The Signal Transduction Mechanism Of Trehalose(Tre) Regulating Cold Stress In Melon

Melon is a kind of thermophilic crop with high economic benefit.It is often subjected to cold injury in early spring,autumn and winter production in China,which seriously affects its normal growth and development,and reduces its yield and quality.Therefore,it is very important to explore the way to alleviate the low temperature stress injury of melon to realize the safe production of melon.Trehalose（Tre）is considered as a green and non-toxic plant growth regulating substance with the reputation of"sugar of life",which can improve a variety of stress resistance of crops.However,there are few reports on its role in regulating cold tolerance of crops,and the specific regulatory mechanism is still not fully understood.Therefore,in this study,cold tolerance of‘IVF571’and‘IVF004’melon seedlings was studied,and the levels of endogenous hydrogen peroxide（H₂O₂）and nitric oxide（NO）in the plants were analyzed.On this basis,we studied the regulation effect of exogenous Tre on the cold tolerance of melon,and whether endogenous H₂O₂,NO and abscisic acid（ABA）induced by exogenous Tre were involved in the cold tolerance of melon as signal molecules,and the related action pathways were identified.The main results are as follows:1.‘IVF571’and‘IVF004’melon were used as test materials,when the third true leaf is fully unfolded,they were treated with normal temperature（28℃/18℃,day/night）and cold stress（15℃/6℃,day/night）.After 24 h of temperature treatment,relevant indexes were sampled and measured.The results showed that under cold stress,the damage of membrane lipid peroxidation of‘IVF571’melon seedlings was significantly lower than that of‘IVF004’melon seedlings,while the endogenous levels of H₂O₂and NO were significantly higher than that of‘IVF004’melon seedlings.The cold tolerance of‘IVF571’melon variety was higher than that of‘IVF004’variety,which was positively correlated with the levels of endogenous H₂O₂and NO.2.Using‘IVF004’melon as test material,the whole plant was presprayed with10 m M trehalose,50 m M sucrose（Suc）,20 m M glucose（Glu）or distilled water when the third true leaf was fully developed.After 12 h,normal temperature treatment（28℃/18℃,day/night）and cold stress treatment（15℃/6℃,day/night）were started,and relevant indexes were sampled at different time points.The results showed that Compared with Suc and Glu,Tre application more effectively increased the maximum photochemical efficiency of PSII and decreased the relative electrical conductivity of melon leaves under cold stress.The activities of superoxide dismutase（SOD）,glutathione reductase（GR）and ascorbate peroxidase（APX）and the ratio of reduced glutathione to oxidized glutathione（GSH/GSSG）were increased.Exogenous Tre could significantly improve the cold tolerance of melon seedlings.3.Using‘IVF004’melon as test material,when the third true leaf was fully developed,the results were pretreated with 10 m M Tre or distilled water,and the relevant indexes were sampled at different time points after treatment.The results showed that exogenous Tre treatment could up-regulate the expressions of respiratory outfall oxidase gene（Cm RBOHD）,nitrate reductase gene 2（Cm NR2）,nitric oxide synthase gene 1（Cm NOS1）,9-cis-epoxide carotenoid plus dioxygenase gene3（Cm NCED3）and plasma membrane intrinsic protein gene（Cm PIP2-1/Cm PIP2-2）in melon leaves,and increase the endogenous levels of H₂O₂,NO and ABA.Exogenous Tre could induce the endogenous production of H₂O₂,NO and ABA in melon seedlings.4.Using‘IVF004’melon as test material,when the third true leaf was fully developed,expanded use 200μM of 2-（4-carboxyl）-4,4,5,5-4-hydroxy methylimidazoline-1-3-oxide（c PTIO,a kind of NO scavenger）and 5 m M dimethylthiourea（DMTU,a kind of O₂^-and H₂O₂scavenger）or spraying on the water melon leaf,after 8 h,with a 10 m M Tre or exogenous spraying water,after 12 h,began to normal temperature processing（18℃/28℃,day/night）cold stress treatment（15℃/6℃,day/night）,Samples were taken at different time points to determine the relevant indexes.The results showed that the removal of endogenous H₂O₂by DMTU weakened the effect of exogenous Tre on the cold tolerance of melon,while the removal of NO by CPTIO almost eliminated the effect of exogenous Tre and H₂O₂on the cold tolerance of melon.H₂O₂and NO participate in Tre to regulate chilling tolerance of melon seedlings.5.Using‘IVF004’melon as test material,when the third true leaf was fully developed,50μM of N-dihydroguaiac（NCED biosynthesis inhibitor,NDGA）or distilled water were used for pretreatment.8 h later,10 m M Tre or distilled water was sprayed.After 12 h,normal temperature treatment（28℃/18℃,day/night）and cold stress treatment（15℃/6℃,day/night）were started,and relevant indexes were measured at different time points.The results showed that,compared with cold treatment alone,the inhibition of endogenous ABA synthesis by NDGA further aggravated the cold stress damage and reduced the relative water content（RWC）of leaves.Exogenous Tre treatment alleviated cold stress and NDGA inhibition,increased leaf relative water content,maintained relatively normal cell morphology and plant photosynthesis.At the same time,virus-induced gene silencing（VIGS）increased the damage of Cm NCED3 gene in melon seedlings under cold stress compared with cold treatment alone,while external application of Tre significantly alleviated the damage.ABA participates in Tre to regulate chilling tolerance of melon seedlings.In conclusion,the chilling tolerance of‘IVF571’melon seedlings was significantly higher than that of‘IVF004’melon seedlings,which may be related to the levels of H₂O₂and NO induced by cold stress.Compared with Suc and Glu,exogenous Tre significantly improved the cold tolerance of melon,suggesting that Tre may have its own unique stress resistance function,rather than the effect of other double dips（such as Suc）or direct decomposition products（Glu）.Exogenous Tre can increase the endogenous levels of H₂O₂,NO and ABA,stimulate the cold response and resistance system of melon,improve the activity of antioxidant enzymes,and finally alleviate the membrane lipid peroxidation damage of melon seedlings under cold stress.In this process,H₂O₂,NO and ABA are involved in the regulation of Tre,and NO may play a role in the downstream of H₂O₂and ABA to maintain the integrity of cell structure,thus improving the photosynthesis and cold tolerance of melon seedlings.