Study On Role Of Melatonin In Regulating And Inducing Chilling Tolerance In Litchi Fruit

Litchi(Litchi chinensis Sonn.)is an important crop with high economic value in tropical and subtropical regions.Litchi fruit after harvest is extremely susceptible to browning and senescence,leading to severe quality deterioration.Refrigeration is one of the most effective strategies to maintain postharvest quality and extend the shelf life in fruits.Nevertheless,due to sensitivity to low temperature,litchi fruit is prone to develop chilling injury(CI)during refrigeration,and CI symptoms rapidly aggravate after removal of the fruit from chilling conditions,which seriously restricts the application of refrigeration technology for litchi preservation.Therefore,exploiting applicable technologies to control CI and unraveling the related mechanisms behind the observed CI prevention in litchi fruit are of great significance for improving the storage quality and increasing the commercial value of the products.Melatonin is an important biologically active compound that is identified as a signalling molecule to regulate various physiological processes in plants.In recent years,the effects of melatonin on physiological properties and quality improvement in harvested horticultural crops have been received much attention.However,there is still a lack of systematic research on regulatory role of melatonin in cold tolerance of litchi fruit and its related mechanism.Towards this objective,in this study,using‘Ziniangxi'litchi fruit as experimental materials,we investigated the controlled influence of exogenous melatonin on CI in litchi fruit during refrigeration;and then the key role of endogenous melatonin in modulating chilling tolerance in cold-stored litchi fruit and the involvement of nitric oxide(NO)in melatonin-mediated cold tolerance were further explored.The related mechanisms were interpreted and discussed.The main results are shown as follow:1.The controlled effect of exogenous melatonin treatment on CI in litchi fruit(1)Compared with the control fruit,0.5 mmol L^-1 melatonin treatment effectively delayed the increase in CI index and decrease in chromaticity a*value in litchi fruit during refrigeration.(2)Exogenous melatonin treatment inhibited the activity of NADPH oxidase(NOX)activity and reactive oxygen species(ROS)generation,while reducing the membrane relative conductivity and malondialdehyde(MDA)content in litchi fruit during cold storage,contributing to maintanence of membrane integrity.Exogenous melatonin treatment reduced the activities of polyphenol oxidase(PPO)and peroxidase(POD)and inhibited the decreases in total phenolics,flavonoids and anthocyanins contents in litchi fruit during refrigeration,thereby ameliorating chilling stress-induced enzymatic browning.(3)Exogenous melatonin treatment enhanced the activities of superoxide dismutase(SOD),catalase(CAT),ascorbate peroxidase(APX),monodehydroascorbate reductase(MDHAR),dehydroascorbate reductase(DHAR)and glutathione reductase(GR),while promoting the increases in contents of ascorbic acid(AsA)and glutathione(GSH),which could lead to in increases in antioxidant activity in litchi fruit during refrigeration.Melatonin treatment induced the enhancement in activities of glucose-6-phosphate dehydrogenase(G6PDH)and 6-phosphogluconate dehydrogenase(6PGDH)in litchi fruit receiving melatonin,contributing to higher NADPH content in melatonin-treated fruit as compared to control fruit,which could sustain the demand of reducing power for chilling resistance-related metabolisms.Furthermore,melatonin treatment up-regulated the expressions of the LcMsrA1,LcMsrA2,LcMsrB1,LcMsrB2,LcTrx2,LcPrx1 and LcNTR1,thereby promoting the repair capacity of ROS-oxidized protein.2.The role of endogenous melatonin in regulating chilling tolerance in litchi fruit(1)Compared with control fruit,0.15 mmol L^-1 p-chlorophenylalanine(p-CPA,an inhibitor of melatonin biosynthesis)exposure increased the CI index and acceletrated the decrease in chromaticity a*value in litchi fruit during refrigeration.The combined treatment of melatonin and p-CPA partially weakened the inhibitory effect of melatonin alone treatment on the CI severity.(2)p-CPA treatment reduced the levels of endogenous serotonin and endogenous melatonin in litchi fruit,which was attributed to down-regulation of of LcTDC,LcT5H and LcSNA expressions in response to p-CPA.(3)p-CPA treatment promoted the increases in NOX activity and ROS level,enhanced the membrane relative conductivity and MDA content,while stimulating the increases in lipoxygenase(LOX)and phospholipase D(PLD)activities in litchi fruit during refrigeration,thereby leading to aggravation of membrane damage.Application of p-CPA to litchi fruit increased the PPO and POD activities,inhibited the PAL activity,and accelerated the declines in contents of total phenolics,flavonoids and anthocyanins.p-CPA treatment inhibited the activities of antioxidant enzymes including SOD,CAT,APX,MDHAR,DHAR and GR,and reduced the contents of AsA and GSH,which could not be conducive to the elimination of cellular ROS.Moreover,p-CPA treatment suppressed the expression levels of genes encoding oxidized protein repair-related genes,including LcMsrA1,LcMsrA2,LcMsrB1,LcMsrB2,LcTrx2,LcPrx1 and LcNTR1,which could weaken the normal function of protein repair system in litchi fruit under chilling stress.3.The role of NO in melatonin-mediated chilling tolerance in litchi fruit(1)Exogenous melatonin(0.5 mmol L^-1)treatment resulted in reduced CI occurrence compared to control fruit,whereas such a CI suppression was markedly impaired by a combined treatment of melatonin(0.5 mmol L^-1)and cPTIO(0.1 mmol L^-1).(2)Exogenous melatonin treatment triggered the higher production of nitric oxide(NO)the activities of nitrate reductase(NR)and NO synthase(NOS).Furthermore,the treatment with combination of melatonin and cPTIO suppressed the accumulation of NO,but did not exert any effect on NR and NOS activities when compared to melatonin treatment.(3)Compared with the control fruit,exogenous melatonin reduced the ROS generation,inhibited membrane lipid peroxidation,increased the activities of antioxidant enzymes(SOD,CAT,APX,MDHAR,DHAR and GR)and the contents of antioxidant(AsA and GSH),as well as promoted the synthesis of NADPH.However,such physiological effect induced by melatonin in refrigerated litchi fruit was significantly negated by treatment with melatonin+cPTIO.