Figure 2

Validation of T-DNA insertion in pap5-1 plants. A, Schematic representation of AtPAP5 (At1G52940); white boxes and solid lines represent exons and introns. T-DNA insertion is represented with a grey arrow and the solid arrows represent the primers used for genotyping and quantitative RT-qPCR. B, Location of the T-DNA insertion and homozygosity of pap5-1 was confirmed by PCR using the gDNA from wild-type and pap5-1 plants (M, 100 bp marker). A 30 cycle PCR reactions was performed with the primer pairs indicated. C, Relative expression of PAP5 transcripts in response to prolonged Pi starvation; For prolonged Pi starvation wild type and pap5-1 seedlings were germinated and grown in 0.5X MS media containing reduced Pi (0.25 mM). After seven days the seedlings were washed with sterile water and transferred to 0.5X MS with no Pi (0 mM). After 9 days the seedlings were harvested for gene expression analysis. Total RNA was extracted from wild-type and pap5-1 plants as described in Methods. Transcript levels of PAP5 was normalized to the expression of GAPDH in the same samples and expressed relative to the normalized transcript levels of Pi starved wild-type plants. The bars represent the mean and standard deviation from two independent experiments. Asterisks represents data sets significantly different from the wild-type data sets (P < 0.05 using one-tailed Student’s t-test).