The nfu4 nfu5 double mutant shows decreased protein lipoylation affecting lipoyl-dependent metabolism. A, Representative images of nfu4 nfu5 double mutant segregants (m) and sibling WT-like (wtl) seedlings, grown on 0.5 MS agar plates under ambient CO₂ and 1% CO₂ in the greenhouse (8/16-h light/dark cycles, variable temperature, ∼90% humidity). Scale bars: 0.5 cm. Additional images in Supplemental Figure S7. B, Concentrations of selected free amino acids and organic acids in 3-week-old seedlings of the indicated genotypes. ACO, aconitase. Values represent the average of three to six biological replicates ± sd. *P < 0.05, **P < 0.01, ***P < 0.001 (Student’s t test, pairwise comparison to WT). See Supplemental Tables S2, S3 for complete data sets. C, Protein blot analysis for lipoyl cofactor (top) and H protein isoforms of GDC (bottom), in WT, nfu4 nfu5 (m), and wtl segregants. See also Supplemental Figure S8.