| Aluminum toxicity is the main limiting factors of plant growth in acidic soils,aluminum ions have significant inhibitory effect of plant root growth,which affect the plant to absorb water and nutrients from soil,eventually the growth of the whole plant is affected.Rice has evolved a variety of aluminum resistant mechanisms in the process of long-term adaptation to acidic soil,therefore,the study on the mechanism of aluminum resistance in rice has a positive effect on understanding the resistance mechanism of rice as well as improving the acid soil adaptability of other crops.In this study,we conducted a large-scale screening of Kasalath mutagenesis library,and obtained an aluminum resistant mutant rail(resistant to aluminum 1)and three aluminum sensitive rice mutants.Using high-throughput sequencing technologies,we have cloned mutanted genes from ral1 and one sensitive mutant sal3(sensitive to aluminum 3),through phenotype analysis and gene function analysis,we found two different mechanisms of rice resistance to aluminum toxicity,the specific results of this study are as follows:(1)Constructed a Kasalath EMS mutagenesis library and figure out an efficient method to perform a large number of screening,after phenotype confirmation in M3 and M4 generation,finally we got an aluminum resistant mutant and three aluminum sensitive mutants.(2)ral1 showed a significant higher RRE under different aluminum concentrations compared with wild type,when grow in neutral soil conditions,root length of ral1 was shorter than wild type,but when grow in acid soil,ral1 showed comparable root length with WT.The expression of known aluminum resistant genes in rice did not change significantly in the ral1,using high-throughput sequencing and map-based cloning,the mutanted gene was cloned,RAL1 encods a 4-Coumarate:coenzyme A ligase enzyme 4 involved in benzene propane and lignin monomer synthesis pathway.RAL1 expressed in the root of the column and epidermal cells.The results of RNA-seq showed that the number of genes in ral1 was less affected by aluminum than in the wild-type,and the genes with significant change was concentrated in various metabolic pathways.(3)Aluminum accumulation is significantly reduced in root tip cell walls of ral1 compared with wild type,these can explain why ral1 showed enhanced aluminum resistance.Lignin content in root tip of ral1 was slightly lower,but the other two lignin monomer metabolic defective mutant c4h2 and cad2 did not showed short root and aluminum resistant phenotype,suggested that lignin was not involved in aluminum adsorption in the cell wall.Further experiments showed that matrix polysaccharide was not the cause of the difference aluminum adsorption in cell wall between ral1 and wild type.Substrates of RAL1 were increased significantly in the cell wall of ral1,the excess of p-coumaric acid and ferulic acid connected with hemicellulose by ester bond.Using feruloyl esterase to remove the combination of p-coumaric acid and ferulic acid with hemicellulose,the aluminum adsorption difference between ral1 and wild type disappeared,suggested that excess p-coumaric acid and ferulic acid modified to hemicellulose prevent aluminum binding to hemicellulose.(4)sal3 showed hypersensitive to both aluminum solution and acidic soil.ER staining showed that aluminum accumulated in root tip meristematic zones significantly and EdU fluorescence showed that cell proliferation was totally abolished in root tip meristematic zones,what’more,Evans blue staining showed that cells of root tip meristematic zone were significantly damaged after aluminum treatment,suggested that aluminum poisoning sal3 mainly concentrated in the root tip meristematic zones.The expression level of ART1 and its downstream genes did not change significantly in sal3.SAL3 encodes a nucler localization transcriptional factor belong to GRAS family,in yeast,N-terminal of SAL3 showed self-activation activity,suggested that SAL3 may regulated an ART1 independent aluminum resistance mechanism. |
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