Cloning Of¹O₂ Marker Genes Of Crofton Weed And Cotton,and Preliminary Functional Study Of¹O₂ Marker Gene SIB1 In Arabidopsis

1O2 has been proposed to trigger different stress responses of plant.At higher concentrations,they cause directly oxidative damage to plants.At lower concentrations1O2 may be perceived as signal molecules that participated in plant response to stress.However,so far,the mechanism of1O2 as a signaling molecule in different plant stresses is remained unclear.Previous research in our laboratory showed that TeA producing by Alternaria alternata(Fr.)Keissler is a photosynthetic inhibitor.TeA could block electron transport from QA to QB by binding to D1 protein.Recent research indicated that TeA could activate 1O2-mediated and EX-dependent signaling pathways in Arabidopsis seedlings.Additionally,TeA can kill many plants,such as crabgrass,crofton weed,Arabidopsis and so on.However,cotton and tobacco are highly tolerant to TeA.This indicates that TeA interacts with different plant species in different action ways.Although it was previously found that the occurrence of TeA induced plant diseases was caused by the activation of EX protein-dependent 1O2 signaling,however,what is the role of 1O2 signaling during the interaction between TeA and host plant crofton weed or resistant crop cotton?We found that normal cotton(Si Kang 1)with high gossypol level shows high insensitivity to TeA,but no glandular cotton(Su Xianwu 152)with extremely low gossypol level shows somhow sensitivity to TeA.This promotes us to think about a question:what is the role of gossypol as a plant protectant in the TeA activated EX protein-dependent and 1O2 mediated signaling pathway?To further probe the mechanism of EX protein-dependent 1O2 signaling in host plants and resistant plants,in this study the geological cloning technology,rapid chlorophyll a fluorescence kinetics,Imaging-PAM and real-time PCR were used to analyse different tolerance between glandular upland cotton and glandless upland cotton to TeA and the expression level of 1O2 marker genes.It is preliminarily clear that TeA could activate EX protein-dependent 1O2 signaling in host plants,leading to cell death and disease development.TeA also activated 1O2 signaling in resistant cotton plants,but gossypol reduced significantly the level of 1O2 generation and suppressed occurance of cell damage and disease.Firstly,the homologous sequence alignment gene cloning method was used to obtain 1O2 maker genes conserved regions of crofton weed such as AaAAA-ATPase and AaWRKY40?which are 728 bp and 470 bp.It provided a basis for qRT-PCR test.After different concentrations of TeA treatments,the data showed that in host plants,TeA can rapidly activate 1O2 signaling.The expression level of 1O2 marker genes is relative to the TeA concentration.The higher TeA concentration could induce a much higher level of 1O2 generation.At the same time,when the leves of crofton weed were infected in vivo with PDA plugs containing actively growing A.alternata,the expression of 1O2 marker genes was gradually increased.The above results indicated that TeA can rapidly activate 1O2 signaling while A.alternata activate 1O2 signaling slower.Our previous studies have found that normal cotton(Sikang 1)is highly tolerant to TeA,however,the Glandless Cotton(Su Xianwu 152)shows somehow sensitivity to TeA.Obviously,the mechanism of interaction between TeA and different plant species is different.In this study,chlorophyll fluorescence induction kinetics of glandular and glandless cotton plants after TeA treatment was conducted and the analysis of JIP-test were carried out.The results showed that the size of lesions between glandular and glandless cotton leaves is significantly different at TeA concentration of 5 mM,10 mM,20 mM and 40 mM.While the glandular and glandless cotton were treated with TeA at 0.25 mM,0.5 mM,1 mM and 2 mM,JIP-test results shows that:(1)J-step in OJIP curve of glandless cotton leaf discs increased after 3 h of treatment with 1 mM TeA.The J-step of the glandular cotton increased after 12 h with 1mM TeA treatment.There was significant difference between glandular and glandless cotton plants after 1 mM TeA treatment of 3 h.(2)When the glandless cotton was treated with 0.25 mM TeA,the J-step had risen.For glandular cotton,a remarkable increase in the J-step was observed at 1 mM TeA.(3)PIabs,?Eo and ?Eo of glandular and Glandless Cotton plants decreased significantly with the increase of concentration,however,the latter showed a faster decrease.The concentration with 50%inhibition for PIabs in glandular cotton is 1.422 mM,while that in Glandless Cotton is only 0.405 mM.As the concentration increased,in glandular and glandless cotton the ?Po values were not decreased significantly.All results suggested that glandular cotton was tolerant to TeA because of gossypol,which reduced the damage of TeA on photosynthetic activity.In order to further clarify the mechanism of TeA-induced 1O2 singnal in cotton,we used high concentration TeA to treat glandular and non-glandular cotton plants.Sequencing of the upland cotton genome provides the basis for cloning the 1O2 marker gene in cotton.The 1O2 marker genes in upland cotton are GhAAA-ATPase,GhVQ,GhWRKY1 and GhWRKY40.After treatment with TeA,the expression level of 1O2 marker genes of non-glandular cotton was significantly higher than that of glandular cotton.The expression of GhAAA-ATPase was the highest at 6 h,which was 3.2 times of glandular cotton.The expression level of GhVQ,GhWRKYl and GhWRKY40 was also the highest at 3 h,which was 10.8,2.2 and 2.4 times of glandular cotton,respectively.The qPCR results of A.alternata infected cotton also showed that the expression of 1O2 marker genes in non-glandular cotton(Su Xianwu 152)was significantly higher than that of glandular cotton(Sikang 1).For Arabidopsis thaliana,the gossypol pretreatment could reduce the expression level of 1O2 marker genes and suppressed the lesion size of TeA-treated leaves.Such results indicate that TeA can activate the 1O2-mediated EX protein-dependent signaling in resistant plant cotton,but gossypol reduced disease development due to hibiting 1O2 production level.In addition,we investigated the function of the 1O2 marker gene SIB1 in Arabidopsis infected by A.alternata.It was found that the expression of JA signal-related marker genes was significantly increased after infection with A.alternata in Col-0,however,the expression level was significantly decreased in SIB1 mutants.The absence of SIB1 reduced the sensitivity of A.thaliana to A.alternata.In conclusion,in this study two 1O2 marker genes(AaAAA-ATPase and AaWRKY40)in crofton weed and four 1O2 marker genes(GhAAA-ATPase,GhVQ,GhWRKY1 and GhWRKY40)in cotton were cloned.It is preliminarily proved that TeA can activate 1O2-mediated and EX-dependent signaling in host plants,leading to cell death and causing leaf lesions.In tolerant plant cotton,TeA also activates executer-dependent 1O2 signaling pathway.However,gossypol can reduce the production level of 1O2 and supress diseases development,resulting to plants resistant to disease.