Figure 4.

SOG1 Works in Conjunction with ATR to Promote Al-Dependent Stoppage of Root Growth.

(A) A sog1-7 atr-4 double mutant was grown for 7 d in the absence or presence of 1.25 mM AlCl₃ (pH 4.2) in a soaked gel environment in order to determine whether the combination of mutations is additive for Al tolerance. Mean ± sd values were determined from 30 seedlings. Asterisk indicates significance at P ≤ 0.01 when comparing Al-treated lines using the Tukey HSD test.

(B) SOG1 expression was found to be localized in part to the Arabidopsis root tip using a SOG1:GUS transgenic line grown for 7 d in either the absence or presence of 1.50 mM AlCl₃ (pH 4.2) in a soaked gel environment, after which seedlings were stained for GUS activity for 1 h. Al treatment resulted in loss of SOG1:GUS in Col-0 wild type but not in an atr loss-of-function mutant, indicating that Al-dependent changes in SOG1 levels are regulated by ATR likely as a part of ATR-dependent terminal differentiation. Bars = 50 μm.

(C) Full-length Arabidopsis ATR protein was produced using a baculovirus protein expression system. Approximately 100 ng of recombinant ATR protein was incubated with either 1 μg of MBP or MBP-SOG1 protein in the presence of [γ-³²P]ATP, after which samples were separated by SDS-PAGE and analyzed by autoradiography.

(D) Bacterially produced MBP-SOG1 protein was tested for its capability to physically interact with the promoter of one of SOG1’s predicted targets, BRCA1. Approximately 50 ng of MBP or MBP-SOG1 was incubated with radiolabeled BRCA1 promoter (−1 to −1500) using a standard EMSA approach. Analysis also included 50 ng of MBP-SOG1^R155G and MBP-SOG1^S206F, as well as MBP-SOG1 in the presence of increasing concentrations of unlabeled BRCA1 promoter. Following separation of samples using an agarose gel, results were examined using autoradiography.