Supplemental Data

Files in this Data Supplement:

• Supplemental Table 1 - Genes Showing Significant Differential Expression in toc132.
• Supplemental Figure 1 - Visible Phenotypes of the toc132-2 and toc132-3 Mutants. Homozygous, mutant plants of the indicated genotypes were grown alongside wild-type plants, under identical conditions, for 26 days. Plants were germinated in vitro and transferred to soil after 15 days growth.
• Supplemental Figure 2 - Immunoblot Analyses of 35S-atTOC120 and 35S-atTOC132 Transformants of toc159. Total protein extracts of untransformed toc159 homozygotes, and of doubly homozygous toc159 plants transformed with 35S-atTOC132 and 35S-atTOC120 constructs, were analyzed by immunoblotting using antibodies against atToc132 (A) and atToc120 (B). Undiluted samples contained 50 μg of protein. Dilution series of the 35S-atTOC120 and 35S-atTOC132 samples were also analyzed, and the dilution steps are given above the blots. Coomassie stained gels of equivalent sample loadings are shown below the blots. By comparing the lanes indicated with asterisks, it can be estimated that the transgenic lines each overexpress the relevant full-length protein (either atToc132 or atToc120) by a factor of ~3- to 4-fold relative to untransformed toc159.
• Supplemental Figure 3 - Protein Import into toc132/toc132; +/toc120 Chloroplasts. ³⁵S-Met-labeled preSSU (A) and preL11 (B) translated in vitro using a wheat germ extract were imported into wild-type and mutant (toc132/toc132; +/toc120) chloroplasts for 1-10 min, as indicated. Ten percent of the translation product added to each import reaction was loaded as a control (10%). Each import experiment was carried out twice, and the data shown are representative. Quantification of imported protein was performed using ImageQuant (Molecular Dynamics), and statistical analyses showed that there is no significant difference in import rate between mutant and wild type, in each case.
• Supplemental Figure 4 - DIGE Analysis of the toc132 Chloroplast Proteome. Total chloroplast protein isolated from 10-day-old seedlings was analyzed using CyDyeDIGE technology. Equal aliquots of the wild-type and toc132 mutant protein samples were pooled, and 50 μg was labeled with Cy2. Separate wild-type and mutant protein samples (50 μg each) were labeled with Cy3 and Cy5, respectively. After labeling, the samples were pooled and run on a single 24-cm, pH 3-10 2D gel and then imaged using parameters appropriate for each fluor. The upper left and upper right panels show corresponding sections (pH 4-8) of the Cy3 and Cy5 images, respectively. The lower panel shows an overlay of the two images following inversion and the application of false colour: Cy3 (wild type) is in green and Cy5 (toc132) is in red. The images were analyzed using DeCyder software (Amersham Biosciences), and the inclusion of a pooled sample in the Cy2 channel allowed the calculation of a standardized abundance volume for each spot. Labels indicate protein spots that were identified by mass spectrometry (see Table 4). The images have been cropped to better illustrate spot resolution, and so spot 11 (SSU), which runs at the base of the gel, is not shown.