| Acid-soil syndrome causes severe inhibition in plant growth for a long time.It has been astimated that more than 40% of the world's potentially arable soils are acidic.Low p H and Al toxicity are recognized as the major factors limiting crop production in acid soils,and both of them can lead to inhibit root elongation rapidly.Howerver,it remains unclear about the mechanisms of low p H and Al toxicity and resistance.In this study,Tandem Mass Tag(TMT)-based quantitative proteomic was used to identify and compare the differentially expressed proteins in response to low p H and Al toxicity.Moreover,60 transcription factors and 40 ubiquitine ligase proteins were overexpressed in Arabidopsis,and then we texted whether or not proteins were induced by Al in transgenic Arabidopsis.The phenotype of AT2G39100(L4975),At5G05910(L0945)and AT1G06170(L2853)transgenic Arabidopsis were significantly changed compared with wild type under Al conditions.Finally,the functional characterization of RING ubiquitine ligase gene AT2G39100(L4975)was analyzed.TMT-based quantitative proteomic was used to identify and compare the differentially expressed proteins in response to low p H and Al toxicity.Through identifying and comparing the differentially expressed proteins in response to low p H and Al toxicity,a total of 509 proteins were identified as Al-responsive,382 proteins were shown to be up-regulated and 127 were down-regulated.In addition,a total of 345 proteins were identified as low p H-responsive,231 proteins were shown to be up-regulated and 114 were down-regulated.There were 196 overlapped proteins responsing to low p H and Al.Besides,149 proteins responsed only to low p H,and 313 proteins responsed only to Al.The expression of responsive protein was regulated in post-transcription,translation and post-translational level under Al stress.Al-responsive proteins were enriched into 66 KEGG pathways and low p H-responsive proteins were enriched into 58 KEGG pathways.Based on functional analysis,we concluded that the adaptive response of Arabidopsis roots to low p H and Al stress might be related to activate the energy and primary metabolism,protein metabolism,signal transduction pathway,antioxidative system,DNA repaire system and transporters.The expression of cell wall and cytoskeleton-associated proteins were severely inhibited by Al,while the damage of the cell wall and cytoskeleton by low p H treatment was relatively small.Protein kinases and phosphatases were considered as the major signaling molecules in response to Al stress.However,there were little kinases and phosphatases proteins responsing to low p H stress.Kinase and phosphatase might not be the major signaling pathways in low p H stress.Therefore,there are many of same toxicity and resistance mechanisms between low p H and Al stress,and their own unique mechanisms also exist at the same time.A total of 60 transcription factor and 40 ubiquitin ligase genes were cloned from Columbia 4(Col 4),and then constructed into plant expression vector p EGAD-LUC.The recombinant vectors were transformed into Col 4 through Agrobacteriummediated dipping flower method.Thus,152 T3 transgenic lines were obtained by basta resistance screening,detection of LUC reporter and genome identification.In addition,327 transcription factor and 420 ubiquitin ligase transgenic seedlings were treated with 25 ?M Al and the expression of proteins were detected by Luminmeter.14 transcription factors protein had both a fold change of more than 1.5 and a P-value of less than 0.01,of which 6 proteins were shown to be up-regulated and 8 proteins were shown to be down-regulated.18 ubiquitin ligase proteins had both a fold change of more than 1.5 and a P-value of less than 0.01,of which 14 proteins were shown to be up-regulated and 4 proteins were shown to be down-regulated.The trend of Al response protein verified by western blot method was consistent with that of Luminometer.Three transgenic Arabidopisis plants with obvious phenotype compared with wild type were screened under Al stress.The relative root elongation of At2G39100(L4975)and AT1G06170(L2853)transgenic plants were significantly longer than that of wild type,whereas the relative root elongation of the At5G05910(L0945)transgenic plants was significantly shorter than that of wild type.L4975 protein may be involved in the repair of DNA in order to increase the Al resistance of transgenic Arabidopsis,and the L2853 protein may increase it by reducing the accumulation of callose at the apical root.The At ATRF1 gene located on chromosome 2 was composed with 6 exons and 5 introns,and encoded a 296 amino-acid predicted protein.Based on the analysis of the predicted domain,a conservative C3HC4 domain was presented at N terminal of At ATRF1.Transient expression of At ATRF1 in Arabidopsis protoplasts indicated that At ATRF1 protein localized to the nucleus.In transcription and translation levels,the expression of At ATRF1 was induced by Al.The At ATRF1-overexpressing transgenic plants displayed the improvement of Al tolerance.There was no significant difference in the expression of known Al tolerance genes(e.g.ALMT1,MATE,STOP1,STOP2,ALS1,ALS3,STAR1,and ALT2)between WT and At ATRF1-overexpressing plants with and without Al treatment.However,compared with WT plants,the expression of ATR exhibited a lower level in At ATRF1 transgenic plants under the conditions of normal and Al treatment.The results demonstrated that At ATRF1 regulated the transcriptional expression of At ATR in Al tolerance of Arabidopsis,and we hypothesized that At ATRF1 may interact and ubiquitinate the transcriptional factor of At ATR in nucleus.In conclusion,the results demonstrated a preliminary understanding of the molecular level regulation network between low p H and Al stress.The phenotype of AT2G39100(L4975),At5G05910(L0945)and AT1G06170(L2853)transgenic Arabidopsis were significantly changed compared with wild type under Al stress.Finally,we hypothesized that At ATRF1 may interact and ubiquitinate the transcriptional factor of At ATR in nucleus. |
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