Effects Of Cations On Cross-talk Between Ethylene And Auxin

Plants need different metal ions including Cu, Zn, Mo, H, Mn et al. for growth. But superfluous ions are harmful to plants. Plants mainly rely on "cationic/H+" to get rid of the redundant ions. H+-ATPase hydrolysis provides energy to drive the whole process. The activity of H+-ATPase is very important for maintaining ions balance under stress in plant cells.Plant hormone auxin and ethylene can response many stress in environment. Moreover, large exogenous ions could effect plant hormone. But there are not specific discusses about how auxin and ethylene to answer ionic stress. We use Atane, Atern and double mutant AtanelAtern for material, research the effect of cation on cross-talk between ethylene and auxin by using the technology of RT-PCR, hybridization, BIFC et al..We identify the homozygous from the series of T-DNA insertion mutants of auxin transport and ethylene biosynthesis which got from Arabidopsis Biological Resource Center, obtain homozygous mutants named Atane (Arabidopsis thaliana Auxin transport mutant not sensitive to ethylene) and Atern (Arabidopsis thaliana gene for Ethylene Response to NaCl). Useing hybridization technique to get homozygous double mutant AtanelAtern. After searching related materials we found that the expression of AtANE and AtERN would be strongly changed under NaCl stress.In contrast to AtERN decrease, the expression of AtANE increase by RT-PCR test under the treatment of 150 mM NaCl, suggesting that the expression of AtANE decrease and AtERN increase in transcription level under Na+ stress.The expression of AtANE and AtERN decline under 65 mM CaCl2 stress, suggesting that the expression of AtANE and AtERN decline in transcription level under Ca2+ stress.We constructed two vectors(AtANE::GUS,AtERN::GUS). After dyeing, we found that both AtANE and AtERN highly expressed from seedling to mature plants. But the expression of AtANE decreased and AtERN increased by treatment with 150 mM NaCl. The expression of AtANE and AtERN decline under 65 mM CaCl2 stress. The result is identical RT-PCR.Observation of the differences of phenotype under ionic stress showed the germination and root growth of Atane and Atern were greatly inhibited when treated with 150 mM NaCl. Atane and Atern were also inhibited when treated with 65 mM CaCl2. But there isn't significantly difference between wild type plants (WT) and mutants. AtANE and AtERN have less effect when after 65 mM CaCl2 treatments.H+-ATPase is very important for preserving balance in plant cells. Assay of H+-ATPase activity showed that the treatment of 150 mM NaCl increased the H+-ATPase activity in Atane (22.9%) and in Atern (32.9%). Under the treatment of 65 mM CaCl2, the H+-ATPase activity in Atane increased 38.5% and in Atern increased 116%. Determination of H+ concentration by pH fluorescent probe marking, the H+ concentration in Atane and Atern promoted by thr treatment with 150 mM NaCl and 65 mM CaCl2. We conjectured that AtANE and AtERN is helpful for extruding the excessive ion through improving H+-ATPase activity, in turn, plant cells improve a relatively stable internal environment.In order to explore the relationship between AtANE and AtERN, we construct some recombination vectors for Yeast two-hybrid. The result showed that AtERN interacting with AtANE. And we also construct some recombination vectors for BIFC. There was yellow fluorescence (interaction indicator) in protoplasts of WT plants transforming with pSPYCE-AtANE vector and pSPYNE-AtERN vector. There experimental results showed that AtANE connecting with AtERN in protein level.In summary, AtANE and AtERN have the relationship, the expression of AtANE depending on the ethylene synthesis. AtANE and AtERN participate in response of plants treat with Na+ and Ca2+. AtANE and AtERN improve plant tolerence to iones stress through H+-ATPase activity.