Identification and characterization of Arabidopsis thaliana genes involved in xylem secondary cell walls

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1 Springer-VerlagTokyohttp:// of Plant ResearchJ Plant ResLife Sciences /s J Plant Res (2006) 119: The Botanical Society of Japan and Springer-Verlag Tokyo 2006 Digital Object Identifier (DOI) /s JPR SYMPOSIUM Ryusuke Yokoyama Kazuhiko Nishitani Identification and characterization of Arabidopsis thaliana genes involved in xylem secondary cell walls Received: November 25, 2005 / Accepted: December 15, 2005 / Published online: March 22, 2006 The Botanical Society of Japan and Springer-Verlag 2006 Abstract The xylem of higher plants offers support to aerial portions of the plant body and serves as conduit for the translocation of water and nutrients. Terminal differentiation of xylem cells typically involves deposition of thick secondary cell walls. This is a dynamic cellular process accompanied by enhanced rates of cellulose deposition and the induction of synthesis of specific secondary-wall matrix polysaccharides and lignin. The secondary cell wall is essential for the function of conductive and supportive xylem tissues. Recently, significant progress has been made in identifying the genes responsible for xylem secondary cell wall formation. However, our present knowledge is still insufficient to account for the molecular processes by which this complex system operates. To acquire further information about xylem secondary cell walls, we initially focused our research effort on a set of genes specifically implicated in secondary cell wall formation, as well as on loss-offunction mutants. Results from two microarray screens identified several key candidate genes responsible for secondary cell wall formation. Reverse genetic analyses led to the identification of a glycine-rich protein involved in maintaining the stable structure of protoxylem, which is essential for the transport of water and nutrients. A combination of expression analyses and reverse genetics allows us to systematically identify new genes required for the development of physical properties of the xylem secondary wall. Key words Arabidopsis Microarray Secondary cell wall Reverse genetics Weight signal Xylem Introduction R. Yokoyama K. Nishitani (*) Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Sendai , Japan Tel ; Fax nishitan@mail.tains.tohoku.ac.jp Xylem appeared relatively early in the evolution of land plants. It provides plants with not only the means for longdistance transport between aerial and ground parts, but also provides mechanical support. This enables land plants to maintain large photosynthetic aerial parts high above the ground. The xylem consists of several distinct types of cells with specialized wall structures. These specific structural features of individual xylem cell walls confer mechanical strength to plant tissues and allow the efficient translocation of water and nutrients within the plant body. Xylem cell walls are dynamic, and typically undergo massive depositions of secondary walls, as well as extensively disassembling primary walls. For example, tracheary elements, which are components of xylem vessels, possess elaborately patterned secondary cell walls with an alternating orientation of cellulose microfibrils and a lignified layer (Fukuda 2004; Oda et al. 2005). Differentiation of tracheary elements involves sequential changes in the cell wall. This includes expansion of primary walls and the laying down of lignified secondary walls, followed by digestion of wall components, such as xyloglucan molecules in the primary wall (Stacey et al. 1995; Bourquin et al. 2002; Matsui et al. 2005). After the cell has stopped growing, the xylem wall is thickened with secondary deposits of cellulose and is further strengthened by impregnation with specific secondary-wall matrix polysaccharides and lignin. The resulting walls provide the tracheary elements with enough strength to withstand high negative pressure within the vessels (Ye 2002), as well as to support the weight of the stem itself. A number of cell-wall structural proteins are also associated with secondary cell wall formation. The function of conductive and supporting xylem tissues is strongly dependent on the structure, composition, and morphology of the secondary cell wall. Thus, there has been a growing understanding of not only the biological importance of the secondary cell wall, particularly regarding xylem differentiation, but also in terms of economic significance. Despite the biological importance of secondary cell walls, their complexity and diversity have hampered the dissection of the molecular mechanism by which the wall structure is generated, assembled, and remodeled during the terminal differentiation of xylem cells. A large number of genes are required for the molecular processes involved

2 190 in this system. Complete genomic sequences and extensive expressed sequence tag library collections allude to the existence of a large number of genes that are likely to be part of these processes (Yokoyama and Nishitani 2004; Aspeborg et al. 2005). Whereas the functions of several genes responsible for xylem wall formation have been elucidated, identifying and determining the function of a large number of genes directly involved in xylem secondary wall formation still remains a major challenge. Forward genetics Forward genetic screens have led to the isolation of several genes involved in secondary cell wall biosynthesis and lignification, as well as in xylem formation. In Arabidopsis thaliana (L.) Heynh., IRX1, IRX3, and IRX5 genes were identified based on recessive mutations that resulted in the collapse of secondary cell walls of xylem and code for catalytic subunits of the cellulose synthase complex specifically responsible for the synthesis of secondary cell walls (Turner and Somerville 1997; Taylor et al. 2003). Another A. thaliana mutant, irx2, which resulted from a point mutation in the KORRIGAN gene, which codes for a β-1,4-endoglucanase, exhibits a cellulose deficiency in the secondary cell wall (Szyjanowicz et al. 2004). In the ectopic deposition of lignin in pith 1 (elp1) mutant of A. thaliana, lignin is deposited ectopically in patches of parenchyma cells in the stem pith, a cell wall type that is not lignified in the wild type (Zhong et al. 2000). The ectopic deposition of lignin correlated with both increased Klason lignin content and the ectopic expression of caffeoyl CoA O-methyltransferase, an enzyme involved in monolignol biosynthesis. In the elp1 mutant, a mutation occurred in the gene coding for a chitinase-like protein, AtCTL1 (Zhong et al. 2002). On the other hand, in rice, the isolation of three cellulose synthase (CESA) genes (OsCESA4, OsCESA7, and OsCESA9) for brittle culm mutations induced by the retrotransposon Tos17 has been reported (Tanaka et al. 2003). These genes are involved in the synthesis of cellulose in secondary cell walls. Functional genomics Although forward genetics is an excellent system for identifying a limited number of genes essential for secondary wall biosynthesis and lignification in plants, the identity of the majority remains elusive. Functional genomics offers the means to identify a large number of genes involved in xylem secondary cell walls and would provide a comprehensive view of these genes. Microarray technologies are now available as powerful techniques for such purposes. With the aid of this technology, it is possible to monitor the expression profiles of hundreds to thousands of genes acting during secondary cell wall formation and to identify a large number of genes involved in secondary cell wall biosynthesis and lignification (Oh et al. 2003; Ko et al. 2004). A system of cluster analyses for many expression data sets from microarray analyses further increases the number of genes found to be involved in secondary cell wall synthesis (Brown et al. 2005; Persson et al. 2005). Tracheary elements differentiated from isolated mesophyll cells of Zinnia elegans have emerged as a good model system for studying gene regulation during xylem differentiation (Fukuda 1997). In the Z. elegans system, secondary wall formation unique to tracheary elements is induced by changes in hormonal balance. Demura et al. (2002) performed comprehensive microarray analyses of gene expression in this cell-differentiation system and identified many candidate genes involved in secondary-wall formation. Comprehensive approach to genes involved in secondary cell wall formation in A. thaliana inflorescence stems As a first step toward characterizing the processes involved in xylem secondary cell wall formation in A. thaliana inflorescence stems, we manufactured a gene-specific 70-mer oligo microarray that consisted of 756 genes classified into 30 putative families of proteins implicated in A. thaliana cell-wall dynamics (Imoto et al. 2005). Using this microarray system, we examined gene expression profiles the genes along the inflorescent stem of A. thaliana. This organ has proved a useful system for studying secondary cell wall formation in that xylem cells form thick and lignified secondary cell walls in the basal region of the stem (Imoto et al. 2005). To classify the expression profiles of individual genes in terms of their preferential expression site along the stem, we defined three types of expression profiles, designated as Type A, B, and C. The Type A profile is defined as that in which the expression in the upper part of the stem is more than twofold higher than that in the basal part. The Type B profile displays expression levels in the middle part of the stem that is more than twofold higher than in both the apical and basal parts. On the other hand, the Type C profile displays expression levels in the basal part that are more than twofold higher than any other region of the stem (Imoto et al. 2005). In this work, we focused on genes with the Type C expression profiles (Table 1). A large number of genes have been identified as belonging to various families of glycosyltransferases (callose/glucan synthase), glycosyl hydrolases (fucosidase, galactosidase, xylosidase, etc.), and structural proteins [arabinogalactan protein (AGP), glycine-rich protein (GRP), cell-wall-associated kinase, and extensin], as well as enzymes implicated in the deposition of cellulose (cellulose synthase) and lignification (peroxidase and laccase). We also identified genes encoding expansin and XTH (Yokoyama and Nishitani 2001), which are implicated in the modification of primary cell walls during the expansion process. This result suggests that different isoforms in a family of proteins have different biological functions in different

3 191 Table 1. Set of genes that exhibit growth-stage-dependent expression profiles defined as Type C (from Imoto et al. 2005) Gene family name No. of genes a Type C b Cellulose synthase 40 3 Callose/glucan synthase 12 1 α-xylosyltransferase 8 0 α-fucosyltransferase 10 0 Glycosyltransferase (GT1*) 19 0 Glycosyltransferase (GT8*) 16 1 Sterol glucosyltransferase 4 0 Epimerase 5 0 Xyloglucan endotransglucosylase/ 33 3 hydrolase β-1,4-glucanase 24 2 Expansin 35 1 α-fucosidase 2 0 β-galactosidase 25 4 α-xylosidase 5 1 β-xylosidase 15 1 β-1,3-glucanase 77 1 Chitinase 23 2 α-arabinosidase 2 0 Mannan-hydrolase 8 0 α-mannosidase 4 0 Pectate lyase 26 0 Polygalacturonase 67 3 Pectinesterase Peroxidase 69 4 Prolyl 4-hydroxylase 10 0 Laccase 17 5 Arabinogalactan protein 25 3 Extensin 38 1 Glycine-rich protein 30 3 Wall-associated receptor kinase 5 1 Total a Number of genes in each family b Number of family members classified as Type C genes, which are defined as those whose expression level in the basal part of the stem is twofold higher than that in the upper part of the stem cell types (for recent review, see Cosgrove 2000; Nishitani 2002; Rose et al. 2002). In fact, some members of these families were recently shown to be involved in secondarywall deposition (Bourquin et al. 2002; Nakamura et al. 2003; Gray-Mitsumune et al. 2004; Matsui et al. 2005). Consequently, Type C gene sets contained a large number of uncharacterized genes, as well as genes previously implicated in secondary-wall deposition of xylem cells during vascular development. Cell-wall genes screened for changes in expression profiles during gravistimulation Changes in gravity conditions have been shown to affect the mechanical properties of cell walls. It is suggested that gravistimulation of A. thaliana inflorescence stems changes the mechanical extensibility of xylem secondary cell walls (Soga et al. 1999, 2002). We examined the expression of 765 cell-wall genes in inflorescence stems during early stages of gravitropic response using our microarray system. Plants were grown on rock wool until the primary inflorescence Table 2. A list of differentially regulated genes in response to gravistimulation Gene family name Up-regulated genes Arabinogalactan protein Gene Type b Fold induction c name a 30 min 60 min AtAGP AtAGP20 A Chitinase CHI Expansin AtEXLA Extensin EXT Glycosyltransferase GT8-12 A XTH AtXTH22 A Down-regulated genes β-1,3-glucanase BGL2 C 2.25 ND β-1,4-glucanase CEL2 C IRX2 C Cellulose synthase IRX3 C IRX5 C Chitinase CTL2 C Galactosidase BGAL4 C 1.24 ND Glycine-rich protein GRP C Laccase Lac1 C IRX12 C Lac17 C Pectinesterase PMT61 C 1.04 ND PMT ND Peroxidase PER42 C PER64 C PER Polygalacturonase PG3 A PG20 C PG43 C 0.83 ND ND the ratio was not determined due to undetectable gene expression a When genes already had published names, we adopted them. Otherwise, the names of the genes we designed in our microarray system are indicated in italics b Type represents a set of genes that exhibit expression profiles. They are defined as A, B, and C (Imoto et al. 2005) c Values are averages of two independent experiments, and expressed as the log 2 ratio. A log 2 ratio of 1 is the same as a fold change of 2 stem reached a length of mm. They were then subjected to gravity stimulation by placing the whole plant on the rock wool block horizontally. After 30 or 60 min of gravistimulation, the apical 40-mm stem region, which ranged from 10 mm to 50 mm below the apex, was excised. Control plants were grown under the same conditions, and the same region was also excised. These excised samples were subjected to RNA extraction according to a conventional SDS phenol protocol. Oligo DNA microarray hybridization and data analyses were performed as described previously (Yokoyama et al. 2004; Imoto et al. 2005). Comparisons of the transcripts present in the control and in the gravistimulated stems revealed that 27 of 765 genes represented on the array exhibited significant expression changes within 60 min of gravitropic stimulation (Table 2). The majority of genes showing differential expression in response to gravitropic stimulation were down-regulated by gravistimulation. Interestingly, most of these down-regulated genes belong to the Type C gene set (Imoto et al. 2005).

4 192 CTL2 GRP Weight signal Gene expression Mechanical support LAC1 IRX12 LAC17 PMT61 Fig. 1. A possible explanation for the regulation of gene expression by weight signaling. The weight carried by the plant body enhances the expression of the key genes involved in secondary cell wall formation and provides mechanical strength to the supporting tissue. Plants are incapable of retaining the signal when placed horizontally PG20 PG43 PER42 In the basal region of the inflorescence stem, mechanical strength of xylem secondary cell walls is required to support the increasing weight carried by the upper portion of the stem. However, when the stem was placed horizontally, the weight carried by the upper portion of the stem was relieved, thereby circumventing the necessity of mechanical support of xylem secondary cell walls. It is suggested that the weight in the apical part may function as a signal for the induction of mechanical strength to xylem secondary cell walls in the basal region of the stem (Fig. 1). Type C genes were preferentially expressed in the basal region, which offers support to the upper portion of the stem (Imoto et al. 2005). Relief from the weight by placing the stem horizontally reduced the expression levels of some Type C genes dramatically. This result may be explained by the relief in weight signaling upon a reduction in the requirement for Type C genes: weight signaling derived from the upper part of the stem ordinarily causes expression of key genes responsible for xylem secondary cell wall formation in the basal region of the stem. However, if the stem is placed horizontally, a lower level of weight-signaling results in less activation of these genes. Recently, Ko et al. (2004) demonstrated that the application of artificial weight induces the production of significant amounts of secondary xylem tissues in A. thaliana inflorescence stems. This result is comparable to the effect of the reduced weight on secondary-wall-related genes investigated in the present study. It also supports our hypothesis that the weight carried by aerial plant parts is a signal for the formation of xylem secondary cell walls with mechanical strength. PER71 Fig. 2. Genomic structure and organization of candidate genes involved in xylem secondary cell wall formation, and locations of T- DNA insertion sites, indicated by solid boxes, in each gene. Open bars and lines indicate exons and introns, respectively Comparisons of two independent sets of microarray data, one from Imoto (2005) and the other from the present study led to the isolation of key genes responsible for the mechanical strength of xylem secondary cell walls. These genes include IRX2, IRX3, IRX5, and IRX12. IRX3 and IRX5 code for catalytic subunits of the cellulose synthase complex during the synthesis of secondary cell walls (Taylor et al. 2003). The IRX2 gene, which encodes β-1,4-glucanase, was shown to participate in secondary-wall cellulose synthesis in the xylem in coordination with cellulose synthase (Nicol et al. 1998). The irx12 mutant was caused by a defect in one of the three laccase genes that were identified in the present study (Brown et al. 2005). The IRX12 gene, as well as two other laccase genes, is the homolog of certain poplar laccase genes demonstrated to be essential for secondary cell wall formation (Ranocha et al. 2002). Similarly, PER42 is the closest A. thaliana relative to a peroxidase from Nicotiana tabacum implicated in lignin biosynthesis (Blee et al. 2003). AtCTL2, a member of the chitinase gene family, encodes proteins highly homologous to AtCTL1, which was previously implicated in the regulation of lignin deposition patterns (Zhong et al. 2002). Thus, it is logical to expect that the collection of uncharacterized genes identified in the present study offers a rich source of new targets for comprehensive studies of xylem secondary cell wall formation. Genes involved in xylem secondary cell wall formation Reverse genetic analyses of gene function To identify the roles of uncharacterized genes in xylem secondary cell walls, we examined phenotypes of T-DNA knockout mutants in which each gene was disrupted (Fig. 2). We identified T-DNA insertion lines for each gene

5 193 in the Salk Institute Genome Analysis Laboratory and isolated their homozygous lines. Insertion of T-DNA into individual genes was confirmed by polymerase chain reaction using primers specific to T-DNA left -border sequences and primers specific to each gene. Although the isolation and confirmation of some mutants is ongoing, no single mutant plant displayed obvious visible phenotypes. An exception was for a gene encoding a GRP. Extracellular enzymes such as pectinesterase, polygalacturonase, laccase, and peroxidase are typically encoded by large gene families in higher plants (Nishitani 2002; Yokoyama and Nishitani 2004). The absence of a phenotype in these mutants indicates functional redundancy among isovariants in the family. Loss-of-function mutations in the GRP gene displayed various morphological phenotypes in inflorescence stems. The GRP was restricted to the walls of protoxylem tracheary elements (unpublished data), and its specific localization suggests that it is involved in the capability of xylem conduits to allow the efficient transport of water and solutes. Recently, it has been shown that some GRPs are localized in the walls of protoxylem in various plant species (Keller et al. 1989; Ryser and Keller 1992; Ryser et al. 1997). A model was developed to illustrate the arrangement of GRPs in the protoxylem. It suggested that the GRPcontaining cell walls of tracheary elements are connected with the walls of surrounding cells, thereby stabilizing the whole protoxylem (Ringli et al. 2001a, b; Ryser et al. 2004). It is likely that the grp mutant has a defect in the stable structure of the protoxylem and reduces the efficient functioning of xylem, leading to the production of various phenotypes in the inflorescence stem. Conclusions A combination of expression analyses and reverse genetics has led to identification of the GRP gene, which may play important roles in the development of tracheary element walls in the xylem. This illustrates the success of combining expression data with reverse genetics to identify rapidly and efficiently the genes required for complex molecular processes, such as xylem secondary cell wall formation. However, no remarkable phenotypes were observed in the mutants of other genes identified as being expressed in supporting tissues. Possible reasons for the invisible phenotypes in most of these genes include functional redundancy among the identified genes and isovariants in the gene family. Thus, it would be difficult to identify each of these isovariants using traditional forward genetic screening. Characterization of double or triple mutants for candidate genes will be a challenge for future research in assigning functions to those genes. 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