The cinnamyl alcohol dehydrogenase gene family in Populus: phylogeny, organization, and expression

Abstract

Background: Lignin is a phenolic heteropolymer in secondary cell walls that plays a major role in the development of plants and their defense against pathogens. The biosynthesis of monolignols, which represent the main component of lignin involves many enzymes. The cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis as it catalyzes the final step in the synthesis of monolignols. The CAD gene family has been studied in Arabidopsis thaliana, Oryza sativa and partially in Populus. This is the first comprehensive study on the CAD gene family in woody plants including genome organization, gene structure, phylogeny across land plant lineages, and expression profiling in Populus.

Results: The phylogenetic analyses showed that CAD genes fall into three main classes (clades), one of which is represented by CAD sequences from gymnosperms and angiosperms. The other two clades are represented by sequences only from angiosperms. All Populus CAD genes, except PoptrCAD 4 are distributed in Class II and Class III. CAD genes associated with xylem development (PoptrCAD 4 and PoptrCAD 10) belong to Class I and Class II. Most of the CAD genes are physically distributed on duplicated blocks and are still in conserved locations on the homeologous duplicated blocks. Promoter analysis of CAD genes revealed several motifs involved in gene expression modulation under various biological and physiological processes. The CAD genes showed different expression patterns in poplar with only two genes preferentially expressed in xylem tissues during lignin biosynthesis.

Conclusion: The phylogeny of CAD genes suggests that the radiation of this gene family may have occurred in the early ancestry of angiosperms. Gene distribution on the chromosomes of Populus showed that both large scale and tandem duplications contributed significantly to the CAD gene family expansion. The duplication of several CAD genes seems to be associated with a genome duplication event that happened in the ancestor of Salicaceae. Phylogenetic analyses associated with expression profiling and results from previous studies suggest that CAD genes involved in wood development belong to Class I and Class II. The other CAD genes from Class II and Class III may function in plant tissues under biotic stresses. The conservation of most duplicated CAD genes, the differential distribution of motifs in their promoter regions, and the divergence of their expression profiles in various tissues of Populus plants indicate that genes in the CAD family have evolved tissue-specialized expression profiles and may have divergent functions.

Figure 1

Distribution of CAD genes on Populus chromosomes. The names of the chromosomes and their sizes (Mb) are indicated below each chromosome. Segmental duplicated homeologous blocks [39] are indicated with the same color. The position of genes is indicated with an arrowhead.

Figure 2

Intron-exon structures of CAD genes from Populus. Exons and introns are indicated by open boxes and lines respectively. Numbers above boxes indicate the exon sizes. The intron sizes are not to scale. The names of CAD genes and intron-exon structure are indicated at the left and right sides respectively.

Figure 3

Maximum Likelihood bootstrap tree phylogeny based on amino acid sequences of CAD genes in various land plants. Numbers above branches refer to NJ bootstrap values. Brackets highlight the three classes of CAD genes. Colors indicate gene groups determined based on their expression in various Populus plant tissues. Red (group 1), green (group 2), and blue (group 3) indicate genes preferentially expressed in xylem, leaves, as well as leaves and xylem respectively. Pink (group 4) represents genes that showed no preferential expression between Populus tissues.

Figure 4

Lignification pattern in Populus tissues selected for qRT-PCR studies. Far left column displays organs and tissues used (Leaf blade, Petiole, bark, Xylem). Middle column shows lignin deposition, visualized under the light microscope after phloroglucine-HCl staining (red color). Right column shows lignin distribution by fluorescent microscopy (autofluorescence). a, b – cross section of leaf vascular bundle, c, d – petiole cross section, e, f – transverse section of stem segment, g, h – secondary xylem from stem. Abbreviations: x – xylem, ph – phloem, s – schlerenchyma. Bars = 100 μm.

Figure 5

Quantitative expression of Populus CAD genes. The name of each gene is indicated at the top of each histogram. Tissues studied are shown at the bottom of the diagrams. Means designated by the same letter do not differ significantly according to Tukey's HSD test; P < 0.05).