To do this, we screened a tilling mutant pool and isolated three pks5 point mutation alleles. Tilling mutants were generated in the Col er105 genetic background , hereafter referred to as the wild type. We backcrossed the mutants three times into Col 0. These mutations are distributed throughout the protein, including in the N terminal kinase domain and in the C terminal regulatory domain . In the pks5 3 mutant, the Ser at position 317 in the FISL motif was mutated to Leu; in the pks5 4 mutant, the Ala at position 168 in the kinase activation loop was mutated to Val; while in the pks5 6 mutant, the Gly at position 219 in the kinase domain was mutated to Ser . Both the kinase activation loop and the FISL motif have been shown to be important for PKS activity . We first tested whether these mutations affect PKS5 activity. PKS5 cDNAs were amplified from Col 0 and pks5 mutants and cloned into the pQE30 vector containing a HIS tag. The fusion proteins were purified from E.
coli using His affinity chromatography, and the purified PKS5 proteins were used in kinase assays. PKS5 has been shown to be an active kinase in both auto and trans phosphorylation mg132 kinase inhibitor . When compared with the activity of the wild type PKS5 protein, recombinant PKS5 3 and PKS5 4 proteins were more active; however, PKS5 6 was less active in both autophosphorylation and Myelin Basic Protein phosphorylation . Next we tested the NaCl sensitivity of the pks5 mutants at alkaline pH. Five day old wild type, pks5 3, pks5 4, and pks5 6 seedlings grown on medium at pH 5.8 were transferred to medium at pH 5.8, pH 7.7 with 75 mM NaCl, or pH 8.1 with 75 mM NaCl. No significant growth differences were detected between the wild type and pks5 mutants on medium at pH 5.8 . On medium at pH 7.7 with NaCl, root growth of pks5 3 and pks5 4 was significantly reduced compared with the wild type . Root elongation in the pks5 3 and pks5 4 mutants was reduced compared with that in wild type, and this reduction in growth was even more pronounced at pH 8.
1 in the presence of 75mM NaCl . However, the root growth of pks5 6 was significantly greater than the growth of the wild type . These results demonstrate that PKS5 activity negatively correlates with root growth on media with salt at alkaline pH. Our previous data suggested that PKS5 is a negative regulator of the PM H ATPase . To further demonstrate the link between PKS5 kinase and PM H ATPase activities, plasma membrane Asarylaldehyde vesicles were isolated from leaves of Col 0, wild type, pks5 1, pks5 3, pks5 4, and pks5 6 plants treated with or without 250mMNaCl, andPMH ATPase activity was measured. Without NaCl treatment, Col 0, the wild type, and the mutants had similar levels of PM H ATPase activity, and salt stress increased their activity but to different levels .