Study On Regulation Mechanism Of Exogenous Melatonin On Leaf Senescence Of Cucumber

Cucumber（Cucumis sativus L.）is one of the main vegetables that plant in greenhouse.Leaf early senescence caused by abiotic stresses or natural leaf senescence reduced photosynthetic efficiency,which has been became one of the main factors threatening high yield cultivation of cucumber.Melatonin（MT）is one small molecule of indoles that participates in the regulation of plant leaf senescence.However,the physiological and molecular mechanism by which MT delays the leaf senescence of cucumber remain unclear.Here,’Jinyou 35’cucumber was used as material and darkness was used to simulate senescence and then we studied the alleviating effect of MT with different concentrations(0,50,75,100,125,150μmol·L^-1)on leaf senescence and selected its optimal concentration.On this basis,we further studied the effects of exogenous MT on the relative expression of senescence-associated genes（SAGs）,chlorophyll degradation,photosynthetic function and active oxygen metabolism of cucumber under darkness,and deionized water（H₂O）treatment was used as the control（CK）.Moreover,we verified the molecular mechanism of MT on delaying cucumber leaf senescence via transient transformation of N-acetyl-5-hydroxytryptamine methyltransferase（ASMT）,which was the key gene for the biosynthesis of MT.The main results are as follows:1.Darkness induced the expression of L-tryptophan decarboxylase（TDC）,tryptophan hydroxylase（T5H）,5-hydroxytryptamine-N-acetyltransferase（SANT）and ASMT in leaves,and the content of endogenous MT was significantly increased.Exogenous spraying of 50,75,100,125 and 150μmol·L^-1 MT could alleviate the decrease of relative chlorophyll content（SPAD）and the increase of electrolyte leakage rate（EL）and malondialdehyde（MDA）content of leaves under dark condition,and delay leaf senescence in a concentration-dependent manner.The leaves treated with 100μmol·L^-1 MT showed the highest SPAD and lowest EL and MDA content among all the treatments,which was selected as the subsequent test concentration.2.Compared with CK,the application of MT significantly down-regulated the expression of senescence related genes SAG12,NAC41 and programmed cell death（PDCD）in leaves under dark condition,and reduced cell death.Meanwhile,the expression levels of Demagnesium chlorophyllin a oxygenase gene（PAO）,Demagnesium chlorophyll hydrolase gene（PPH）and Chlorophyll b reductase gene（NOL）,which are key genes for chlorophyll degradation,were down-regulated,and the chlorophyll content was significantly increased.The chlorophyll a and b contents of leaves treated with water decreased by 32.9%and 35.8%,respectively,and those of MT treatment only decreased by 20.8%and 22.1%,respectively after 5 days of darkness.In addition,after 5 days of darkness,the chloroplast structure was deformed,and the number of stromal lamellae increased,and the dissolution of stromal lamellae was disordered,and the chloroplast structure of MT treatment was more normal,in term of the clearly visible stromal lamellae and the lower accumulation of stromal lamellae.3.The application of MT could obviously increase the photosynthetic rate（P_n）,the actual photochemical efficiency(Φ_PSⅡ),the maximum photochemical efficiency（F_v/F_m）,the comprehensive photosynthetic performance index(PI_ABS),the light energy capture(TR₀/Cs₀andφ_Po),photosynthetic electron transport(φ_Eo and ET₀/CS₀)of photosystemⅡ（PSⅡ）as well as photosystemⅠ（PSⅠ）activity（ΔI/I₀）during the aging process of cucumber seedlings.Moreover,MT increased the activities and m RNA expression of of ribulose-1,5-diphosphate carboxylase/oxygenase（Rubisco）and Rubisco activase（RCA）and also enhanced the protein stability of Rubisco,RCA,and PSⅠcore complex protein subunit PSⅠcore complex protein subunit（Psa D）and PSⅡphotochemistry center（D1）,PSⅡcomplementary pigment complex（LHcaⅡ）complex of leaves under darkness.The data implied that MT can improve the photosynthetic function and delay the leaf senescence induced by darkness through promoting photosynthetic carbon assimilation,the activities of PSⅡand PSⅠreaction center as well as the levels of photosynthesis-associated protein.4.With the extension of dark time,the contents of EL,MDA,Hydrogen peroxide（H₂O₂）and superoxide anion（O₂.^-）gradually increased and the activities and gene expression of SOD,CAT,POD,APX and GR as well as the contents of Reduced Ascorbic acid（As A）and Reduced Glutathione（GSH）in cucumber seedlings also promoted.However,leaves of MT-treated showed obviously lower EL and MDA and ROS contents and higher activities and gene expression of SOD,CAT,POD,APX and GR as well as the contents of As A and GSH,compared with the control,suggesting that MT can up-regulate the antioxidant capacity and reduce the accumulation of ROS to delay the leaf senescence induced by darkness.5.Overexpression of ASMT can significantly reduce the MDA content,down-regulate the expression levels of key chlorophyll genes PAO and PPH,increase chlorophyll content,F_v/F_m andΦ_PSⅡ,and also up-regulate the activities and gene expressions of SOD and CAT to decrease the accumulation of ROS and alleviate cell death in leaves under dark condition.The results further prove that MT can alleviate the dark-induced senescence of cucumber leaves.