| Heavy metal accumulation causes serious environmental problem,particularly in agricultural ecosystem which has deteriorative effects on yield and quality of crops.Individual copper(Cu)and chromium(Cr)effects have been investigated extensively in plants,however,co-contamination of Cu and Cr induced stress on Brassica napus L.is still unclear.Brassinosteroids(BRs)are a group of plant steroidal hormones which was first found in Brassica species,and it plays important roles in regulating plant development in response to stress condition.The current study was performed to investigate the physio-biochemical responses of two B.napus cultivars(’Zheda 622’ and ’ZS 758’)under co-contamination of Cu2+ and Cr6+,and the ameliorating role of BR under these metal stress treatments.We also used RNA-Seq method to analyze the expression of genes under BR-induced positive effects on plant growth under stress condition.The major results are as follows:1.In first experiment,interactive effects of Cu and Cr were studied in two B.napus cultivars(’Zheda 622’ and ’ZS 758’)in growth chamber.Results showed that the application of Cr was more toxic than Cu,and their combined stress had shown a significant adverse effect on plant growth.Biomass and photosynthetic pigments were decreased remarkably under all metal treatments.Individual treatments of Cu and Cr and their combination caused the accumulation of ROS and lipid peroxidation.Moreover,the activities of antioxidant enzymes and their mRNA transcription levels,such as catalase(CAT),ascorbate peroxidase(APX),glutathione reductase(GR),superoxide dismutase(SOD),and peroxidase(POD),were increased,especially when treated with Cr alone or under Cu+Cr combined treatment in both cultivars,except the CAT activity which was decreased in both leaf and root of sensitive cultivar ’Zheda 622’ as compared with their respective controls.Additionally,non-enzymatic antioxidants like reduced glutathione(GSH)showed a differential activity pattern in roots and leaves of both cultivars.More pronounced modification in chloroplast ultrastructure was observed in both cultivars under Cu+Cr treatment followed by Cr and Cu alone treatments.Furthermore,synergistic effects of Cu and Cr were prominent,that may be due to the enhanced metals uptake under combined treatment which suggest that Cr and Cu interaction is not competitive rather it is additive and genotypic dependent.2.Co-contamination of Cu and Cr to observe their effects on plant growth,photosynthetic parameters and specially the subcellular distribution of these metals in leaves and roots were investigated in the green house.The results showed that exposure to Cu and Cr cause decline in plant growth,including biomass and plant height.Significant decrease in pigment concentration and the photosynthetic activity[photosynthetic rate(Pn),stomatal conductance(Gs),and transpiration rate(E),maximal quantum yield of photosystem Ⅱ(Fv/Fm)]in leaves were also observed.Results of subcellular distribution of metals showed that Cu and Cr were predominantly distributed in cell wall and soluble fraction of roots and leaves.Moreover,Cu and Cr in cellular fractions showed a synergistic accumulation pattern under combined metal stress treatment.Both cultivars showed increased levels of reactive oxygen species(ROS),i.e.hydrogen peroxide(H2O2)and superoxide radical(O2·),and significant modulation in the activities of antioxidant enzymes(SOD,CAT,POD,APX)under Cu/Cr alone or their combined treatments.Similarly,expression levels of defense related genes,such as BnCat,BnApx,BnPrx,BnSod,were also generally up-regulated compared with control.Electron micrographs(TEM)of the mesophyll and root tip cells indicated prominent alterations both in cellular and organelle levels.Additionally,Cr was found to be more toxic than Cu but less than their combined effect,as revealed by enhanced production of oxidative stress and a reduction in biomass production and photosynthetic activity.The present results also suggest that cultivar ’ZS 758’is more resistant to Cu/Cr than ’Zheda 622’,due to better adapted metabolism and maintenance of structural integrity under metal stress.3.The effects of BR on the growth,physiology and biochemistry of B.napus seedlings under Cu and Cr stress were discussed.It was found that different concentrations of exogenous BR could promote the growth of B.napus plants by increasing the biomass and net photosynthetic rate,and 0.05 μM BR treatment had the most obvious effect on the growth of B.napus plants.In addition,single Cu,Cr and Cu+Cr treatment significantly inhibited the growth of the plants,showing the decreased biomass,but increased ROS(H2O2 and O2·)and MDA accumulation.However,exogenous application of BR under heavy metal stress could effectively alleviate the damage caused by Cu and Cr to B.napus plants.Compared with the heavy metal treatment,after application of BR,the fresh and dry weight of the plants increased significantly,and the accumulation of ROS and MDA decreased significantly.Furthermore,the activities of antioxidant enzymes(SOD,POD,CAT,and APX)were increased significantly when plants were applied with BR.These results suggested that exogenous application of BR can effectively enhance the plant tolerance to heavy metals and alleviate damage caused by heavy metal stress on B.napus seedlings.4.The gene expression patterns of B.napus under heavy metal and BR pre-treatment were investigated by transcriptome analysis.When treated with BR under Cr,Cu,and combined stress,RNA-Seq detected 1175,799,2437 and 2481,1208,1793 genes were up-regulated;1040,2479,1961 and 3596,1437,1246 genes were down-regulated in cultivars’Zheda 622’ and ’ZS 758’ respectively.These differentially expressed genes(DEGs)were enriched in MAPK signaling pathway-plant,plant hormones signal transduction,phenylpropanoid biosynthesis,plant-pathogen interaction,glutathione metabolism,flavonoid biosynthesis,tryptophan metabolism and so on.In cultivar ’Zheda 622’,total of 274 DEGs were found in the overlap regions of these three comparisons,which indicated these genes were differentially expressed at all treatments under BR application.Similarly in ’ZS 758’,the overlap region of these three comparisons under BR application had 250 DEGs.When analyzing the function of these DEGs,only 92 and 24 genes were selected which were all up-regulated in three comparision groups,and they are mostly involved in amino acid and lipid metabolism,transcriptional regulation and stress-related protein.Especially the genes about defensin-like protein,proline-rich protein,glutaredoxin,lipoxygenase,osmotin,transcription factor,which were related to detoxification,were up-regulated under BR treatment.indicating that BR could alleviate the stress damage through regulating the gene expression of some defense enzymes and transcription factors. |
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