| Dark septate endophytes(DSEs)are soil fungi,which prevalently colonize in the roots of plants growing in the heavy metal stressed environments.Our previous researches revealed that inoculation of DSE enhanced the biomass of plant and reduced the Cd concentrations of plant tissues.But it is still unclear why the tolerance of maize(Zea mays)to Cd can be enhanced by DSE(Exophiala pisciphila)colonization.In the present study,maize was inoculated with DSE and cultured under different concentrations of Cd for 30 days.Then activities of antioxidants and antioxidative enzymes in maize leaves were analysed.The impacts of DSE on the Cd distribution in subcellular level and their varied chemical forms in maize were investigated.And the expressions of the genes ZIP,PCS and MTP were also tested by fluorogenic quantitative PCR to reveal the cytological,physiological and molecular mechanism of the maize tolerance to Cd when the plant is inoculated by DSE.Results are as follows1.DSE can successfully colonize in maize roots and develop typical DSE structures after 30 days.With the treatments of different concentrations Cd(0,10,50,100 mg/kg),the colonization intensities of DSE in maize roots ranged from 19%to 31%on the 30th day after inoculation.The biomass of plant shoots was significantly increased(1.86-fold)in comparison of the control under the treatment of 100 mg/kg Cd2.With the increase of Cd concentration in the culture medium,the Cd accumulation into plant roots and shoots was also increased.However,the majority of Cd was accumulated in the root both in inoculated and non-inoculated maize.This trend was not altered by DSE inoculation,while the concentration of Cd in the maize was reduced by inoculating DSE3.Maizes exposure to Cd resulted in oxidative burst(increased malondialdehyde contents),which was accompanied by decreased membrane stability.However,the increasing level of glutathione(GSH)and the activity of antioxidant enzymes like superoxide dismutase(SOD),catalase(CAT)and peroxidase(POD)suggested that all these parameters were synergistic with Cd induced oxidative stress.DSE alleviated oxidative stress through enhancing the production of GSH as well as upregulating the antioxidant enzyme activities4.Inoculation of DSE remarkably increased Cd proportion in the cell walls of roots and leaves,but decreased the proportion of soluble Cd after 30 days compared to control.The results also showed that the Cd proportion of poisonous forms in maize roots and leaves were significantly reduced.The Cd proportion of low poisonous or non-toxic forms was remarkably increased.These results suggested that the toxic free Cd(Cd2+)in maize roots would be transformed into non-toxic or weak poisonous state of Cd when inoculated with DSE,thus the toxicity of Cd to maize was reduced.5.Molecular responses to cadmium(Cd)stress were studied in inoculated and non-inoculated maize.Real-time reverse transcriptase-polymerase chain reaction(qRT-PCR)showed that genes involved in the pathways of Cd detoxification varied in response to DSE inoculation and Cd stress.Expression of the gene encoding a zinc-regulated transporter,iron-regulated transporter-like protein(ZIP),was reduced strongly in roots and leaves by Cd stress and DSE inoculation.However,expressions of a gene encoding a phytochelatin synthase(PCS)and another one encoding metal tolerance protein(MTP)were both increased strongly in roots and leaves by Cd stress and DSE inoculation.Results of the present study indicated that both the biomass and antioxidant system of maize were enhanced by colonization of DSE.Subcellular distribution and chemical forms of Cd in maize were changed by DSE colonization under Cd stress.The expressions of genes involved in heavy metal tolerance in maize were also changed by DSE inoculation.These results provided some arguments for the mechanism that DSE colonization enhanced Cd tolerance of maize from physiological,cytological and molecular levels. |
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