Article Figures & Data
Figures
- Fig. 1.
The effect of plant growth on MCCase activity. MCCase specific activity was determined in extracts of wild-type orbio1 mutant seedlings between 3 and 28 d after sowing. Seedlings were grown either with or without the exogenous addition of 1 mm biotin. Data are the mean ±se from four replicates.
- Fig. 2.
The effect of biotin on the biotinylation status and accumulation of the MCCase subunits. Protein extracts were prepared from seedlings (A–C) or excised cotyledons (D) of wild-type andbio1 Arabidopsis seedlings at the indicated DAP. Aliquots of extracts containing equal amounts of protein (150 μg) were subjected to SDS-PAGE, followed by western-blot analysis with either125I-streptavidin to detect the biotinylated MCC-A subunit (A) or immunological detection with antibodies to MCC-A (B) or MCC-B (C and D). Where indicated, exogenous biotin (0.25 mm) was provided to the bio1 seedlings 2 d before harvest. The data presented were gathered from a single experiment; five replicates of this experiment, with two different batches of bio1 seeds, gave similar results.
- Fig. 3.
The effect of biotin-depletion on MCCase gene transcription. Northern-blot analysis of MCC-A(A) and MCC-B (B) mRNA accumulation in wild-type and bio1 Arabidopsis seedlings. RNA was isolated from wild-type and bio1 seedlings grown to 20 DAP in the absence of exogenous biotin. Equal amounts of isolated RNA (50 μg) were subjected to electrophoresis in formaldehyde-containing agarose gels, and MCC-A or MCC-B mRNAs were detected by hybridization with respective32P-labeled probes. Reporter gene expression studies of the MCC-A andMCC-B genes. GUS activity was determined in protein extracts from transgenic Arabidopsis seedlings of either wild-type (wt) or bio1 genetic background and carrying anMCC-A::GUS (C) orMCC-B::GUS (D) reporter transgene. Seedlings were grown without exogenous biotin to the indicated DAP. Data are the means ± se from three replicates.
- Fig. 4.
The effect of biotin-depletion on the metabolic regulation of MCC-A andMCC-B gene transcription. GUS activity was determined in protein extracts from wild-type (wt) or bio1Arabidopsis seedling carrying anMCC-A::GUS (A) orMCC-B::GUS (B) reporter transgene. Seedlings were grown to 13 DAP on Murashige and Skoog agar medium without biotin, followed by 2 d of additional growth either in the absence (−) or presence (+) of exogenous biotin. In these last 2 d of growth, seedlings were grown either under constant illumination (white bars), or transferred to darkness (black bars), or CO2-free air (dotted bars). Data are the means ± se from three replicates.
- Fig. 5.
Time course of the biotin dependence of the metabolic regulation of MCC-A andMCC-B gene transcription. GUS activity was determined in protein extracts from wild-type (wt) or bio1Arabidopsis seedlings carrying anMCC-A::GUS (A) orMCC-B::GUS (B) reporter transgene. Seedlings were grown in the absence of exogenous biotin to the indicated DAP. These seedlings were maintained in constant illumination until the last 2 d of growth, at which stage they were transferred to total darkness. Data are the means ±se from three replicates.
- Fig. 6.
Electrophoretic characterization of MCCase. Protein extracts from Arabidopsis, soybean, and pea seedlings (A) and wild-type (wt) and bio1 mutant Arabidopsis seedlings (B) were subjected to exhaustive electrophoresis (for 14,400 V h−1) in gels composed of a linear gradient of 5% to 30% polyacrylamide according to the method of Hedrick and Smith (1968). After western blotting, MCCase was immunologically detected by reacting the membranes with anti-MCC-B serum (identical results were obtained with anti-MCC-A serum; data not shown). The native molecular mass of MCCase was determined by comparing its migration to standard proteins (apoferritin dimer, 886 kD; apoferritin monomer, 443 kD; urease dimer, 545 kD; and urease monomer, 272 kD). The position of MCCase is indicated by arrows. C, Analysis of MCCase charge isoforms. Aliquots of protein extracts from wild-type (wt) and bio1 Arabidopsis seedlings at the indicated DAP, containing equal amounts of MCCase activity, were subjected to electrophoresis at 70 V for 17 h in a linear 5% to 20% gradient polyacrylamide gel (Lambin and Fine, 1979). After western blotting, MCCase was immunologically detected by reacting the membranes with anti-MCC-B serum (identical results were obtained with anti-MCC-A serum; data not shown).
