| In the nature animals and plants unavoidable suffer from biotic stress and abiotic stress.The first stress reaction of plants to these stresses is the burst of reactive oxygen species(ROS).Singlet oxygen is the inevitable byproduct during the photosynthesis process,especially thought to be the main ROS produced in the chloroplasts.Because of singlet oxygen's high activity and short life,also produced simultaneously with others ROS,it is very difficult to study singlet oxygen's characters and functions.Until the emersion of condition mutant flu,make it possible to separate signlet oxygen from others ROS and do further researches.The previous researches showed that single oxygen was the only ROS produced in the chlroloplast of flu after dark to light cycle.The generation of singlet oxygen in flu caused growth retardation and cell death even the death of the whole plant.All these phenomenons were attributed to 1O2-mediated and EXECUTER1(EX 1)and EXECUTER2(EX2)dependent signaling in the flu,but not the toxicity of 1O2.The two proteins were encoded by cell nucleus and localized in chloroplasts.Tenuazonic acid(TeA)is secreted by Alternaria alternata(Fr.)Keissler isolated from an invasive plant Croftonweed,which is one of the three worst plant pathogenic fungi in nature.TeA is a novel photosynthesis inhibitor with several action sites,which mainly interrupts photosystem II(PSII)electron transport beyond QA(primary quinone acceptor)by competing with QB(secondary quinone acceptor)for QB-niche of the D1 protein.The initial production of TeA-induced ROS was restricted to chloroplasts and accompanied with a certain degree of chloroplast damage.TeA can kill quickly most monocotyledon and dicotyledon weeds at a low concentration and has a potential to be developed into a new bioherbicide.In Arabidopsis seedlings treated with TeA,1O2-mediated and EX-dependent signalling was activated as indicated by the rapid and transient up-regulation of 1O2-responsive genes in wild type,and a clear decrease in exlex2 mutant.Since TeA activated 1O2-mediated and EX-dependent signaling,the left question was that A.alternata might can do.In this research Arabidopsis flu and flu/exlex2 or Col-0 and exlex2 mature plants were treated with A.alternata in order to confirm weather the 1O2-meidated and EX-dependent signaling is involved in the disease development.Arabidopsis mutant flu and flulexlex2 were incubated with spores solution of A.alternata.After the treatment,flu and flu/exlex2 showed remarkable phenotypic difference.The result of TBD showed that cell death occured in flu after 48 h treatment,with the time increase,the blue zone namely disease spots constantly got bigger and deepener.While until 144 h after the treatment cell death emerged in flu/exlex2.The treatment induced up-regulation of 1O2-responsive genes in flu and flu/exlex2,but the 1O2-responsive genes rised remarkable higher in flu than in flu/exlex2.WhenA.thaliana plants were treated with spores solution of A.alternata,the spores needed to germinated firstly.Instead of spores solution,Arabidopsis plants were treated with the PDA plugs containing actively growing A.alternata hyphae.This treatment would skip the germination process.Arabidopsis mature plants treated with PDA plugs showed the same result relative to spores treatment.However,the latter caused Arabidopsis disease spots with a bigger size and earlier occurrence.The relative electrolyte leakage was the ratio of the value obtained in treated samples to control(without A alternata)samples.Electrolyte leakage was measured after Arabidopsis leaves were treated with A.alternata for 48 h.Electrolyte leakage showed a significant increase at the time poit,meaning the death of some cells have occurred.The relative electrolyte leakage in flu plants increased to 4.3 times compared with control,and increased to 1.4 times in flu/exlex2 plants.All these results show that 1O2-mediated and EX-dependent signaling was activated during disease development of Arabidopsis flu mature plants treated with A.alternata.Then Arabidopsis Col-0 and exlex2 plants were incubated with PDA plugs containing actively growing A.alternata hyphae.After the treatment,Col-0 and exlex2 showed remarkable phenotypic difference.The relative electrolyte leakage showed a significant increase after Arabidopsis leaves were treated with A.alternata for 48 h.It's suggested that a clear cell death have occurred.The value of relative electrolyte leakage in Col-0 plants is 3.4 times relative to control,and is 1.7 times of control in exlex2 plants.TBD staining experiments showed that cell death occured in Col-0 after 12 h treatment,with the time increase,the blue zone namely disease spots constantly got bigger and deepener.While 12 h after the treatment cell death occured in exlex2,but at particular moment the disease spots in Col-0 were bigger and deepener than exlex2's.The results of Imaging-PAM indicated that the injury of photosynthetic function of Col-0 plants had occurred after 12 h treatment,showing worse injury by increasing time of A.alternata treatment.Although the photosynthetic damage also emerged after 12 h treatment in mutant exlex2,disease symptom was lighter than Col-O's at specific time.The fluorescence parameters Fv/Fm and ETR showed the similar current.A.alternata treatment induced up-regulation of 1O2-responsive genes in Col-0 and exlex2,but the level of 1O2-responsive genes rised in Col-0 was remarkable higher than exlex2.And the H2O2-responsive gene maintained unchanged,showing no difference between Col-0 and exlex2 after the treatment.The results indicated that 1O2 play a central role during the disease development,not H2O2.It is concluded that that 1O2-mediated and EX-dependent signaling was activated by A.alternata during disease development of Arabidopsis Col-0mature plants.After Arabidopsis Col-0 and exlex2 plants were pre-treated under high light/low temperature stress(600 ?mol photons s-1 m-2,12 ?)for 24 h,plants were transfered to the growing condition(100 hmol photonss-1m-2,22 ?)for 12 h recovery.Subsequently,Col-0 and exlex2 plants were inocubated with PDA plugs containing actively growing A.alternata hyphae.Col-0 plants with pre-stressed revealed much smaller disease spots than the non-pre-stressed ones after A.alternata treatment.The relative electrolyte leakage showed a significant increase after Arabidopsis leaves were treated with A.altern.ata for 48 h.The value of relative electrolyte leakage in Col-0 plants without pre-stress is 3.4 times relative to control,and is 1.7 times of control in exlex2 plants without pre-stress.However,the relative electrolyte leakage in pre-stressed Col-0 and exlex2 plants is around 1.3 times of control.The up-regulation level of 1O2-responsive genes in pre-stressed Col-0 plants was remarkable decreased compared with the non-pre-stressed ones.Such results indicated that acclimation of Arabidopsis mature plants exposed to high light stress suppressed the expression of 1O2-mediated and EX-dependent maker genes induced by A.alternata.While in mutant exlex2,the 1O2-mediated and EX-dependent signaling could't be activated due to the lack of EX proteins.The pre-stressd couId't lead to the stress acclimation in exlex2.It is further demonstrated that 1O2-mediated and EX-dependent signaling is involved during disease development of Arabidopsis mature plants treated with A.alternata.Arabidopsis Col-0 and edsl were inocubated with PDA plugs containing actively growing A.alernata hyphae.After the treatment a remarkable phenotypic difference was found between Col-0 and edsl plants.The size of disease spots in edsl was remarkable bigger than Col-O's.TBD staining showed that cell death occured in Col-0 and edsl after 12 h treatment,but at particular moment the disease spots in edsl were bigger and deepener than Col-O's.The results of Imaging-PAM indicated that the injury of photosynthetic function of edsl and Col-0 plants had occurred after 12 h treatment,showing worse injury by increasing time of A.alternatatreatment.And disease symptom in Col-0 plants is lighter than eds plants at the indicated time.The fluorescence parameters Fv/Fm and ETR showed the similar current.The treatment induced up-regulation of 1O2-responsive genes in Col-0 and edsl,but the 1O2-responsive genes rised remarkable higher in edsl than in Col-0.And the H2O2-responsive gene maintained unchanged,showing no difference between Col-0 and edsl after the treatment.The results indicated that 1O2 play a central role during the disease development,not H2O2.The action mechanism of disease spots formed in edsl and edsl's role during the treatment is expected further studies.In conclusion,102-mediated and EX-dependent signaling is activated during the process of A.alternata causes disease of Arabidopsis mature plants.There might be a cross-talk between 1O2-mediated and EX-dependent signaling and EDS 1 mediated signaling. |
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