| Pseudostellaria heterophylla is the Caryophyllaceae herbaceos perennial,one of the Chinese herbal medicine with high medicinal value,mainly produced in FuJian,AnHui,GuiZhou,JiangSu,ShangDong provinces,etc.However,consecutive monoculture of this plant causes a series of problems that the growth of the overground and underground parts become deterioration,the root tuber can’t normally expand and the soil-borne disease aggravated.Consecutive monoculture leads to a serious decline in the yield and quality.Some researches suggested that the consecutive monoculture of medicinal plant R glutinosa could result in the imbalance of rhizosphere microbial communities,the increase of calcium concentration in the cytoplasm and then caused the differential expression of the calcium signal-related genes.Previous studies have shown that the calcium signal system participated in perceiving,conducting and magnifying processes of consecutive monoculture problems of R.glutinosa and other plants.Our previous study found that consecutive monoculture of Pseudostellaria heterophylla could also lead to the deterioration of the rhizosphere microenvironment,and especially the large increase of pathogenic fungus Fusarium oxysporum.So far,the researches on Pseudostellaria heterophylla on the molecular level were rarely reported because of the limitation of this plant’s genetic background information.Meanwhile,there were no reports revealing the relationships between the calcium signal system and the consecutive monoculture problems under the infection of soil-born pathogen,i.e.Fusarium oxysporum.Therefore,Fusarium oxysporum isolated from the consecutively monocultured soil and the "Zhengshen Ⅱ" radix pseudostellariae were used as the experimental materials.Tissue culture seedlings of radix pseudostellariae infected by Fusarium oxysporum were used for RNA-Sequencing at three different stages to study the responding mechanism to stress.Real-time quantitative PCR was used to verify the differential expression of genes from certain key metabolic pathways and calcium signal system.Besides,the changes in these calcium signal-related genes including CaM.CDPKs,CIPK,CBL were studied under the treatment with different concentrations of calcium channel blocker(i.e.heparin sodium(HS)and trifluoperazine).The results of RNA-Seq and qPCR showed that the key genes including the phenylalanine ammonia-lyase(PAL)and caffeoyl-CoA O-methyltransferase were up-regulated,while the cinnamate 4-hydroxylase gene(C4H),acetaldehyde dehydrogenase gene(ALDH)and the peroxidase gene were all down-regulated in the phenylalanine metabolism pathway and phenylpropanoid biosynthesis pathway,which catalyze the transformation of phenolic acids into aldehydes,alcohols and even lignin,It resulted in the accumulation of the phenolic acids in the plant which was strongly linked to the consecutive monoculture,this may also explain that why there were so many phenolic acids in the consecutive monoculture soil;However,the accumulation of glutathione and taurine,along with the decline of flavonoids suggested that radix pseudostellariae could initiate the innate immune systems to defense the infection of harmful fungi and also remove the excrescent reactive oxygen species(ROS).Besides,we found that the calcium signal-related genes such as CDPK,CML,CaM were up-regulated in plant-pathogen interaction pathway.The results from qPCR confirmed that CaM,CDPKs,CIPK,PLC and CBP genes in the calcium coding system were up-regulated,but Annexin,Glutamate receptor 3.3 in the calcium decoding system were down-regulated.Furthermore,the calcium signal-related genes of monocultured plants were up-regulated to some degree at different growth stages.We also found that the calcium channel blocker(heparin sodium(HS)and trifluoperazine(TFP))could obviously weaken the expression of the key genes CDPK,CDPK1,CDPK3 related to the calcium signal when the plants were infected by Fusarium oxysporum.Moreover,the inhibition rate became more distinct when the blocker’s concentration became higher.However,pathogen infection could lead to the hypersensitivity(HR)of the plant.A lot of genes related to the programmed cell death(PCD)were up-regulated,including Metacaspase,HSPs,Ascorbate peroxidase and PR,which might be the key factor of consecutive monoculture problem.In conclusion,radix pseudostellariae plant could precept and transfer the signal under the infection of pathogens,and then cause the cascade reaction,together with the differently expression of the calcium signal genes,the changes in expression of resistance genes,stress-inducible gene and innate immunity genes in monocropping system.The changes in these key genes led to the disorder of the secondary metabolism,the increase of the autotoxins,and finally result in the serious consecutive monoculture problems of radix pseudostellariae. |
