Supplemental Data

Files in this Data Supplement:

• Supplemental Figure 1 - Wild-type, atcnfU2-1, and atcnfU2-2 plants were grown under continuous light without sucrose for 24 days. Detached leaves were used for measurements of rate of O₂ evolution at a saturating concentration of CO₂ (~10%) at 25^oC as described in the Methods section.
• Supplemental Figure 2 - Semiquantative RT-PCR analysis of ferredoxin gene expression in 23-day-old wild-type and atcnfU2 mutant plants grown photoautotrophically under continuous light (A) or under long-day conditions supplemented with sucrose (B). Numbers of cycles of PCR amplification are indicated. As a control, expression of a cytosolic elongation factor 1α (EF1α) gene, which is known to be expressed constitutively under various conditions, was also analyzed.
• Supplemental Figure 3 - (A) Total protein extracts (10-μg proteins) of 23-day-old plants grown under long-day condition supplemented with sucrose were analyzed by Western blotting. Relative protein contents were quantified densitometrically and are shown in (B). Protein content found in wild-type extracts was set to 100%. Fd, ferredoxin (2Fe-2S); SiR, sulfite reductase (4Fe-4S); PsaC, a component of photosystem I (4Fe-4S); cpHsp70, a stromal molecular chaperone as a non-iron-sulfur cluster-containing protein.
• Supplemental Figure 4 - Holo-ferredoxin formation assay using wild-type or atcnfU2-2 stromal extracts was performed as shown in Figures 8C and 8D, with the exception that more reducing conditions (i.e., in the presence of 1 mM dithiothreitol) were used for incubation.
• Supplemental Figure 5 - Holo-ferredoxin formation assay using atcnfU2-2 stromal extracts was carried out as shown in Figure 8C in the presence of externally added holo-FdI. We estimated the ferredoxin content in the wild-type stromal extracts to be 0.5-1% of total stromal proteins. Since the total amount of stromal extract used per assay was estimated to be 50 μg of protein, 1 μg of the purified holo-FdI corresponds to a twofold excess over the endogeneous ferredoxin in the wild-type stromal extracts.
• Supplemental Figure 6 - Three micrograms of purified AtCnfU-V was incubated with [³H]-labeled apo-Fd I either at 25^oC (upper panel) or 4^oC (middle panel) for the indicated incubation time and was analyzed as shown in Figures 6C and 6D. The quantity of holo-ferredoxin formed at 25^oC at 30 min was set at 1.0.
• Supplemental Figure 7 - [³H]-labeled apo-Fd I, which was synthesized in vitro as described in Methods, was incubated in the buffer containing 50 mM HEPES-KOH, pH 7.5, 50 mM KOH for 60 min at 25^oC in the presence of the indicated concentration of dithiothreitol and was analyzed using our standard nondenaturing PAGE (A) (Williams and Resisfeld, 1964, Ann. NY Acad. Sci. 121, 373-381) or using the widely used Laemmli system, except that SDS was omitted (B) (Laemmli, Nature, 227, 680-685.).
• Supplemental Figure 8 - In vitro holo-ferredoxin formation with the stromal extracts prepared from either wild-type of atcnfU2-2 chloroplasts shown in Fig. 8C was repeatedly analysed. For detail, see the legend to Fig. 8.
• Supplemental Figure 9 - (A) The purified AtCnfU-V (20 μg) was incubated in the buffer containing 50 mM HEPES-KOH, pH 7.5, 50 mM KOH, 1 mM dithiothreitol at 25^oC for 60 min with or without 100 μM ferrous ammonium citrate and 100 μM sodium sulfide in the presence of the indicated concentration of EDTA and was analyzed as shown in Figure 9D. (B) [³H]-labeled apo-ferredoxin (TP, inputted translated products of maize ferredoxin Fd I) was incubated with the stromal extracts prepared from wild-type chloroplasts in the presence of the indicated concentration of EDTA at 25^oC. After the indicated incubation time, conversion of apo-ferredoxin to the holo-form was analyzed as shown in Figure 8C.