The Protective Function Of Melatonin In Malus Under Light Stress

Apple is one of the most important fruit trees in the world,which occupies an important position in fruit production.The yield and cultivation area of apple in China account for more than 50%of the world's total.The Northwest Loess Plateau region is the largest high-quality and world-famous apple production base in China.The light stress caused by high light and UV-B is one of the most important limiting factors in the apple industry development of high altitude production areas in Northwest of China.The excess light energy can damage the photosynthetic apparatus of the leaves,reduce the photochemical efficiency,cause photoinhibition,and even cause sunburn in severe cases.Moreover,light stress inhibits the growth and development of apple trees,reduces yield and fruit quality as well as economic value.Therefore,it is of great significance to find ways to improve apple resistance to light stress and to study its mechanism.Melatonin is widespread in plants and this compound plays an important impact in plant growth regulation,circadian rhythm regulation and free radical scavenging.However,there are few reports about the relationship between melatonin and light stress in Malus.Apple rootstocks,apple varieties,and two transgenic apple plants overexpressing melatonin synthasegenes?humanarylalkylamine-N-acetyltransferase,AANATand hydroxyindole-O-methyltransferase,HIOMT?were used in this study as the experimental materials.The function of melatonin under high light stresses such as methyl viologen?MV?,UV-B and high light was characterized from the two perspectives by exogenous melatonin and endogenous melatonin.The study provided new germplasm resources and theoretical basis for stress research of apple and provide some help for the healthy growth and development of plants.The main results are documented as follows:1.Exogenous melatonin significantly mitigated the detrimental effect of oxidative stress induced by MV in apple leaves.The leaves of‘Naganofuji No.2'apple?Malus domestica Borkh.cv.Naganofuji No.2?were used as experimental materials.The effect of different concentrations of melatonin pretreatment?0,0.01,0.1,1,10 mM?on photooxidative stress alleviation was studied by using detached apple leaves.The results indicated that the effect of melatonin depends on the pretreatment concentration,and the 1 mM melatonin mitigated the detrimental effects of oxidative stresses most effectively.Under MV treatment,melatonin alleviated the decrease in chlorophyll concentrations and maximum potential Photosystem II efficiency?F_v/F_m?while also mitigating membrane damage and lipid peroxidation.The melatonin-treated leaves also showed higher activities and transcripts of antioxidant enzymes.In addition,the expression of genes for those enzymes was upregulated.Melatonin-synthesis genes MdTDC1,MdT5H4,MdAANAT2,and MdASMT1 were also upregulated under oxidative stress in leaves but that expression was suppressed in response to 1 mM melatonin during the MV treatments.It is indicated that the exogenous melatonin mitigates the detrimental effects of MV stress,perhaps by slowing the decline in chlorophyll concentrations,moderating membrane damage and lipid peroxidation.2.Exogenous melatonin significantly mitigated the detrimental effects of UV-B stress in Malus hupehensis Rehd.In this study,seedlings pretreated with melatonin showed significant reduction in plant growth and root system development under UV-B stress.However,the addition of 1?M melatonin markedly alleviated these effects,especially at the higher dosage of UV-B radiation.The inhibitory effects of UV-B radiation on photosynthetic parameters,chlorophyll fluorescence parameters,stomatal apertures,chlorophyll levels and damage to leaf membranes were also markedly alleviated with melatonin application.Melatonin treatment was also associated with higher activity and expression of genes encoding antioxidant enzymes?ascorbate peroxidase,APX;catalase,CAT;and peroxidase,POD?and greater decline of hydrogen peroxide?H₂O₂?content in leaves exposed to UV-B.Moreover,exogenous melatonin treatment and UV-B stress increased the concentration of endogenous melatonin.The content of several phenolic compounds,including chlorogenic acid,phloridzin and quercetin-3-galactoside,increased under UV-B stress,and these were further elevated significantly with melatonin addition.3.The AANAT and HIOMT overexpressing lines showed better resistance under UV-B stress.The human melatonin synthetic genes AANAT and HIOMT were transferred into‘GL-3'apple plants.The study found that the melatonin content in the transgenic lines was higher,and the melatonin content of HIOMT overexpressing lines was significantly higher than that of AANAT overexpressing lines.Then,the effects of UV-B treatment were studied by using AANAT and HIOMT overexpressing lines and wild-type?WT?plants.The study found that overexpression of AANAT and HIOMT enabled the transgenic lines maintaining higher P_n and F_v/F_m than WT plants in UV-B stress,and reducing the effect on stomatal aperture of UV-B.Under UV-B stress,the total phenolics content in the transgenic lines was increased compared with the WT,and the content of benzoic acids and hydroxycinnamic acids,dihydrochalcones and flavanols were increased.In particular,the content of chlorogenic acid,phlorizin and proanthocyanidin B1 were increased significantly.Under UV-B stress,the overexpressing lines not only promoted the expression of genes related to phenolic synthesis,but also increased the accumulation of phenolics by inhibiting the expression of phenolic degradation-related gene PPO.4.The HIOMT overexpressing lines showed enhanced resistance against high light stress compared with WT plants.By using the HIOMT overexpression lines,we tested the role of melatonin under high light stress and under high light combined with drought stress.The results showed that under shading conditions,the light saturation point of the transgenic lines was higher than that of WT plants.Under high light stress,the maximum photosynthetic rate and light compensation point of the transgenic lines were significantly higher than that of WT plants.High light stress damages plants,while drought can increase the degree of damage.The transgenic lines significantly alleviated photodamage,and H₂O₂,superoxide anion?O₂^-?,malondialdehyde?MDA?content,relative electrolyte leakage?REL?.Studies on photosynthesis and fluorescence parameters revealed that the transgenic lines could maintain higher photosynthetic capacity,and reduce the inhibition of PSII after high light stress.Overexpression of HIOMT also alleviated the decrease of F_v/F_m,F_v'/F_m',ETR and inhibited the increase of NPQ and Y?NPQ?.Under high light and drought stress,overexpression of HIOMT increased melatonin content and alleviated the degradation of Rubisco.Moreover,overexpression of HIOMT increased the antioxidants content in AsA-GSH as well as enhanced the activity of related antioxidant enzymes.In addition,overexpression of HIOMT promoted the accumulation of sorbitol,glucose,malic acid and starch by regulating the expression of related genes.