Figure 1.

NaCAD genes cluster with bona fide AtCAD genes (AtCAD4 and AtCAD5) from Arabidopsis known to be involved in stem lignification. A, An unrooted phylogenetic tree of the NaCADs and AtCAD gene family. The dendrogram was generated with MEGA software (Tamura et al., 2007) using ClustalW and the neighbor-joining method, and bootstrap values are shown at each branch. Bootstrap tests were performed using 1,000 replicates. Gene accession numbers are shown in Supplemental Table S1. B, Schematic representation of N. attenuata wild-type plant parts analyzed for transcript expression. C and D. Mean ± se NaCAD (C) and NaCAD1 (D) transcript levels quantified by qRT-PCR in wild-type plants (n = 5). Insets present silencing efficiency of NaCAD (C) and NaCAD1 (D) in 10-cm basal stems of two independently transformed inverted-repeat homozygous lines (ir-CAD-206 and ir-CAD-225) at the early elongating stage of development (n = 5). Elongation factor, a housekeeping gene, was used to normalize all qRT-PCR assays. Relative transcript abundance was calculated with respect to wild-type (WT) stem levels, which were set to 100. Asterisks indicate significant differences among the genotypes (one-way ANOVA: *** P < 0.001).