Physiological Mechanism Of Melatonin And Zinc Oxide Nanoparticles In Alleviating The Cobalt Stress In Brassica Napus

Cobalt（Co）,a hazardous metal,has a detrimental effect on agricultural crops.Most of the studies related to Co toxicity in plants focused on physiological and biochemical attributes with little information at molecular level.The excessive Co accumulation in soil-plant system severely impairs the crop yield.Melatonin（MT）,phytohormone,facilitates abiotic stress tolerance.Recently,MT has been shown as a pleiotropic molecule with several physiological actions in plants such as growth traits,seed germination,root functioning and photosynthesis.Nanotechnology is an emerging field in agriculture due to its stress mitigate functionalities and key roles in handling the environmental stresses.Therefore we investigated the protective roles of MT and/or ZnO NPs in attenuating the Co toxicity in Brassica napus.For that,we used two cultivars of B.napus cvs.ZS 758（metal-tolerant）and Zheda 622（metal-susceptible）to apprehend MT and/or ZnO NPs cross-talks at physio-biochemical,cellular,and molecular levels against Co stress.The main findings are as follows:1.The present study was aimed to investigate the protective mechanisms of exogenously applied MT（0,50 and 100μM）under Co（0,100,200 and 300μM）stress by focusing on morpho-physiological,biochemical,and cellular characterizations of B.napus plants.Cobalt（300μM）alone drastically inhibited the stomatal opening,plant height（45%）,leaf area（30.4%）,free amino acid content（139.06%）,relative electrolyte leakage（109.81%）,and total soluble sugars content（71%）,compared with the control.The exogenous supply of MT notably minimized the oxidative damage,lipid peroxidation and maintained the membrane integrity under Co-toxicity by restricting the overproduction of ROS markers(H₂O₂and O₂^·?)and MDA in leaves and roots,respectively.Melatonin significantly enhanced the activities of ROS-scavenging antioxidant enzymes,secondary metabolism-related phenylalanine ammonia lyase（PAL）,polyphenol oxidase（PPO）,stress-responsive genes（heat shock protein-HSP-90,methyl transferase-MT）,and regulated the Co-transporters,especially in roots.These findings indicated that an exogenous supply of MT improved the plant morphology,photosynthetic apparatus,osmotic adjustments,and antioxidant defense systems by enhancing the Co-detoxification in B.napus plants.2.In this study,an attempt has been made at the transcriptome level to assess the molecular changes in B.napus.Oilseed rape is one of the candidate plants that can absorb a considerable quantity of toxic metals from the soil.We also performed large-scale transcriptome sequencing of B.napus plants under Co+MT using the Illumina sequencing technology.Twelve c DNA libraries constructed from m RNAs of control,Co and Co+MT leaf for ZS 758（metal-tolerant）and Zheda 622（metal-susceptible）were sequenced by Illumina technology.The sequence of read assembly displayed total clean reads as 44.86,44.99,44.69 Mb and 44.90,44.99 and 44.87 Mb,total clean reads ratio as 89.36%,89.61%,89.02%and 89.44%,86.68%,87.52%for CK,Co and Co+MT in ZS 758 and Zheda 622 cultivars,respectively.On average,total raw reads（50.20/50.74 Mb）,total clean reads（44.84/44.92 Mb）,total clean base（6.72/6.73Gb）,and clean read ratio（89.33%/88.55%）were observed in ZS 758/Zheda 622 cultivars,respectively.After clean filtering,clean reads are mapped to reference genome.DGE and TPA techniques were adopted to uncover the response of leaf genome of B.napus（Zheda 622 and ZS758）cultivars.In Zheda 622,the total number of up-regulated DEGs were 6517,7915,and 24,while down-regulated DEGs were 6551,9553,and 110 under CK vs Co,CK vs Co+MT,and Co vs Co+MT treatments when CK and Co were taken as constants,respectively.In ZS 758,the total number of up-regulated DEGs were 7548,5192,and 79,while down-regulated DEGs were 9297,7674,and 339 under CK vs Co,CK vs Co+MT,and Co vs Co+MT treatments when CK and Co were taken as constants,respectively.Some of the DEGs identified may be candidates for future research aimed at detecting Co bioaccumulation-responsive genes and will be useful for understanding the molecular mechanisms of Co tolerance in the leaves of B.napus in relation MT.3.This study sought to ascertain the role of ZnO NPs and MT in mitigating the phytotoxicity induced by Co stress.Root length（47%）,entire plant height（51%）,photochemical efficiency（46%）,net photosynthetic rate（56%）,and stomatal opening were considerably declined under alone Co（300μM）in comparison to the control.This study revealed that ZnO NPs+MT significantly improved the plant biomass,photosynthetic efficacy,and plant growth regulation in B.napus under Co stress.ROS(H₂O₂（42%/35%）and O₂^·?（27%/39%）)and MDA（27%/18%）accumulation was lowered by the ZnO NPs and MT in leaf/root,respectively.More significantly,compared to their alone treatments,ZnO NPs+MT greatly decreased the adverse effects of Co by lowering its bio-accumulation and conferred stability to plant ultra-cellular structures.Additionally,ZnO NPs and/or MT treatments exhibited a higher accretion of nutritional contents and antioxidant defense enzymes.Collectively,we revealed the first findings to validate the alleviation of Co toxicity in B.napus seedlings through alone and collective influence of ZnO NPs and MT.