Functional Analysis Of AtMYB50 And AtMYB61 Transcriptional Factors Which Are Implicated In The Regulation Of Heavy Metals, Low Phosphorus And Osmotic Response In Arabidopsis

We have undertaken a systematic reverse genetic approach to study functions of R2R3-MYB genes in Arabidopsis, particularly in terms of defence response. Here, we describe the functional,characterization of AtMYB50 and AtMYB61 together based on the extended degree of sequence similarity especially within their highly conserved MYB DNA-binding domains. In this study, the focus is on the involvement of AtMYB50 and AtMYB61 genes in the abiotic stress of lead (Pb), low phosphorus (LP) and osmotic. The T-DNA insertion mutants atmyb50, atmyb61-1 and atmyb61-2 were confirmed to be function deficiency mutants, which showed enhanced tolerance to Pb(â…¡), LP and osmotic stress compared with the wild type.AtMYB50 and AtMYB61 genes were strongly repressed by Pb(â…¡) treatment in both short-term and long-term inductions. Decreased Pb(â…¡) contents in AtMYB50 and AtMYB61 knockout plants relative to wild type were detected when subjected to Pb(II) treatment. It may be, at least in part, a result of increased expression level of AtPDR12 gene, which is considered as a pump located in the cell membrane have the ability to exclude Pb(â…¡) and/or Pb(â…¡)-containing toxic compounds from the cytoplasm. However, the expression level of GSH1, a gene involved in GSH synthesis to chelate heavy metals and sequester them into inactive organelles, was not changed, suggesting enhanced Pb(â…¡) resistance of AtMYB50 and AtMYB 61-knockout mutants was glutathione (GSH)-independent. This implied that these two R2R3 MYB genes regulated Pb(â…¡) tolerance through a GSH-independent but AtPDR12-mediated pathway. Also, atpdr12atmyb50 and atpdr12atmyb61 double mutants were costructed to further test this mechanism, appearance similarity coupled to alike Pb(â…¡) content with the atpdr12 mutant indicates that AtMYB50 and AtMYB61 proteins may act as up-stream TFs in regulating Pb response. Also, increased resistance of atpdr50atmyb61 double mutant from phenotype as well as decreased Pb content relative to atmyb50 and atmyb61 single mutant when subjected to Pb(â…¡) treatment suggests that these two proteins may work independently in Pb(â…¡) response. These results imply that knockout of AtMYB50 and AtMYB61 genes lead to enhanced Pb(â…¡) tolerance coupled with lower Pb(II) accumulation, which may be useful for the development of crop plants that grow in heavy metal contaminated soil. Interestingly, both atmyb50 and atmyb61 plants were neither CdCl2, CuSO4, CaCl2, MnCl2, AgNO3, FeSO4, FeCl3, NiSO4, ZnSO4, CoCl2 nor H2O2 resistant, which suggests that AtMYB50 and AtMYB61 may specificly implicated in Pb tolerance. Take together, the mutant appearance and their Pb(II) contents, as well as the identification of the AtPDR12 and GSH1 expression levels, and characterastics of the double mutants atmyb50atpdrl2 and atmyb61atpdr12, suggest that AtMYB50 and AtMYB61 transcription factors may participate in regulation of the AtPDR12 pump to exclude Pb(â…¡) and/or Pb(â…¡)-containing toxic compounds from cytoplasm. However, it is still unclear whether they directly or an indirectly regulates the expression of AtPDR12.AtMYB50 and AtMYB61-knockout mutants also exhibit insensitive to low phosphorus stress. Results indicated that P contents in the mutants were not changed significantly, while anthocyanidin contents decreased obviously. However, both WT and mutant displayed increased root hair numbers in low phosphorus condition though the later showed much longer primary roots. LP-related gene expression analyse suggested that AtPTl, AtACP5 and AtPAPl were varied significantly in the mutant under LP condition. Enhanced low phosphuros resistance of AtMYB50 and AtMYB61-knockout mutants may be assaciated with optimized in vivo hormone synthesis.In addition, we also found atmyb50 and atmyb61 mutants displayed enhanced osmotic and drought tolerance. In terms of osmotic stress, we utilized NaCl, KC1, LiCl and Mannitol to test the phenotype. Results indicated that all mutants showed increased resistance to these osmotic mediators, no matter in seed germination ratio or in seedling volumn. Finally, we confirmed the increased tolerance of atmyb50 and atmyb61 mutants to drought stress in realative to WT. Thus, we conferred that the enhanced resistance of atmyb50 and atmyb61 to drought stress may be closely related to osmotic stress.The innovations of this thesis can be summarized as follows:1. MYB TFs is the biggest gene family in Arabidopsis, which is maily involved in the regulation of plant growth and development and defence responses. However, little is known about their roles in the rugulation of heavey metal resistance. Our results suggest that AtMYB50 and AtMYB61 play important roles in heavy metals (particularly Pb) resistance.2. We chosed two R2R3 MYB genes AtMYB50 and AtMYB61, which had the most close relationship in the whole MYB family. Our results indicated that these two factors really possess many similar functions, which may be a result of sequence similarity.