Study On The Relationship Between TeA,¹O₂ Signaling And Cell Death During Necrotrophic Fungus Alternaria Alternata-Induced Disease Development Of Arabidopsis Mature Plants

In the natural environment,when the growth of plants suffers from biotic or abiotic stress,the first reaction of plants to these stresses is the burst of reactive oxygen species(ROS).Singlet oxygen(1O2)is an inevitable by-product during the photosynthesis process,which is especially thought to be the main ROS produced in the chloroplasts.However,in the past,there are fewer studies on 1O2 than that on other ROS because of its high activity,extremely short life span and the difficulty in separating it from other ROS.Till 18 years ago it was found that mutant flu could produce 1O2 alone in plant cells after simple conversion from dark to light treatment,it was possible to study 1O2 alone.Tenuazonic acid(TeA)can be secreted by Alternaria alternata(Fr.)Keissler isolated from an invasive plant Crofton weed,which is one of the three worst pathogenic plant fungi in nature.TeA is a toxin of herbicidal activity so it can be developed as a biological herbicide.It is a novel photosynthesis inhibitor with several action sites,which mainly interrupts electron transport from QA(primary quinone acceptor)to QB(secondary quinone acceptor)by combining with D1 protein in the photosystem ?(PS?),causing over-energization.Then the initial production of TeA-induced ROS is restricted to chloroplasts,further accompanied with a certain degree of chloroplast damage.Moreover,previous studies show TeA could activate the EXECUTER-dependent 1O2 signaling in Arabidopsis thaliana seedlings by,so it was speculated that A.alternata might also be able to activate the 1O2 signal in Arabidopsis thaliana mature plants by secreting toxin TeA.Under this precondition,in this research Arabidopsis wild-type mature plants Col-0 were infected by A.alternata wild-type(New)(WT),TeA-deficient mutant HP001(MU),mutant binding to TeA(MU+TeA)and TeA in order to investigate the relationship between TeA production and the1O2 signaling,and disease development during the interaction between A.alternata and Arabidopsis mature plants.The results of ultra-high performance liquid chromatography-mass spectrometry(UPLC-MS)showed that TeA content in the wild type is 4.2 times that in the mutant.And the concentration of TeA in the leaf cells after wild type infection was more than that of the mutant.The wild type and mutant of A.alternata were used to infect Arabidopsis Col-0 mature plants,The fluorescent quantitative analysis of1O2 marker genes showed that WT treatment caused up-regulation of1O2 marker genes in Col-0 starting after 6h and reaching peak at 12h,and resistance gene reaching peak at 6h.The scope and speed of1O2 marker genes and resistance gene expression level rising in WT treatment were remarkably wider and higher than that in MU treatment.In addition,the rising scope and speed of their expression level after adding 0.2 ?g TeA to MU-infected Arabidopsis were also significantly wider and higher than that in MU and TeA treatment alone both after 6h and 12h.Meanwhile,there was no significant difference in the expression level of H2O2-labeled genes between the four treatments and the expression level was not high,which indicated that the effect of 102 was the main factor in the process of infection.Lesion observation showed that the lesion formed by the infection of the WT was significantly larger than that of the MU.Specifically,the former was 19.6 times the size of the latter.The lesion in Arabidopsis formed by MU+TeA infection is 3 times larger than that by MU.The relative electric conductivity(REC)showed a significant increase after Arabidopsis leaves were treated with WT,MU and MU+TeA for 48h.It suggested that a clear cell death had occurred.REC of the WT infection site was 6 times that of the MU one.REC of Arabidopsis infested by MU+TeA was also higher than that by MU alone after 48h.In summary,A.alternata induced Arabidopsis mature plants to secrete TeA which entered plant cells to activate rather1O2 signal than H2O2 signal and to further promote disease development.However,the1O2 signaling mechanism to promote disease development was still not clear Especially that if 1O2 signal had interaction with Salicylic acid(SA)signal and Jasmonic acid(ET/JA)signal needed to be further investigated.Increased hormone synthesis is a common response of plants to pathogen infection and is also the basis for plants to produce resistance to saprophytic attack.It is generally believed that SA signal is responsible for positive regulation of parasitic fungi and facultative parasitic fungi resistance,and ET/JA signal is responsible for positively regulating saprophytic resistance,so elevated SA increased the sensitivity of plants to saprophytic bacteria.It has also been reported that these three hormone signals can make a positive contribution to plant immune rot fungi,i.e.SA induces early resistance to infection,and ET/JA is responsible for late resistance.Yet,many studies have suggested that the function mechanism of SA and ET/JA was very complex during interaction between different pathogenic bacteria and plants.This paper also explores content change of SA and JA and its relationship with cell death in the A.alternata-induced process of Arabidopsis in order to further investigate if there were cross-talks between 1O2 signal and SA and JA signal.The result of fluorescent quantitative analysis showed that WT and MU treatment induced rising expression level of SA-related genes in Col-0,but its rising speed was significantly lower than that of1O2 marker genes.When Arabidopsis which had been applied into exogenous 3-methyl salicylic acid for 24h was inoculated to WT,the rising amplitude and rate of1O2 marker genes were lower than that of the control group,but the expression level of resistance gene and SA-related gene increased significantly higher than that of the control group.The lesion of Arabidopsis sprayed by 3-methyl salicylic acid and then infected by WT for two days was significantly smaller than that of the control group,which was 1.5 times the size of the former according to statistics.The results above indicated that exogenous SA treatment could restrain1O2 gene expression but increase resistance gene expression level.Therefore,a preliminary conclusion could be reached that during A.alternata-induced disease development of Arabidopsis which was secreted by pathogenic bacteria could later induce the expression of SA gene level,but SA would antagonize against1O2 signal.It needs further study on specific mechanism.Similarly,the fluorescent quantitative analysis also showed that both the WT and MU treatments caused up-regulation of JA-related genes in Col-0 starting after 6h and reaching peak at 12h,which was similar to the rising tendency of1O2 marker gene,but the rising range and rate of JA-related genes level in WT were remarkably larger and higher than that in MU.The up-regulation range and rate of JA-related genes in MU-infected Arabidopsis after adding 0.2 ?g TeA were significantly larger and higher than those in MU and TeA treatment separately both after 6h and 12h.These results preliminarily indicated that A.alternata-induced infection process of Arabidopsis can also activate the expression of JA genes and help secrete TeA to further promote disease development,but mechanism of cross-talks between JA and1O2 signal was still unknown.In conclusion,A.alternata induced mature Arabidopsis plants to secrete TeA entering plant cells to activate1O2 signaling pathway and to finally promote disease development.Meanwhile,JA and 1O2 signal had a same action direction,but SA and 1O2 signal antagonized against each other.