Figure 4.

Distribution of GUN4 in Lysed and Fractionated Chloroplasts That Were Either Fed or Not Fed with PPIX.

(A) Immunoblot analysis of lysed and fractionated chloroplasts using anti-GUN4 antibodies. Chloroplasts were purified from gun4-1, F191A-14, R211A-2.2, cch, gun5, gun5-101, and cs. Purified intact chloroplasts (200 μg) were either fed (+) or not fed (−) with 20 μM PPIX. Chloroplasts were then fractionated into soluble (S) and membrane-containing pellet (P) fractions of equal volume. Equal volumes were analyzed by SDS-PAGE and immunoblotting with anti-GUN4 antibodies. Representative immunoblots are shown.

(B) Proportions of GUN4 in soluble and pellet fractions. The chemiluminescence that was emitted from the immunoreactive bands described in (A) was quantified. The percentage of GUN4 in the pellet (white bars) and supernatant (light-gray bars) fractions derived from chloroplasts that were not fed PPIX and the percentage of GUN4 in the pellet (medium-gray bars) and supernatant (dark-gray bars) fractions derived from chloroplasts that were fed PPIX are indicated for wild type (Col-0) and each mutant and transgenic line. Results from at least five independent experiments for Col-0 and four independent experiments for all mutants and transgenic lines are shown. Error bars indicate se. A single asterisk indicates a statistically significant difference in the levels of GUN4 in the membrane-containing pellet fractions between Col-0 and the gun4 mutants gun4-1, F191A-14, and R211A-2.2 (P < 0.0005) and between Col-0 and the chlH/gun5 mutants cch (P < 0.01), gun5, gun5-101, and cs (P < 0.04). A double asterisk indicates a statistically significant difference in the percent GUN4 in the membrane-containing pellet fractions derived from chloroplasts that were fed PPIX and not fed PPIX and were purified from Col-0 (P = 0.009), F191A-14 (P = 0.002), R211A-2.2 (P = 0.002), gun5 (P = 0.04), and gun5-101 (P = 0.007).