Preliminary Study On Mechanisms Of Reducing Heavy Metal Contents In Nicotiana Tabacum Leaves Conferred By Piriformospora Indica

Endophytic fungi which exist in plants,can coevolute with host,and form symbiotic relationship with their hosts.As reported,endophytic fungi have positive effect on increasing plant resistance against various abiotic and biotic stresses.Piriformospora indica,isolated from Indian Thar desert,performs the coincident influences on plant.Nicotiana tabacum is one of China's important economic crops and model plants.It is reported that the heavy metal contents in P.indica root-colonized tobacco leaves were significantly lower than that of uncolonized ones,while the heavy metal contents in the roots were just the opposite.On the basis of the research,this paper carried on the mechanisms of reducing heavy metal contents in Nicotiana tabacum leaves conferred by P.indica,including the enrichment of P.indica hyphal under heavy metals stress,fixtion of heavy metals in P.indica-colonized roots,antioxidant levels,and heavy metal-related genes induced by P.indica under heavy metal stresses.The main results were as follows:1 The enrichment of the fungal mycelia on heavy metalsThe enrichment of heavy metals by mycelia is conducted to investigate.The results showed that P.indica still can grow on high concentrations of 4 mg·L-1 Cd,30 mg·L-1 Cr and 5 mg·L-1 Pb respectively.Using microwave digestion and graphite furnace atomic absorption spectrometry to analyze the heavy metal absorbtion capability of P.indica.The Cd,Cr and Pb contents in the P.indica mycelia were all significantly higher compared to the controls.The results showed that P.indica performed excellent tolerance against high concentrations of heavy metals,and enrich function on heavy metal Cd,Cr and Pb.2 The effects of P.indica on agronomic traits of tobacco under Cd,Cr and Pb stressesUnder Cd,Cr and Pb stresses,the agronomic traits(the leaf dry weight,stem circumference,maximum leaf length and leaf width)of tobacco which were colonized by P.indica were significantly better than that of uncolonized tobacco,indicating that P.indica can enhance the tobacco resistance against heavy metal stress.3 The effects of P.indica on Cd,Cr and Pb contents in tobaccoHydroponic experiment results showed that there is nearly significant differences of Cd,Cr and Pb contents in the roots and leaves of colonized tobacco without any exposure in heavy metal.However,after treated with 1 mg·L-1 of Cd,5 mg·L-1 of Cr and 5 mg·L-1 of Pb for two weeks,the Cd,Cr and Pb contents were significantly lower in colonized tobacco leaves compared with the controls,while it's just the opposite in the roots.4 The research of fixtion of P.indica on heavy metals in tobacco root system by Raman spectroscopy technologyVia the confocal micro Raman spectroscopy,we can see that the root epidermis and cortex cell of control plants were severely damaged under 5mg·L-1 Cr stresses,and abundant black blocks flocked in the column cells,while the root cell morphology of colonized tobacco was kept normal and closely packed.The black blocks distributed randomly in the epidermis,cortex and column cells.In order to understand what the black blocks were in the roots,we used the Raman spectral technology to confirm that they were Cr-complex.The results showed that P.indica colonization was beneficial to fix heavy metal in the tobacco root,reducing the toxicity of heavy metal,blocking heavy metal transportation to the ground.5 The effects of P.indica on antioxidative in tobacco leaves under heavy metal stressesUnder Cd,Cr and Pb stresses,the AsA,GSH contents and CAT,SOD activities in the leaves colonized by P.indica were significantly higher than those in uncolonized ones.Collecting the antioxidants changes in P.indica mycelia,we concluded that P.indica can improve the resistance of tobacco against stresses,enhance the ability of tobacco roots to heavy metals.6 The effects of P.indica on the expression level of heavy metal-related genes in tobacco under heavy metal stressesAnalyze the expression level of genes phytochelatins synthesis(Hmt1,Hmt2,CAD1),MT2 and pathogenesis-related proteins(PR2 and Chitinase)between plants colonized and uncolonized by P.indica.The results showed that P.indica could make the root cells produce a series of structural changes which are favorable to improve tolerance of root system,for example,cell walls became thicker obviously,cell color became darker,substance deposition occurred on cell walls;P.indica could reduce Cd,Cr and Pb contents in tobacco leaves in hydroponic experiments;the AsA,GSH contents and CAT,SOD activities in the leaves colonized by P.indica were significantly higher than that in uncolonized ones;qRT-PCR analysis showed that the expression level of genes Hmtl,Hmt2,CAD1,MT2,PR2 and Chitinase in colonized plants was 4.04,1.68,2.02,1.98,4.48,1.87 under Cd stress,and was 2.82,1.52,1.55,1.25,2.51,1.59 folds and 2.65,2.01,1.42,1.32,2.17,1.53 folds respectively under Cr and Pb stresses.