Dirigent Proteins in Conifer Defense: Gene Discovery, Phylogeny, and Differential Wound- and Insect-induced Expression of a Family of DIR and DIR-like Genes in Spruce (Picea spp.)

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Abstract
The outer stem tissues of conifers provide a durable constitutive and inducible defense barrier consisting of suberized or lignified periderm, sclereids, a network of terpenoid-filled resin ducts, and phenolic phloem parenchyma cells. Microarray gene expression profiling of Sitka spruce (Picea sitchensis) bark attacked by stem-boring weevils (Pissodes strobi) or through mechanical wounding demonstrated significant accumulation of transcripts resembling dirigent protein (DIR) genes. To investigate this gene family and its spatial and temporal patterns of expression in conifer defense, we isolated cDNAs representing 19 unique DIR and DIR-like genes from Sitka spruce, white spruce (P. glauca), and interior spruce (P. glauca × engelmannii). Sequence alignments also identified a large number of DIR-like proteins in other plant species, which share several conserved protein motifs with known DIR proteins. Phylogenetic analysis of 72 DIR and DIR-like proteins suggests five distinct subfamilies, DIR-a and four DIR-like subfamilies (DIR-b, DIR-c, DIR-d and DIR-e). Previously characterized members of the DIR-a subfamily direct stereoselective phenolic coupling reactions in the formation of lignans and possibly lignins. The spruce genes identified here are members of the DIR-a and DIR-b subfamilies. Using gene-specific quantitative real-time PCR we measured constitutive expression for six DIR-a genes and three DIR-like genes in different stem tissues, green shoot tips, and roots of Sitka spruce. DIR-like genes revealed ubiquitous high expression in all tissues. In contrast, the six DIR-a genes showed a gradient of transcript abundance in stem tissues with highest levels in the outer cortex and lowest levels in the inner xylem. Gene-specific transcript profiling of six DIR-a genes confirmed rapid and strong accumulation (up to 500-fold) in wound- and weevil-induced stem bark and xylem. These findings suggest a role for spruce DIR genes in constitutive and induced phenolic defense mechanisms against stem-boring insects.

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