| Nearly 50%of arable land is acid soil,where aluminum toxicity is one of the most restrict factors affecting the yield of crops.In the acid soil,free aluminum ions seriously inhibit the growth and development of plant roots.Current studies revealed that aluminum stress could induce the secretion of organic acids to reduce the aluminum toxicity to plants and recent research also showed that auxin,ethylene and cytokinin are involved in the aluminum stress inhibition on Arabidopsis root elongation.In order to isolate new regulatory factors involved in aluminum stress regulated root elongation,we use TAA1:GFP transgenic line under aluminum treatment to get GFP-labeled root transiton zone cells with flow cytometry technique and isolate differentially expressed genes through RNAseq.Finally,we screened two genes differentially expressed in the transition zone,TEZ2 and TEZ3.We got the mutants tez2-1,tez2-2,tez3-1 and tez3-2 by using the CRISPR/Cas9 gene editing technology and analyzed their phenotype.We found that when tez2 mutants were exposed to 6 ?M AlCl3,they showed Al resistance phenotype and displayed a greatly reduced root growth inhibition.To explore the molecular mechanism of TEZ2 involved in the Al resistance process,we studied the possible effect of auxin and ethylene on TEZ2 expression in the transition zone under Al stress.The results showed auxin could enhance Al-induced TEZ2 expression in the transition zone,but not ethylene.To elucidate whether the Al resistance phenotype of tez2 is related to organic acid,we detected the expression of MATE?ALMT1 and STOP1 in tez2 mutants.The results showed that these three genes are all downregulated,which is not consistent with the Al-resistance phenotype of tez2,implying that Al-resistance of TEZ2 is not due to this organic acid regulatory system.To further implore the Al-resistance mechanism of TEZ2,we screened a TEZ2 interacting protein ACO1 through yeast two-hybrid system.ACO1 encodes an aconitase that can catalyze the conversion of citrate to isocitrate through a cis-aconitate intermediate.The function loss of TEZ2 may affect the transformation of the citric acid to isocitric acid.In order to confirm this speculation,we measured the content of citric acid in the tez2 mutant using high performance liquid chromatography.The results showed that the citric acid content in tez2 mutant was higher than that in wild type.The increased citric acid may be the cause of Al resistance in tez2.tez3 mutants did not display Al-resistance phenotype when treated with 6 ?M AlCl3.But phenotypic analyses revealed there are fewer lateral roots in tez3 mutants.Expression analyses showed TEZ3 is mainly expressed in the pericycle and stele.Yeast two-hybrid experiments showed that ARF7 and LBDs do not interact with TEZ3.However LBDs expression are downregulated in tez3 mutant.More evidence is needed to elucidate molecular mechanism of TEZ3 on the regulation of LBDs and on the development of lateral root.In summary,this thesis tries to illustrate the molecular mechanisms of TEZ2 participating in aluminum resistance in plants,and how TEZ3 regulates lateral root development. |
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