Detection Of Melatonin And Its Roles In Defensing Abiotic Stresses In Grapevines

Melatonin?MT?as a type of indoleamine with little molecule exists ubiquitously in animals and plants.Biosynthesis and function of MT have been well studied in humans and animals,whereas the systematic and further researches are needed in plants.The existing researches show that MT plays an important role in improving plant antioxidant capacity and enhancing stress resistance.In this study,the method for detecting content of MT was established,and we determined the distribution of MT in different grape organs and the dynamics of MT accumulation in fruits of different cultivars.Additionally,the metabolic patterns of MT were detected under Na Cl,drought and high temperature stresses.Moreover,the roles of MT in alleviating the stress injure were investigated.The main results were as follows.1.The method of quantitative detection of MT,5-HT and 2-OHMel in grapes was established by using assistance of ultrasonic wave crushing and solid phase extraction combined with triple quadrupole LC/MS.Solid phase extraction conditions:the supernatant was filtered through C₁₈solid phase extraction column,and 5 m L of methanol,ultrapure water,sample,5%methanol solution and 90%methanol solution were added in sequential order for activation,equilibration,loading,leaching and elution;the speed was controlled to 1mL/min;the eluent was set to 1 mL finally.The binary mobile phases consisted of methanol?A?and water with 0.05%?v/v?of acetic acid?B?delivered at 0.3 mL/min.The C₁₈ gradient was started at an initial composition of 80%B,held for 0.8 min,then 1.4 min linear gradient to 40%B,held for 3 min.A return to the initial conditions was accomplished by a 0.1 min gradient to 80%B,where it was held for2.9 min.Thus,the total chromatographic run time was 8.0 min.Detection conditions:a column temperature of 25°C,a capillary temperature of 300°C,a spray voltage of 3000 V,an auxiliary pressure of 15 V and a sheath pressure of 35 V.2.The detection of MT contents in the skin of 12 grape varieties showed the significant differences among grape varieties.MT content were less than 1ng/g DW in most detected grape cultivars.Merlot had the highest content of MT,followed by Baker and Vidal while Kyoho Early and Chambourcin had the lower content.3.The first peak of MT content in Kyoho Early and Cabernet Sauvignon appeared before veraison,i.e.,on July 13?1.26 ng/g DW?and July 8?6.86 ng/g DW?,respectively;the second peak appeared on July 28?1.29 ng/g DW?and August 27?5.15 ng/g DW?.In contrast,there were four MT content peaks in Moldova,with two major peaks on July 18?10.4 ng/g DW?and September 21?15.68 ng/g DW?.The changing pattern of 5-HT?MT precursor?content was W-type in Kyoho Early,which was consistent with the M-type shape of MT.The MT peak of Cabernet Sauvignon appeared on August 31 and the double peaks of Moldova appeared on August 22 and September 6.The accumulation of 2-OHMel?the main metabolites of MT?reached peak value at the early stage of fruit development,and then gradually declined.The highest 2-OHMel content occurred on June 29 in Moldova and the lower 2-OHMel content was on July 8 in Cabernet Sauvignon.4.The MT contents in the organs of one-year-old Vidal were determined in September.The MT content in new roots?3.84 ng/g DW?was higher than in the new shoots and leaves.5-HT content was distributed in a way similar to MT.The content of 2-OHMel was determined in the order:new shoots>leaves>new roots;the content of 2-OHMel in the new shoot?3.72 ng/g?was 14.3 times as high as in the new roots.5.The content of endogenous MT in the roots increased sharply after 7 days of NaCl treatment,which was 0.4 times higher than in the control;2-OHMel in the new shoot and leaves was largely accumulated after 14 days of NaCl treatment,and the 2-OHMel content under NaCl+MT treatment was increased 4.3 times and 0.48 folds than the control and NaCl stress,respectively.6.The contents of MT and 5-HT in leaves were significantly increased by exogenous spraying and watering MT under drought stress.The MT content peak of leaves was 47 ng/g DW under drought plus MT watering,the amount of 2-OHMel in stems with drought plus MT spraying treatment was 19.4 times higher than the treatment with drought plus MT watering.2-OHMel content under drought stress was 1.4 times higher than the control.2-OHMel content in new root with drought and MT irrigation treatment was 28.2 and 15.7 times as high as under drought alone and drought plus MT spraying treatment,respectively.7.The increasing extent of K point on the PSII donor was alleviated by watering 100 nmol·L^-1MT under NaCl,drought?irrigation and spray?and heat stresses.The excitation efficiency of electron transport beyond QA??_O?was increased 5.8%-32%compared to the control.Density of reaction centers?RC/CS_m?were significantly increased by 26%,17%and 17.7%under NaCl,drought and heat stresses,respectively;the donor side limitation of PSI Y?ND?decreased under NaCl and drought stresses;in contrast,the acceptor side limitation of PSI?Y?NA??and the electron flow through PSI ETR?I?were significantly increased.