In Arabidopsis thaliana Columbia ecotype, 33 open reading frames (ORFs) potentially encoding proteins homologous to XTH proteins have been identified based on the genome sequence database (Arabidopsis Genome Initiative 2000) (Nature, 408:796-815) and the cDNA sequence data. All 33 XTH ORFs represent functional genes. Thus "Arabidopsis" there are good grounds for a systematic genome-based nomenclature for the Arabidopsis XTH gene family (Genes and Clones).
The 33 XTH genes are dispersed across all five chromosomes of Arabidopsis, with one third of the genes occurring as clusters consisting of two to four members (Fig. 1) and (Yokoyama and Nishitani 2001b), resulting from genome duplication and gene reshuffling
(Blanc et al. 2000). For example, At-XTH28 located on chromosome I and At-XTH27 on chromosome II share one ancestral gene, while At-XTH16 on chromosome III and XTH15 on chromosome IV also share a common ancestor. The cluster of At-XTH14 and At-XTH23 on chromosome IV corresponds to the cluster of At-XTH12, At-XTH13, At-XTH25, andAt-XTH22 on chromosome V. Based on the order and orientation of these 6 genes, At-XTH14 on chromosome 4 corresponds to an ancestral gene that might have been further duplicated to generate At-XTH12 and At-XTH13 on chromosome 5. Similarly, At-XTH23, which is located next to At-XTH14 on chromosome 4, corresponds to the ancestral gene for At-XTH22 andAt-XTH25 on chromosome 5 (Yokoyama and Nishitani 2001b). These gene-duplications are believed to have resulted from large-segment gene-duplication (Arabidopsis Genome Initiative 2000). However, in addition, two pairs of solitary XTHgenes have been identified as probably having been duplicated by the action of transposons: At-XTH17 and At-XTH18 share almost identical sequences in their promoter regions, while At-XTH19 and At-XTH20 are identified as duplicates by phylogenetic analysis (Fig. 2).
Figure 1: Genomic structure and phylogenetic relationship between the members of the Arabidopsis XTH gene family. Physical map showing the distribution of the Arabidopsis XTH genes (see alsoTable1, "Genes and Clones") among the five chromosomes with arrows indicating duplicated genes (Rose et al. 2002).
The Arabidopsis XTH gene family can be divided into three major phylogenetic groups, or subfamilies (Fig. 2) and (Yokoyama and Nishitani 2001b), in good agreement with other phylogenetic studies of XTH genes from a broad range of plant species (Nishitani 1997, Schünmann et al. 1997, Campbell and Braam 1999, Catalá et al. 2000; Uozu et al. 2000). This classification is based not only on the dendrogram shown in Fig. 2, but also on the structure and organization of individual genes, as outlined in Fig. 1. Most members of Group 1 contain 4 exons, whereas XTHs in Group 2 have two or three exons, with the exception of At-XTH26, which has 4 exons. On the other hand, members of Group 3 are composed of 4 or 5 exons and posses characteristic amino acid sequences, particularly in their C-terminal regions. All XTHs have a conserved DEIDFEFLG sequence, a motif that is considered to function as catalytic site for either hydrolase or transferase activity (Okazawa et al. 1993).This motif is located in the third exon in Group 1 and 3, while in Group 2 it is located in the second exon, again with the exception of At-XTH26. Another structural features common to the XTH family, except for At-XTH6, is the sequence in the beginning of the first exon, encoding a potential signal peptide that has been shown to direct secretion into the apoplast (Yokoyama and Nishitani 2001b).
The group numbering here is consistent with that used in previous studies, where the members of the EXGT-type of XTH belong to Group 1 and the nasturtium hydrolase/XET NXG1 aligns most closely with Group 3 (Campbell and Braam 1999). It should be noted that some of these previous reports identified a fourth phylogenetic group when XTH sequences from monocotyledons such as barley (Hordeum vulgare) were included. An obvious consideration is that this phylogenetic divergence reflects the evolution of XTH subgroups with different biochemical mechanisms of action, such as transglycosylation versus hydrolysis. Indeed, individual members of Groups 1 and 2, includingAt-XTH4 (formerly EXGT-A1); (Okazawa et al. 1993), and At-XTH22 (formerly TCH4); (Xu et al. 1995), respectively, have been demonstrated to exclusively mediate molecular-grafting reactions between xyloglucans in vitro(Nishitani and Tominaga 1992, Xu et al. 1996), while members of Group 3, as represented by At-XTH31 (formerly ATXG); (Aubert and Herzog 1996) have been shown to catalyze xyloglucan hydrolysis (Fanutti et al. 1993, DeSilva et al. 1993, Tabuchi et al. 2001). Thus, the XTH gene family has apparently undergone diversification into two groups in terms of acceptor-substrate specificity. However, while this suggests a trend, it is important to note that the phylogenetic grouping has not been definitively shown to reflect any particular biochemical characteristics or mechanism of action and exceptions have been reported(Schröder et al. 1998).
Figure 2: Genomic structure and phylogenetic relationship between the members of the Arabidopsis XTH gene family. Dendrogram generated using CLUSTALW and TreeViewPPC software, based on the predicted amino acid sequences of all 33 Arabidopsis XTH genes. For both parts A and B of the figure, genes from groups 1, 2 and 3 are shown in red, green and blue, respectively. The physical map and dendrogram are revised from versions published in (Yokoyama and Nishitani 2001b, Rose et al. 2002)
Furthermore, within each XTH subfamily, no clear diversification is found with respect to enzyme action (Campbell and Braam 1999). The presence of multiple genes encoding similar enzymatic activities raises the question of whether individual members, particularly those in each subfamily, are redundant in terms of their physiological roles, or whether there is simply a functional distinction that has not yet been discerned. To address this question, a comprehensive expression analysis of the Arabidopsis XTH gene family was conducted, using of real-time PCR to accurately quantify mRNA levels, and to distinguish between gene products with similar nucleotide sequences (Yokoyama and Nishitani 2001b). This analysis has revealed that many members of the XTH gene family exhibit distinct organ- or tissue-specific expression profiles. For example, within Group 2, At-XTH17, -18, -19, and -20 are predominantly expressed in roots, At-XTH21and At-XTH22 in siliques and At-XTH24 in stems. Furthermore, individualXTHgenes respond differently to different plant hormones:At-XTH17, -18, -19, and -20, which exhibit the same organ-specific expression profile, are differentially by hormones, including auxin, gibberellins, brassinolide and abscisic acid (Yokoyama and Nishitani 2001b). These results are consistent with those reported by (Xu et al. 1996) and (Akamatsu et al. 1999) using RNA blot analyses. In conclusion, given that the limited range of regulatory factors and spatial and temporal variables that have been tested experimentally have already uncovered great variability among members of the Arabidopsis XTH family, functional redundancy is not likely to be common. The expression and regulation of each XTH gene is therefore likely to have a unique "fingerprint" reflecting a unique physiological function, which will be revealed as the degree of experimental sophistication and complexity increases.
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