Figure 9.

Complementation Test Using Genes Encoding Mutant Variants of CB5D.

(A) qRT-PCR analysis of gene expression levels in T2 transgenic lines expressing pC4H:CYB5DΔ1, pC4H:CYB5DΔ2, or pC4H:CYB5DΔ1Δ2 in the cb5d-1 background. Two-week-old T2 seedlings were used for RNA extraction. Approximately 12 seedlings were pooled as one biological replicate. The data represent means ± sd of three biological replicates, each with three technical repeats.

(B) Relative content of sinapoyl malate in 4-week-old rosette leaves of the Col-0 wild type, cb5d-1, and T1 transgenic lines harboring pC4H:CYB5DΔ in the cb5d-1 background. The data represent means ± sd of all biological replicates and are expressed as relative values compared with the Col-0 wild type. The data for the Col-0 wild type and cb5d-1 were obtained from three independent plants representing three biological replicates; the data for pC4H:CYB5DΔ transgenic plants were obtained from an average of 10 T1 independent transgenic lines per transgene. Different letters indicate significant differences (P < 0.05; one-way ANOVA, Tukey’s HSD test) between the different genotypes.

(C) Histochemical observation of S-lignin in the Col-0 wild type, cb5d-1, and two independent T2 transgenic lines of pC4H:CYB5DΔ1, pC4H:CYB5DΔ2, and pC4H:CYB5DΔ1Δ2 in the cb5d-1 background with Mӓule staining. Cross sections of 7-week-old stems (1 cm from the bottom, 50 µm thick) were used. Bars = 30 µm.