Figure 4.

T-DNA insertions and gene expression in 5ptase13 mutant lines. A, T-DNA insertions in the 5PTase13 gene. Exons are shown as dark gray boxes; light gray arrows indicate primers used to amplify the LB of the T-DNA; dark gray arrows indicate positions of gene-specific primers. RB, Right border. B, PCR performed with gene-specific and LB primers confirms that both lines are homozygous. Gene-specific primers that flank the T-DNA insertion (primers 13-1for [F] and 13-1rev [R]) in conjunction with the T-DNA LB primer amplify 0.4- and 2.3-kb fragments in 5ptase13-1 mutants, indicating the presence of two LB sequences in proximity to the 5PTase13 gene. C and D, Expression of genes in 5ptase13 and wild-type seedlings. Total RNA (1–2 μg) was isolated, and semiquantitative RT-PCR was performed with the indicated primers (Supplemental Table S4). C, Verification of the loss of 5PTase13 expression in 2-week-old leaves from soil-grown plants. D, SnRK1 and 5PTase expression from 7-d-old wild-type and 5ptase13 (13-1 and 13-2) seedlings grown on 0.5× MS salts and 0.8% agar in the dark. The experiment was independently repeated two times.