Several PopFLAs are highly and specifically represented in tension wood
Recently, Schultz et al. (2002) classified 47 Arabidopsis glycosylphosphatidylinositol (GPI)-anchored AGPs into four groups: classical AGPs, AG-peptides, basic AGPs and fasciclin-like AGPs. Compared with the three other groups, the FLAs group is quite heterogeneous as it includes AGPs with one or two AGP-like domains and one or two fasciclin-like domains. Johnson et al. (2003) proposed to subdivide the Arabidopsis FLAs into groups named A to D. In this study, we reported 15 poplar full-length cDNA encoding fasciclin-like AGPs, named PopFLA1 to PopFLA15. Nine of these PopFLAs were similar to the group A of FLAs from Arabidopsis. These protein sequences were predicted to have a N-terminal secretion signal, a GPI anchor and one fasciclin-like domain flanked by two AGP-like regions.
The 15 PopFLAs were identified among a set of 10 062 ESTs sequenced from four different cDNA libraries prepared from different wood areas (CZ, DX-TW, DX-OW and MX) (Déjardin et al., 2004). Among the 15 PopFLAs, six were closely related sequences, classified into six different contigs within the cluster 1592, whereas the others originated from nine different clusters. The examined PopFLAs genes were highly expressed during wood formation. Interestingly, none of these PopFLAs transcripts were detected in CZ samples. CZ samples have been harvested by scraping the bark surface with a scalpel and they contain not only cambium cells but also very young expanding xylem and phloem cells. This suggests that PopFLAs are most likely expressed during xylem differentiation once the xylem cells have started to build their secondary cell wall. Several AGP genes were also shown to be highly expressed in differentiating xylem from loblolly pine (Loopstra & Sederoff, 1995; Loopstra et al., 2000; Zhang et al., 2003). As with pine AGPs, the observed specific expression of the PopFLAs in differentiating and mature xylem suggests an important but still undetermined function for these proteins during xylogenesis and secondary cell wall thickening.
Moreover, expression analyses revealed that 10 of the PopFLAs genes, PopFLA1 to PopFLA10, were very strongly expressed in DX-TW and MX-TW, while no or very low expression could be detected in the opposite wood (Fig. 2). This expression profile was consistently accessed by using independant approaches such as ESTs distribution in cDNA libraries, Northern blot hybridisations, cDNA microarray or PCR. In addition, RNA accumulation for these 10 PopFLAs except PopFLA9, appears slightly higher in mature xylem cells than in differentiating xylem (Fig. 2). In loblolly pine, several AGP genes also appeared to be regulated in compression wood (Loopstra & Sederoff, 1995; Zhang et al., 2000). Indeed, based on the analysis of ESTs distribution in pine cDNA libraries, three of the six available pine AGP genes were over-represented in compression wood (Whetten et al., 2001). Compression wood has a mechanical function similar to tension wood, however, it is very different chemically and anatomically. It remains to be elucidated how AGPs act during secondary cell wall differentiation.
Could the specific expression of these 10 PopFLAs be related to features specific to tension wood? TW differs mainly from normal wood by an increased rate in cambial cell division and the differentiation of G-fibres. As PopFLAs are almost not expressed in cambial cells, it is likely that they play a role in the differentiation of G-fibres.
Although PopFLA11–15 are closely related to the other 10 PopFLAs sequences, they exhibit different expression patterns. These five PopFLAs are also specifically expressed in xylem tissues, as are the others; however, their expression wasn't regulated in tension wood. In addition, the expression of four of them, PopFLA11, 13, 14 and 15 (PopFLA15 has been previously published as Pop14A9; AF183809), appears higher in differentiating xylem than in mature xylem. It remains to be determined whether this specific expression pattern accounts for a specific function during cell wall differentiation (Fig. 2). From sequence alignment, we observed that PopFLA11 and 13 were very similar and appeared to merge to the Arabidopsis FLA12 and to the cotton GhAGP1 (Fig. 1). Furthermore, PopFLA11–15, FLA12 and GhAGP1 have a very short proximal AGP-like domain. Pop14A9 was isolated from differentiating poplar xylem and the accumulation of GhAGP1 was reported to increase as soon as 5 d post anthesis during cotton fibre elongation and differentiation (Ji et al., 2003).
Rocket electrophoresis indicated that AGPs accumulated specifically in TW compared with OW either from DX or MX samples (Fig. 4). These results correlate well with the expression pattern of PopFLA1–10. Moreover AGPs appear more abundant in fraction A (Fig. 4), which contains most likely the cytoplasmic cell compartment together with the plasma membrane-bound proteins. AGPs may also be imbedded within the cell wall, or weakly attached to the cell wall.
We investigate the cellular localization of those AGPs with the JIM14 antibody known to recognize epitope from AGPs associated to the cell wall (Knox et al., 1991). This antibody reacts with a carbohydrate epitope that is potentially present on various protein backbones of AGPs. Therefore, it is not specific of the fasciclin-like AGPs, and in poplar wood samples, it mainly binds two polypeptides of high molecular weight (Fig. 5). As the two signals increase markedly in DX-TW and MX-TW compared with OW samples and because the PopFLAs are among the most accumulated transcript in TW, we can reasonably assume that some TW-regulated PopFLAs are recognized by JIM14. It should be pointed out that with JIM14 antibody, the stronger signal obtained in the Western blot analysis occurs in the TW fractions composed of cell wall-bound proteins (Fig. 5 Fraction B). In agreement with this, immunocytolocalization study indicates that JIM14 decorates the inner surface of the G-layer. Nevertheless, recognition of TW-regulated PopFLAs by JIM14 remains to be unequivocally demonstrated. The labelling observed in the middle lamella, the primary cell wall and the ray parenchyma cells may correspond to other AGPs like PopFLA11–15 or even other uncharacterized AGPs. RNA expression analysis, biochemical investigations and immunolocalization support the hypothesis of the high expression of some PopFLAs in tension wood and raise the question of their function in the formation of this tissue.
The strong differences in the expression pattern of the different PopFLAs probably reflect different cellular functions that remain to be elucidated. Based upon their predicted structure, potential role of these proteins during tension wood formation may be related to adhesion properties and signalling.
From deduced structure to predicted function for PopFLAs in tension wood formation
Nine PopFLAs proteins belong to the subclass of FLAs with a single fasciclin domain bordered by two domains predicted to be highly glycosylated usually found in arabinogalactan proteins (AGP-like domains) and a potential anchoring to the glycosylphosphatidylinositol (GPI). Presently, direct evidence for GPI anchoring is still lacking for a number of predicted GPI-anchored proteins and most of them are inferred from sequence analyses. GPI anchoring is believed to enable a great mobility of the protein at the surface of plasma membrane and permits localized delivery of the protein after GPI cleavage by C phospholipases. In silico analysis indicated that all but six PopFLAs are suitable candidates for plasma membrane-bound GPI-anchored proteins. The six FLAs without GPI anchorage (PopFLA3, 4, 6, 8, 9 and 10) are up-regulated in tension wood. These PopFLAs are likely secreted or cell wall-bound proteins and are potential candidates for the AGPs detected by JIM14 in the border of the G-layer. Although many plant GPI-anchored proteins have been documented (Borner et al., 2002; Borner et al., 2003), very few have been functionally studied. However, functional data have been recently reported for three Arabidopsis GPI-anchored proteins encoded by cobra, sku5 and sos5 (Schindelman et al., 2001; Sedbrook et al., 2002; Shi et al., 2003) and for a rice COBRA-like protein (Li et al., 2003). All these GPI-anchored proteins seem to be involved in the orientation of cell elongation and/or in cell-to-cell adhesion. In our study, JIM14 decorates the plasma membrane region and the inner side of the G-layer. This suggests that some GPI-anchored PopFLAs, namely PopFLA1, 2, 5 and 7, are mobilized even transiently to the G-fibre plasma membrane. Their possible function may be related to the establishment of connections between cell wall and cytoskeleton during fibre expansion and elongation.
All the deduced PopFLAs proteins share a central β-immunoglobulin (Ig)-H3/fasciclin domain known to occur as multiple repeats in a limited number of proteins including Drosophila fasciclin I and TGF-β induced protein Ig-H3 domain. β-Ig-H3/fasciclin domain is present in Algal-CAM, a Volvox glycoprotein, and seems to play an important role in cell adhesion (Huber & Sumper, 1994). All 15 PopFLAs exhibit two highly conserved motifs, namely H1 and H2, on the edges of the fasciclin domain (Kawamoto et al., 1998, Fig. 1). Flanking these two motifs, several residues have been shown to be important for mediating adhesion properties (Kim et al., 2000; Kim et al., 2002). In human cells, DI and YH motifs are thought to interact with several components of the extracellular matrix such as integrins. PopFLAs do not contain a DI motif, which is not surprising as this motif is only present in Arabidopsis FLAs from group B (Johnson et al., 2003). However, as in group A of Arabidopsis FLAs, FH (Phe His) residues are often found in PopFLAs in place of the YH (Tyr His) conserved motif. There is still lack of experimental evidence in plants for FLAs adhesion properties. Recently, Shi et al. (2003) published the first report on the functional analysis of a fasciclin-like protein in an Arabidopsis mutant. The mutated gene, FLA4 (also named sos5), encodes a putative cell surface adhesion protein containing two fasciclin-like domains. The sos5 mutant is characterized by abnormal root cell expansion and defective cell wall structure. From this phenotype it has been suggested that FLA4 functions through noncovalent ionic interactions between its fasciclin domains and other FLAs (Johnson et al., 2003). In order to regulate cell expansion, such interactions may occur at the plasma membrane and/or in the cytoplasm.
Alternatively, FLAs like AGPs containing N-acetylglucosamine can be a substrate for chitinase (Rojas-Herrera & Loyola-Vargas, 2002) leading to the release of oligosaccharide signal molecules. Endochitinase activity may either operate through the inhibition of specific AGP signalling molecules, and/or upon the liberation of oligosaccharides acting as Nod factor (Domon et al., 2000). In such a system, already described on a plant embryogenesis model (McCabe et al., 1997), oligosaccharin transmits a signal necessary to induce somatic embryo formation. A similar process may occur during tension wood formation as suggested by the high expression of an endochitinase gene in the DX-TW library (Déjardin et al., 2004). Such a signal could potentially be transmitted through the ray parenchyma cells toward the cambium.
This study demonstrates that many FLAs are expressed in woody tissues, whereas some of them are specifically regulated during tension wood formation and are among the most highly expressed genes in this reaction wood. Likewise, preliminary studies at the protein level revealed an accumulation of AGPs in the tension wood zone. Moreover, high-resolution localization studies with an antibody specifically directed against AGPs indicated that some AGP accumulation occurs close to the plasma membrane on the internal side of the G-layer, a characteristic feature of tension wood fibres. Immunocytological studies with FLAs specific antibodies help delineate the specific localisation of the different PopFLAs. Toward this end, antibodies against specific domains of the protein backbone could be raised, although the extensive glycosylation of these proteins may interfere with recognition between such antibodies and native FLAs in poplar wood. Alternatively, antibodies directed toward wood purified FLAs fractions could circumscribe this problem. Finally, strategies are currently being explored to investigate the function of PopFLAs during wood formation, through the production of transgenic poplars with altered PopFLAs expression.