| Heavy metal pollution in soils is a worldwide environmental problem,and it’s especially true for the unreclaimed mine tailing sites,with a high content of heavy metals,low level of macronutrients and acidity in abandoned substrates,which are the major constraints to restoration.While,it is considered a scientific hot spot using the combined bioremediation of plants and their root-associated endophytes,as a safe and feasible alternative to be applied in metal-contaminated soils.Our previous studies find that dark septate endophytes(DSEs),one of the most common fungal colonizers of roots,significantly improve the tolerance of plants to heavy metal stress and promote the phytoremediation of heavy metal contaminated soils.Accordingly,ammuculating evidence supports that DSEs with the morphological characteristics of dark and septate hyphae and the formation of microsclerotia in plant roots,prevalently colonize in the roots of plants growing in mining area,and exhibit the strong heavy metal accumulation capability and tolerance,but there is little knowledge on the mechanisms against metal stress.In addition,the formation of heavy metal veins is usually accompanied by sulfide,and sulfur(S),which is an essential elementand plays a very important role in the adaptation of various organisms to heavy metal stress.In this thesis,DSE strain(Exophiala pisciphila,H93),isolated from the roots of Arundinella bengalensis growing in Huize ancient Pb/Zn slag heap,Yunnan,southwestern China,were cultured under the cadmium stress(EC50:111.2 mg/L Cd)with three different sulfur concentration supplements(low sulfur(2 mg/L),normal sulfur(40 mg/L)and high sulfur(600 mg/L)),and H93 cultured under with normal sulfur without Cd was also evaluated as positive control.The aims of this study were to investigate the the roles of S in the tolerance of DSE to heavy metal stress under different S regimes and to reveal the cytological,physiological and molecular mechanisms of sulfur when the DSE were stressed with Cd.The main results were list as follows:1.With the increasing sulfur concentration supplements(ie.sulfur increased from 2 mg/L to 40 mg/L and 600 mg/L),the more S was uptake and accumulated in the hyphae of DSE,while Cd concentrations were significantly reduced in DSE hyphae,and the biomass was increased accordingly.In comparison with the low sulfur supplement(2 mg/L),the dry weight of H93 hyphae was increased significantly with the addition of sulfur nutrition.It was noteworthy that the biomass of H93 hyphae was decreased at high sulfur(600 mg/L)compared with normal sulfur(40 mg/L),but was still higher than that of low sulfur(2 mg/L),Therefore,moderate sulfur nutrition plays an important role in alleviating Cd toxicity and promoting H93 growth and development.2.S enhanced the adaptability of H93 to Cd stress by promoting the synthesis of antioxidants,antioxidant enzymes and sulfides.The contents of malondialdehyde(MDA)and oxygen free radical(OFR)was decreased,while GSH,MT,phytochelatins(PCs),non-protein thiols(NPT)and total sulfhydryl compounds was increased in H93 hyphae with the increasing sulfur concentration supplements under EC50 Cd stress.These bioaugmented substances were identified as effective antidotes to heavy metal poisoning,suggesting that S enhanced the tolerance of H93 to Cd stress by affecting the S-related substances.3.Data from the subcellular distribution of Cd in H93 showed that Cd was mainly distributed in the cell wall and soluble components(ie.heavy metal detoxification area),while it was less in the organelles(ie.metal-sensitive factions,MSF).Moreover,a large amount of Cd was accumulated in the cell wall when low sulfur was supplemented.With the increase of sulfur concentrations under EC50 Cd stress,the concentrations of Cd in cell soluble fraction was also increased,which was consistent with the increase of the Cd binding ability in cells under the elevated sulfur concentration supplements.At the same time,the chemical forms of Cd in H93 were also assessed and found that the water extractable Cd,NaCl extractable Cd and acetic acid extraction Cd mainly consisted of Cd accumulated in H93.Thereinto,the highest proportion was NaCl extractable Cd,followed by water extractable Cd.With the increasing sulfur concentration supplements,the proportion of NaCl extractable Cd,i.e.the pectate and protein-bound Cd,was increased.Moreover,the proportions of both acetic acid and hydrochloric acid extractable Cd were positively correlated with the increasing sulfur concentration supplements,which was considered as the lowest metal mobility compared with the other Cd chemical forms.These results suggested that sulfur relieved the toxicity of Cd stress by altering the chemical forms and subcellular distribution of Cd in H93.4.Based on the transcriptional data of H93 under EC50 Cd stress,eight genes related to sulfur metabolism and heavy metal stress were selected,including metallothionein(MT),glutathione S-transferase(GST),sulfate assimilation(biosynthetic process),SRP-dependent cotranslational protein targeting to membrane and superoxide dismutase(SOD)encoding genes.Data from real-time reverse transcriptase-polymerase chain reaction(qRT-PCR)showed that the distinct expression of six genes to Cd stress under three different sulfur concentration supplements was observed,except no apparent change for one GST encoding gene and one SOD encoding gene.And even different encoding genes of the same protein family,we laso noted the different response to the heavy metal and sulfur concentration supplements.For example,compared with the positive control under the normal S nitrition without Cd stress,the increasing sulfur concentration supplements under EC50 Cd stress(ie.sulfur increased from 2 mg/L to 40 mg/L and 600 mg/L)significantly up-regulated the expression of MT encoding genes,but did not significantly affect the expressions of one GST encoding gene and one SOD encoding gene,comparing with the expressions of the other GST and SOD encoding genes regulated by both the heavy metal Cd stress and sulfur concentrations.Compared with the positive control,SOD encoding gene was also significantly up-regulated under normal MMN medium(30 mg/L Mg2+),while it was significantly down-regulated under the higher Mg2+ concentration(450 mg/L).At the same time,sulfate assimilation(biosynthetic process)encoding gene,sulfate transporter,and SRP-dependent cotranslational protein targeting to membrane were also regulated by the sulfur concentration supplements,and.The maximum expression of the first two genes occurred at normal sulfur concentration(40 mg/L),but the sulfate synthesis encoding gene was down-regulated under the higher sulfur concentration and higher Mg2+ concentration(600 mg/L,450 mg/L)with the positive control,while the expression of sulfate transporter encoding gene reached the maximum at the normal sulfur concentration(40 mg/L)and high Mg2+ concentration(450 mg/L).The results showed that the regulated expression of genes related to sulfur metabolism and heavy metal stress contributed to Cd tolerance of H93 to Cd stress.These results showed that different sulfur concentration supplements had a significant effect on the tolerance of H93 to Cd stress.In general,the tolerance of H93 to Cd was also enhanced with the increasing sulfur concentration supplements,which involved the cytological,physiological and molecular mechanisms related to sulfur metabolism and heavy metal stress.In this thesis,the preliminary study laid a foundation for the enhancing Cd tolerance of fungi by the increasing sulfur supplements. |
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