guava fruit during low temperature storage were investigated. The fruit were treated with 0 and 200 mM of trehalose for 30 min and kept at 8 °C for 14 d followed by 4 d of shelf storage at 25 °C. The fruit from each group was randomly taken as previously described to analyze soluble sugar contents, energy sensing-mediated gene expression and activity of sucrose non-fermenting-1-related protein kinase 1 (SnRK1) as well as gene expression and enzyme activity of energy production. It was shown that SnRK1 gene expression and activity increased after trehalose treatment, reaching maximum within 1-2 d. The trehalose treatment increased the expression of energy producing genes and activity of ATP producing enzymes such as ATP synthase, NADH dehydrogenase, succinate dehydrogenase and cytochrome c oxidase, accompanied by higher energy charge and lower CI throughout storage. The increase in SnRK1 activity by exogenous trehalose was closely associated with an initial transient increase of sucrose during the first 2 d of storage. The SnRK1 activation was also induced by 50-200 mM exogenous sucrose within 18 h after treatment. These results suggest that beside low intracellular sucrose, SnRK1 is also activated in response to an early transient sucrose generation by exogenous trehalose. Thus, this sucrose could trigger the activation of SnRK1 in regulating the expression of genes encoding enzymes of sugar metabolism for maintaining energy homeostasis of ‘Kim Ju’ guava fruit during cold storage. It is concluded that exogenous trehalose could cause alteration of the soluble sugars via the sugar metabolism for providing sufficient energy and signal-activated energy sensing involved in energy homeostasis regulation, consequent improved membrane stability which led to the amelioration of CI in ‘Kim Ju’ guava fruit during exposure to chilling stress.