ITRAQ-based Proteomics Profiling Of Aluminum Toxicity And Tolerance Responses In Duckweed(Lemna Minor L.)

In the present study,the widely distributed aquatic plant Lemna minor was chosen as the experimental material,the aluminum(Al)was chosen.as the stress factor,the response mechanisms of L.minor to Al stress were studied systematically using physiological and biochemical assays,iTRAQ technology,quantitative real time polymerase chain reaction(qRT-PCR)and western-blot technology.1.The accumulation of Al and its influence on the morphology,growth rate,reactive oxygen species(ROS),enzymatic and non-enzymatic protection systems,isoenzyme expression and osmoregulation substances were evaluated in L.minor exposed to 0,100,300,500 ?M Al for 15 d.The results as follows:(1)With the increase of Al treatment concentration,the content of Al in L.minor increased significantly.Meanwhile,the leaf appeared chlorosis,the contents of photosynthetic pigments and soluble protein were significantly reduced,the levels in the highest concentration groups were 72%(Chl a),67%(Chlb),84%(Car)and 37%of these in controls,respectively.However,the soluble sugar content was increased first and then decreased,the content in the 300 ?M Al treatment group reached the maximum,which was 2.4 times higher than the control group.(2)Histochemical staining showed that with the increase of Al treatment concentration,the accumulation of hydrogen peroxide(H2O2)and superoxide anion(O2·-)increased in L.minor fronds.Meanwhile,the balance of antioxidant enzyme system was broken,the activity of superoxide dismutase(SOD)was gradually decreased,and the activity of catalase(CAT)increased initially and decreased afterwards.In the highest concentration group,SOD activity reduced by 33%,CAT activity reduced by 44%.But peroxidase(POD)activity was induced to raise,the level in the highest concentration group was 1.5 times higher than the control.The isoenzymic spectrum analysis demonstrated that SOD,CAT and POD isoenzymic bands number did not change under Al stress.Meanwhile,SOD and CAT isozymes were down-regulated expression,POD isozymes were up-regulated expression with the increase of Al level.(3)The activities of key enzymes APX,glutathione reductase(GR)and the content of ascorbic acid(AsA)decreased first and then increased in AsA-GSH cycle under A1 treatment.In the highest concentration group,the activities of APX and GR were 1.7 times and 1.4 times higher than the control group,respectively,but the content of AsA was still lower than the control.With the increase of A1 treatment concentration,dehydroascorbate reductase(DHAR)activity was inhibited,the activity in the-highest concentration group was 78%compared with the control group.While the contents of dehydroascorbic acid(DHA)and reduced glutathione(GSH)were gradually increased,and the former was 1.5 times higher than the control in the highest A1 treatment group.The content of oxidized glutathione(GSSG)increased initially and decreased afterwards with the increase of stress.(4)After Al treatment,both the contents of non-protein thiols(NP-SH)and phytochelatins(PCs)observed to increase at first and then decrease.The peak values of NP-SH and PCs in L.minor were 1.5 times and 3.4 times higher than the control group when the concentration of Al was 300 ?M.The contents of NP-SH and PCs were slightly decreased in the highest Al treatment group,but still higher than the control group.2.The Al toxicity mechanisms at proteomic levels were evaluated by iTRAQ technology in L.minor exposed to 300?M Al for 15 d.The results showed:261 differential proteins including 99 up-regulated proteins and 162 down-regulated proteins were successfully identified and quantified through iTRAQ technology.The differential proteins were analyzed by COG classification and KEGG pathway enrichment analysis.The COG classification mainly classified the differential proteins into information storage and processing related proteins,cellular processes and signaling related proteins,metabolism related proteins and unknown function proteins.KEGG pathway enrichment analysis further confirmed that metabolism was the most affected by Al stress in L.minor.The level of photosynthetic proteins expression in energy metabolism decreased significantly,indicating that photosynthetic systems have been severely damaged by Al in L.minor.The mRNA levels and quantification of some proteins mapped in the photosynthesis were further analyzed by qRT-PCR and western blot,suggesting that the proteomic data are reproducible and reliable.Up-regulated proteins involved in antioxidative enzymes and OA synthesis related to carbohydrate and amino acids metabolisms might play a functional role in Al adaptation for L.minor.