Grafting And Melatonin Application Improves Nitrogen Use Efficiency And Vanadium Stress Tolerance Of Watermelon Seedlings

Watermelon is an important crop cultivated across the world at a commercial scale.The growth and development of watermelon is affected by the environmental factors.Limited availability of nutrients particularly nitrogen?N?and presence of heavy metals such as vanadium in the soil restricts plant growth and development.Grafting is an important agro-technical procedure utilized to enhance the capacity of plants to tolerate biotic and abiotic stresses in horticultural crops;and melatonin?N-acetyl-5-methoxytryptamine?is a widely studied biomolecule that acts as an antioxidant and a signaling molecule and enhances the abiotic stress tolerance of plants.In this study,we utilized grafting and melatonin application to improve nitrogen use efficiency?NUE?and vanadium stress tolerance of watermelon.The main results are as following:1.The first experiment was aimed to investigate how pumpkin grafting?PG?improves N use efficiency of watermelon.A commercial watermelon cultivar Zaojia 8424 was self-grafted and grafted onto pumpkin?Cucurbita maxima � C.moschata?rootstock cv.Qingyan Zhenmu No.1.The grafted plants were exposed to two levels of N?9 m M and 0.2 m M?under hydroponic conditions.PG improved the N use efficiency of watermelon scion through the vigorous root system of pumpkin rootstock that enhanced the uptake and accumulation of N,P,K,Ca,Mg,B,and Mn of watermelon.Relative expression of nitrate reductase?Cla002787,Cla002791,and Cla023145?and nitrite reductase?Cla013062?genes was increased,promoting N assimilation.Mesophyll thickness and SPAD index?relative chlorophyll content?were also improved.Furthermore,pumpkin rootstock also enhanced the supply of zeatine riboside?ZR?and isopentenyl adenosine?i PA?in the leaves,promoting shoot growth.All these lead to improved plant growth and nitrogen use efficiency of pumpkin rootstock-grafted watermelon plants.2.The second experiment was focused on genome-wide expression profiling of leaves and roots of watermelon in response to low N.Plants were grown at 0.2 m M,4.5 m M,and 9 m M N for 14 days,the result showed that dry weight and photosynthetic assimilation at low N?0.2 m M?was reduced by 29% and 74% compared with high N?9 m M?.The photochemical activity?Fv/Fm?was also reduced from 0.78 at high N to 0.71 at low N.At low N,the concentration of N in the leaf,stem,and root of watermelon was reduced by 41%,51%,and 27% respectively,compared with high N.In leaf tissues of watermelon grown at low N,9,598 genes were differentially expressed compared with high N,these included 4,533?47.22%?up-regulated genes and 5,065?52.78%?down-regulated genes;similarly,in the root tissues 3,956 genes were differentially expressed compared with high N,these included 1,605?40.57%?up-regulated genes and 2,351?59.43%?downregulated genes.Our results suggest that leaf tissues are more sensitive to N deficiency compared with the root tissues.Functional annotation showed that the availability of N significantly affected 19 biological processes,8 cell component metabolic pathways,and 3 molecular functions in the leaves;and 13 biological processes,12 molecular functions,and 5 cell component metabolic pathways in the roots of watermelon.The low affinity and high affinity nitrate transporters,ammonium transporters,genes related with nitrogen assimilation,and chlorophyll and photosynthesis were expressed differentially to the availability of N.Three nitrate transporters?Cla010066,Cla009721,and Cla012765?substantially responded to low nitrate supply in the root and shoot tissues.A large number of transcription factors?1,365?were found involved in adaptation to low N availability.The major transcription factor families identified in this study includes MYB,AP2-EREBP,b HLH,C2H2 and NAC.The candidate genes identified in this study?Cla010066,Cla009721 and Cla012765?for nitrate uptake and transport can be targeted and utilized for further studies in watermelon breeding and improvement programs to improve N uptake and utilization efficiency.3.The third experiment was conducted to assess the role of melatonin to modify the root architecture and nitrogen uptake of watermelon.Different concentrations of melatonin?0 ?M,0.1 ?M,1 ?M,2.5 ?M,5 ?M,and 10 ?M?were applied to watermelon seedlings grown at 9.0 m M and 0.2 m M nitrogen?N?under hydroponic conditions.Application of melatonin altered the root growth,root morphology and nitrogen uptake of watermelon seedlings.Our results revealed that application of melatonin reduced the length of primary root.However,root length,root surface area,root volume,average root diameter,number of root forks and root crossings were increased.The N concentration of the shoot was not affected by the application of melatonin up to 5 ?M at 9 m M N whereas;at 10 ?M melatonin it was slightly reduced.Interestingly,at 0.2 m M N,the root and shoot N concentration was improved in melatonin-treated watermelon seedlings compared with non-treated seedlings.4.The fourth experiment was focused to investigate the response of watermelon seedlings to V stress,and the potential of melatonin to enhance V stress tolerance of watermelon.Melatonin pretreatment lowered leaf and stem V concentrations by reducing V transport from root to shoot.Melatonin pretreatment enhanced superoxide dismutase?SOD?and catalase?CAT?activities,and reduced the hydrogen peroxide?H₂O₂?and malondialdehyde?MDA?contents of watermelon seedlings,by regulating melatonin biosynthesis and gene expression for superoxide dismutase,peroxidase,ascorbate peroxidase,glutathione peroxidase and glutathione S-transferase.The reduced V concentration in the leaf and stem and improved antioxidant capacity of seedlings increased the relative chlorophyll content?SPAD index?,photosynthetic assimilation and plant growth compared with non-melatonin pretreated seedlings.5.The fifth experiment was aimed to assess the potential of bottle gourd and pumpkin rootstock to improve the vanadium stress tolerance of watermelon,and the mechanism involved.V application?50 mg/L?substantially reduced the growth of watermelon plants,however,grafting of watermelon onto bottle gourd and pumpkin rootstock improved V stress tolerance of watermelon by reducing the V concentration in leaf tissues,improving the relative chlorophyll content?SPAD index?and photosynthetic assimilation,up-regulating the expression of SOD?Cla008698,Cla0012125,Cla009820 and Cla001158?,glutathione S-transferase?Cla013224?and glutathione peroxidase?Cla021039?genes in the leaves,and enhancing the activities of antioxidant enzymes?SOD,CAT?.