Melatonin Reduces Cadmium And Aluminum Damage In Rapeseed And Induces The Germination Of Secondary Dormant Seeds Of Cabbage

Chapter 1:Seed dormancy is a prevailing condition in which seeds are unable to germinate,even under favorable environmental conditions.Harvested Brassica oleracea seeds are dormant and normally germinate（poorly）at 21°C.In 2004,scientists isolated3-methyl-2H-furo[2,3-c]pyran-2-one from the smoke of burning plant material.Afterward,an analogy such as 3-methyl-2H-furo[2,3-c]pyran-2-one was discovered,and these analogies were referred to as karrikins.This study investigated the connections between ethylene,nitric oxide（NO）,and karrikin 1（KAR1）in the dormancy release of secondary dormant B.oleracea seeds.NO and KAR1 were found to induce seed germination,and stimulated the production of ethylene and1-aminocyclopropane-1-carboxylic acid（ACC）,and both ethylene biosynthesis enzyme ACC oxidase（ACO）and ACC synthase（ACS）.In the presence of NO and KAR1,ACS and ACO activity reached maximum levels after 36 and 48 h,respectively.The inhibitor of ethylene 2,5-norbornadiene（NBD）had an adverse effect on B.oleracea seed germination（inhibiting nearly 50%of germination）in the presence of NO and KAR1.The benefits from NO and KAR1 in the germination of secondary dormant B.oleracea seeds were also associated with a marked increase in reactive oxygen species（ROS）(H₂O₂ and O^2-)and antioxidant enzyme activity at early germination stages.Catalase（CAT）and glutathione reductase（GR）activity increased 2 d and 4 d,respectively,after treatment,while no significant changes were observed in superoxide dismutase（SOD）activity under NO and KAR1 applications.An increase in H₂O₂ and O^2-levels were observed during the entire incubation period,which increasing ethylene production in the presence of NO and KAR1.Abscisic acid（ABA）contents decreased and gibberellic acid（GA）contents increased in the presence of NO and KAR1.Gene expression studies were carried out with seven ethylene biosynthesis ACC synthases（ACS）genes,two ethylene receptors（ETR）genes and one ACO gene.Our results provide more evidence for the involvement of ethylene in inducing seed germination in the presence of NO and KAR1.Three out of seven ethylene biosynthesis genes（BOACS7,BOACS9 and BOACS11）,two ethylene receptors（BOETR1 and BOETR2）and one ACO gene（BOACO1）were up regulated in the presence of NO and KAR1.Consequently,ACS activity,ACO activity and the expression of different ethylene related genes increased,modified the ROS level,antioxidant enzyme activity,and ethylene biosynthesis pathway and successfully removed（nearly 98%）of the seed dormancy of secondary dormant B.olereace seeds after 7 days of NO and KAR1 application.Chapter 2:The relationship between ethylene and cyanide（HCN）and karrikin 1 （KAR1）in dormancy release was studied in secondary dormant B.oleracea seeds.Freshly harvested seeds of B.oleracea usually have poor germination potential.Karrikin1（KAR1）and cyanide（HCN）are able to stimulate seed germination.However,the stimulatory effects of these two chemicals depend on the activation of the ethylene biosynthesis pathway and on ethylene perception.In this study,KAR1 and HCN application increased the activity of ethylene and of two ethylene biosynthesis enzymes,ACC synthase（ACS）and ACC oxidase（ACO）.KAR1 and HCN collectively promoted the accumulation of 1 aminocyclopropane-1-carboxylic acid（ACC）.In the presence of NO（nitric oxide）and KAR1,ACS and ACO activities reached their maximum levels after 36 and 42 h,respectively.Ethylene inhibitors suppressed seed germination by approximately 55%,whereas the respiratory inhibitors SHAM and Na N3 inhibited seed germination by 5–10%in the presence of HCN and KAR1.KAR1 and HCN collectively reduced the abscisic acid（ABA）content in seeds,increased the gibberellic acid（GA）content and released seed dormancy.The expression of ethylene biosynthesis genes and ethylene receptor genes（BOACO1,BOACS1,BOACS3,BOACS4,BOACS5,BOACS7,BOACS9,BOACS11,BOETR1 and BOETR2）provided further evidence of the involvement of ethylene in KAR1 and HCN-induced germination.BOACO1,BOACS1,BOACS5,BOACS7,BOACS9,BOACS11,BOETR1 and BOETR2 genes were up regulated in the presence of KAR1 and HCN,while the remaining genes were down regulated.The expression of various ethylene biosynthesis and ethylene receptor genes suggested functional diversification and variations in seed sensitivity in the presence of KAR1 and HCN.Therefore,in the current study,KAR1 and HCN application effectively induced the germination of B.oleracea seeds（approximately 97%germination rate）after six days by modifying the ethylene biosynthetic pathway.Chapter 3:Melatonin has emerged as an essential molecule in plants,due to its role in defense against metal toxicity.Aluminium（Al）and cadmium（Cd）toxicity inhibit rapeseed（B.napus）seedling growth.In this study,we applied different doses of melatonin（50 and 100μM）to alleviate Al（25μM）and Cd（25μM）stress in rapeseed seedlings.Results show that Al and Cd caused toxicity in rapeseed seedling,as evidenced by a decrease in height,biomass and antioxidant enzyme activity.Melatonin increased the expression of melatonin biosynthesis-related B.napus genes for caffeic acid O-methyl transferase（BnCOMT）under Al and Cd stress.The genes BnCOMT-1,BnCOMT-5 and BnCOMT-8 showed up-regulated expression,while BnCOMT-4 and BnCOMT-6 were down-regulated during incubation in water.Melatonin application increased the germination rate,shoot length,root length,fresh and dry weight of seedlings.Melatonin supplementation under Al and Cd stress increased superoxide dismutase,catalase,peroxidase,ascorbate peroxidase,proline,chlorophyll and anthocyanin content,as well as photosynthesis rate.Both Cd and Al treatments significantly increased hydrogen peroxide and malondialdehyde levels in rapeseed seedlings,which were strictly counterbalanced by melatonin.Analysis of Cd and Al in different subcellular compartments showed that melatonin enhanced cell wall and soluble fractions,but reduced the vacuolar and organelle fractions in Al-and Cd-treated seedlings.These results suggest that melatonin-induced improvements in antioxidant potential,biomass,photosynthesis rate and successive Cd and Al sequestration play a pivotal role in plant tolerance to Al and Cd stress.This mechanism may have potential implications in safe food production.In the first two chapters,I learned how to induce germination in dormant seeds successfully,based on the successful germination results of dormant seeds using Kar1,NO,and HCN.Then,I move ahead and studied impact of heavy metals on seed germination in the presence of melatonin.In order to relieve heavy metal stress,melatonin showed important results.