In cross sections of the root, PKS5P:GUS was mainly observed in phloem , which is consistent with previous findings , while the J3P:GUS signal was observed in epidermal cells, the cortex, phloem, and xylem parenchyma cells ; this expression pattern is similar to that of AHA2P: GUS . We also analyzed the tissue specific expression of PKS5 and J3 using quantitative real time PCR. Total RNA was extracted from roots, stems, rosette leaves, cauline leaves, flowers, and siliques of 40 d old Col 0 plants. Both PKS5 and J3 were constitutively expressed in all tissues with highest expression in reproductive and root tissues . To learn more about the interaction between PKS5 and J3, we determined the subcellular localization of the two proteins. The green fluorescent protein reporter was fused to both proteins at their N termini under the control of the 35S promoter, and the resulting plasmids were transformed into the Arabidopsis Col 0 genetic background. Transgenic plants in the T2 generation were tested for GFP localization using confocal microscopy. GFP J3 was detected at the cell membrane, in the cytoplasm, and in the nucleus ; however, no GFP PKS5 signal was detected in 100 35SP:GFP PKS5 transgenic lines.
We then fused yellow fluorescent protein to the C terminus of PKS5 under the control of a dexamethasone inducible promoter , and the T0070907 selleck chemicals YFP signal was analyzed in transgenic plants treated with 10 mM dexamethasone. As was found for GFP J3, PKS5 localized to the cell membrane, in the cytoplasm, and in the nucleus . To further analyze the subcellular localization of PKS5 and J3 in plant cells, we fused a 33FLAG tag at the N terminus of PKS5 under the control of a dexamethasone inducible promoter and 33FLAG tag at the N terminus of J3 driven by the 35S promoter. The resulting plasmids and 35SP:GFP J3 were transferred into their corresponding mutants and the mutant phenotypes were rescued by the transgenes . We then isolated nuclei, a plasma membrane enriched fraction, and a soluble fraction from the transgenic plants expressing 35SP:33FLAG J3 and DexP:33FLAG PKS5 and analyzed the immunoblots with anti FLAG antibody.
As shown in Figure 2Q and 2R, the tagged PKS5 and J3 proteins were detected Diosmetin in all three fractions. These results are consistent with our PKS5 YFP and GFP J3 results . Using the same protein samples, as expected, MITOGEN ACTIVATED PROTEIN KINASE3, the PM H ATPase, and histone H3 were found in the soluble, plasma membrane enriched and nuclear fractions, respectively. To further determine the purity of the phase partitioned membrane fractions, anti Arf1 and anti Sar1 antibodies were used to investigate the presence of endoplasmic reticulum and Golgi membranes in the plasma membrane enriched fraction. Both proteins were at undetectable levels in the plasma membrane enriched fraction.