Mutation of the chitinase-like protein-encoding AtCTL2 gene enhances lignin accumulation in dark-grown Arabidopsis seedlings

Abstract

Several genes that encode a chitinase-like protein (called the CTL group) have been identified in Arabidopsis, rice, pea, and cotton. Members of the CTL group have attracted much attention because of their possible role in the biosynthesis of the cell wall in plants. The hot2 mutation in the CTL1 (AtCTL1) gene of Arabidopsis thaliana causes multiple defects in growth and development. The Arabidopsis genome possesses the AtCTL2 gene, which exhibits 70% similarity to AtCTL1 at the amino acid level. We showed that the AtCTL2 gene was predominantly expressed in stems, which was in contrast to the presence of AtCTL1 transcripts in most organs of Arabidopsis. In addition, β-glucuronidase (GUS) staining was detectable in all tissues of the stem in transgenic plants expressing the AtCTL1::GUS construct, while GUS activity under control of the AtCTL2 promoter was significantly restricted to the xylem and to interfascicular fibers in stems. The phenotypes of atctl2 single mutant and of hot2, atctl2 double mutant plants were significantly similar to those of wild-type and of hot2 single mutant plants, respectively. The expression levels of CESA1 and CESA4 transcripts were not affected in the two single mutants or corresponding double mutant plants, compared with the levels in wild-type plants. The accumulation of lignin in etiolated hypocotyls, however, was increased by mutation of AtCTL2. These findings suggest that AtCTL2 is required for proper cell wall biosynthesis in etiolated seedlings of Arabidopsis.

Introduction

Chitin is the second most abundant glycopolymer of N-acetyl-d-glucosamine in nature (after cellulose) and constitutes the integument of many species, such as insect exoskeletons, shells of crustaceans, and fungal cell walls (Neville et al., 1976; Debono and Gordee, 1994). Chitinases are ubiquitous, chitin-fragmenting hydrolases and are produced by a vast array of organisms, including those that do not contain chitin. According to the glycosyl hydrolase classification system, chitinases fall into two groups: glycosyl hydrolase families 18 and 19 (Henrissat, 1991). The differences in amino acid sequence, structure, and chitin-degrading mechanism of chitinases in both families imply that they have arisen from different evolutionary origins. They are further classified into five different classes (I–V), based on amino acid sequence similarity (Neuhaus et al., 1996). Chitinases in classes I, II, and IV belong to family 19, whereas chitinases in classes III and V are family 18 glycosyl hydrolases.

Although it is generally believed that plants do not possess an analogous chitin in their cell walls, chitinase genes have been identified in all plants analyzed to date (Graham and Sticklen, 1994). Keyword-based searches of Arabidopsis annotation databases and subsequent analysis of all accessions have shown that 24 chitinase genes are distributed throughout all five chromosomes of the Arabidopsis genome (Passarinho and De Vries, 2002). The comparison of their amino acid sequence has also revealed that Arabidopsis possesses several members of all five chitinase classes. Based on the presence of numerous chitinase genes in virtually all plants, we may predict multiple functions associated with the different types of chitinases.

Early researches revealed that chitinases serve as a defense mechanism against infection of chitin-containing pathogens by attacking chitin, which is not found in the cell wall of plants (Mauch et al., 1988; Arlorio et al., 1992; Jach et al., 1995). In addition, the spatial and temporal appearance of chitinase isoforms reflects their involvement in plant growth and development (Hanfrey et al., 1996; Takakura, 2000; Passarinho et al., 2001). Furthermore, De Jong et al. (1992) demonstrated the chitinase-mediated rescue of a carrot mutant during somatic embryogenesis, which suggests a critical role for these enzymes in somatic embryonic development. Lipochitin oligosaccharides (LCOs) are signal molecules that were discovered during the study of the root nodulation process in leguminous plants. In addition to active chitinases, Arabidopsis, rice, pea, and cotton express a number of genes that encode chitinase-like proteins (CTLs), which lack chinolytic activity (Zhang et al., 2004). For example, two homologous cotton (Gossypium hirsutum L.) CTL genes (GhCTL1 and GhCTL2) are expressed preferentially during secondary cell-wall deposition in cotton fiber cells (Zhang et al., 2004). The AtCTL1 gene is highly co-regulated with three cellulose synthase A (CESA) genes (CESA1, 3, and 6), which are largely responsible for cellulose biosynthesis during primary cell-wall formation. The AtCTL2 transcripts are coexpressed with three other CESA genes (CESA4, 7, and 8), which are required for cellulose biosynthesis during secondary cell-wall formation in vascular tissues (Brown et al., 2005; Persson et al., 2005). Furthermore, genetic analyses showed that mutations in AtCTL1 cause severe defects in various aspects of Arabidopsis development under optimal conditions, which include cellulose synthesis, ethylene production, and responses to abiotic stresses (Zhong et al., 2002; Kwon et al., 2007).

To gain a deeper understanding of the biological roles played by the members of the CTL subgroup in plants, we performed molecular and genetic analyses of the AtCTL1 and AtCTL2 genes in Arabidopsis. We introduced promoter-GUS fusion constructs of the AtCTL1 and AtCTL2 genes into Arabidopsis. The analysis of transgenic Arabidopsis plants expressing each of the 5′ promoter regions fused to the GUS reporter gene revealed different expression patterns for the two genes. The T-DNA insertion of the AtCTL2 gene had little effect on development and abiotic stress tolerance in Arabidopsis. We also found that the double-mutant phenotypes for both genes were similar to that observed for the AtCTL1 mutant. However, mutation of the AtCTL2 gene resulted in an increase in lignin accumulation in etiolated seedlings. These results suggest that AtCTL1 and AtCTL2, which are members of the CTL subgroup, play common roles during cell wall synthesis in Arabidopsis.