Mechanisms Of Plant Immunity Triggered By Copper Ions In Arabidopsis

Copper,one of the essential micronutrients in all the living organisms,is an important cofactor for many enzymes,such as cytochrome c and copper-zinc superoxide dismutase.Copper deficiency causes organisms growth delay or even death.In plant,copper ions participate in photosynthesis,electron transportion,ethylene recognition and other processes,affecting plant’s growth and developmemt.Acting as heavy metal ions,copper ions are toxic to microorganisms,which inhibite the enzyme proteins’activity and the germination of fungal spore.In agricultural production,copper formulations are widely used to protect plant from various plant diseases,such as Bordeaux mixture.Bordeaux mixture has been applied for nearly 200 years in agriculture and plays an important role in controling plant diseases.Although there are pathogenic microorganisms being resistant to copper ions,copper formulations have effective suppression on plant diseases.The phenomenon suggests that an unknown mechanism may be exist except heavy metal toxicity.Based on the fact that cadmium ions can trigger plant innate immunity,we proposed the hypothesis that copper ion is an elicitor.And further research highlights the defense response and underling mechanisms triggered by copper ions.We performed our experiments in the interaction system between model plant Arabidopsis thaliana and Pseudomonas syringae pv tomato DC3000（DC3000）to investigate the innate immunity triggered by copper ions.Main results are as followes.（1）The application of CuSO₄ whose concentraton is safe to DC3000（100μM or as low as 10 nM）could enhance the disease resistance of Arabidopsis to DC3000.Copper ion is the key factor in enhancing disease resistance by comparing CuSO₄,CuCl₂ and MgSO₄.（2）Copper ions can activate a serious of physiological resistance responses,including MAP kinase protein phosphorylation,accumulation of reactive oxygen species,callose deposition,and up-regulation of disease related genes.And the disease resistance activated by copper ions is dependent on the salicylate and ethylene signaling pathways.（3）RNA-seq analysis was performed at 2 and 24 hours post treatment with CuSO₄ in Col-0.And the compared analysis of the genes expression profiles with flg22（correspoding to22 ammino acids located in the ammino terminus conserved part in the flagellin protein）and elf26（the elongation factor EF-Tu）revealed the undering mechanisms.There are 517differentially expressed genes（DEGs）identified same with flg22 or elf26,and 370 of them are up-regulated by copper ions,flg22 and elf26.These results indicated that the innate immunity triggered by copper ions was similar to PAMP-triggered immunity（PTI）.（4）A number of ethylene biosynthases genes are up-regulated by CuSO₄,including ACS2,ACS6,ACS7,ACS8 and ACS11.The conversion of SAM to ACC catalyzed by ACS protein enzyme is the rate-limiting step,so the ACS is the key factor in the ethylene biosynthesis.The ethylene production was measured with gas chromatograph.CuSO₄triggered the rapid production of ethylene within 30 minites in Arabidopsis,and the rate of ethylene production peaked within 2 hours.The ethylene accumulation significantly decreased in acs8 mutant,and disease resistance is also compromised.However,there is no significant difference in accumulation of ethylene triggered by copper ions in acs2-1/acs6-1and acs1-1 acs2-1 acs6-1 acs4-1 acs5-2 acs9-1 acs7-1 acs11-1（acs1/2/6/4/5/9/7/11）octuple mutant at 2 hours post treatment.These results indicated that disease resistance triggered by copper ions was relied on ACS8.（5）In order to explore the molecular mechanisms of ACS8 induced by copper ions,the promoter of ACS8 was analyzed by transient expression in Nicotiana benthamiana and trangenic expression in Arabisopsis.We have identified the copper resposne element（CuRE）which was located at the region between-1155 and-901 in the promoter of ACS8.With the aid of cis-elements forcast website,we found that CuRE cis-element is necessary in response to copper ions for ACS8.Similar results were also achieved in transgenic Arabidopsis.So we can conclude that CuRE is necessary for expression of ACS8 in response to copper ions.（6）To investigate the upstream mecahnisms of Arabidopsis percepting and transporting copper ions,we solo silenced COPT1,2,3,4,5 gene in Arabidopsis by means of RNA interfering technology.Disease resistance triggered by copper ions is compromised in the COPT4-RNAi transgenic plants.The defense responses such as up-regulation of PR1 and ERF1 and callose deposition are compromised in COPT4-RNAi transgenic plants compared to the wild type triggered by copper ions.All these results indicated that the copper ions triggered plant immunity is dependent on COPT4.We constructed the transgenic Arabidopsis expressing GUS gene driven by native promoter of COPT4 to explore the tissue specific expression pattern.The chemical staining results showed that COPT4 mainly expressed in the vascular bundle of Arabidopsis root,steam and leaves.In summary,our research demostrated that copper ion acted as an elicitor and elicited disease resistance by activating salicylate-and ethylene-dependent signaling pathways.Except inhibiting growth of pathogens,copper formulations confer plant resisitance to variety diseases by activating plant innate immunity.And the theretical conclusion revealed the molecular mechanisms of copper formulations’durability and broad spectrum.The rapid emmition of ethylene is essential for copper triggered disease resistance,which is dependent on the up-regulation of ACS8 by binding to the CuRE cis-element in the promoter region.This conclusion provides a theoretical basis for the side effects of copper formulations on defoliation of fruit trees and the damage to young fruits.We also tried to highlight how the Arabidopsis sensing coppper ions and converting it to disease resisitance signal,and plant immunity triggered by copper ions may be dependent on COPT4,which needs further research.