Consistent with a previous study , these proteins were detected in total membrane and soluble fractions . Together, these data demonstrate that the gene expression and protein localization of J3 and PKS5 overlap during Arabidopsis development. j3 Mutants Have Increased Sensitivity to Salt in Alkaline Conditions To determine if J3 and PKS5 have similar functions, we obtained two J3 T DNA insertion lines from TAIR . The positions of the T DNA insertions are shown in Figure 3A. Homozygous T DNA lines, j3 1 and j3 2, were identified using T DNA left border primers and J3 gene specific primers. To determine if expression of J3 is abolished in these two lines, total RNA was extracted from 10 d old Col 0, pks5 1 , and j3 1 and j3 2 seedlings and analyzed using RNA gel blots. The expression of J3 could not be detected in j3 1 and j3 2; however, it is present in Col 0 and pks5 1 . We have previously shown that PKS5 is a negative regulator of the PM H ATPase and that PKS5 loss offunction mutants are resistant to high pH in the external medium . When we monitored j3 mutant seedling growth in response to alkaline pH, no consistent, significant difference was detected between Col 0 and mutant plants.
In nature, soil alkalinity is often associated with increased soil salinity partly due to application of fertilizers and irrigation water Wortmannin selleck . Alkaline conditions significantly increased the salt sensitivity of Arabidopsis . To determine if the j3 mutants have increased sensitivity to salinity in alkaline conditions, 5 d old seedlings of Col 0, j3 1, and j3 2 grown on Murashige and Skoog 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 difference in growth was detected between Col 0 and the j3 mutants on medium at pH 5.8 . On medium at pH 7.7 with 75 mM NaCl , root elongation in the j3 mutants was reduced compared with that of Col 0, and this reduction in growth was even more pronounced at pH 8.1 in the presence of 75 mM NaCl . By contrast, when we grew Col 0 and pks5 1 seedlings on the same media, primary root elongation in pks5 1 seedlings was less sensitive to NaCl in alkaline conditions relative to the growth of wild type plants .
This result is consistent with our previous finding that pks5 1 is more tolerant to alkaline pH than Col 0 . When we amlodipine tested the sensitivity of the j3 1 transgenic plants expressing 35SP:J3 to salinity in alkaline conditions, we found the mutant phenotype was rescued by the transgene . J3 Positively Regulates PM H ATPase Activity Because J3 interacts with PKS5 and loss of function j3 and pks5 1 mutants have opposite responses to salinity in alkaline conditions, we determined if J3 also has an effect on PM H ATPase activity.