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Wood – the hard, fibrous, lignified secondary xylem tissue in the stems of woody plants – is of major environmental and economic importance. As the most biologically important sink for excess atmospheric CO2, it can contribute to reducing global warming (Plomion et al., 2001; Boudet et al., 2003), and as a renewable natural resource that forms the basis of a major global industry producing fibre, timber and energy, it is the fifth most important product of world trade.
Wood formation, or xylogenesis, begins when cells in a lateral meristematic tissue called the vascular cambium divide and differentiate to give rise to secondary xylem. The term ‘cambium’, in its strictest sense, refers to one or several layers of cells called initials (Lachaud et al., 1999). Periclinal divisions of these initials produce phloem or xylem mother cells that undergo several rounds of cell division before differentiating (Lachaud et al., 1999). These layers of undifferentiated cambial cells and xylem or phloem mother cells are referred to the ‘cambial zone’. The differentiation of xylem mother cells involves processes fundamental to plant development such as cell elongation, cellulose deposition, deposition of lignin in secondary cell walls and programmed cell death (Ye, 2002).
To provide insights into the genetic control of xylem formation, many large-scale transcriptome analyses of isolated vascular tissues or cultured differentiating tracheary elements have been performed in model plants such as arabidopsis (Oh et al., 2003; Brown et al., 2005; Zhao et al., 2005) and zinnia (Demura et al., 2002) as well as in commercially important species, including poplar (Sterky et al., 2004), pine (Allona et al., 1998; Hertzberg et al., 2001; Whetten et al., 2001; Lorenz & Dean, 2002) and eucalyptus (Paux et al., 2004, 2005; Foucart et al., 2006; Gallo de Carvalho et al., 2008; Novaes et al., 2008). Transcript profiling of thousands of genes in vascular tissues has uncovered a large number of genes expressed preferentially in xylem, but most of the regulatory genes devoted specifically to xylogenesis still remain to be identified.
While attempting to characterize candidate genes potentially involved in xylogenesis in eucalyptus, we isolated a partial sequence encoding a Rac-like small GTPase from a subtractive Eucalyptus gunnii cDNA library enriched in sequences preferentially expressed in secondary xylem (Paux et al., 2004, 2005; Foucart et al., 2006). Small GTPases are monomeric guanine nucleotide-binding proteins that act in eukaryotic cells as molecular switches (Yang, 2002; Vernoud et al., 2003; Nibau et al., 2006); they are activated by binding GTP and inactivated by hydrolysis of the bound GTP to GDP (Bourne et al., 1991). The balance between GTP- and GDP-bound forms is maintained by the action of several regulatory molecules: GTPase-activating proteins (GAPs) inactivate small GTPases by stimulating GTP hydrolysis whereas guanine nucleotide exchange factors (GEFs) activate them by favouring the binding of GTP.
In eukaryotes, the Ras superfamily of small GTPases contains five families: the Ras, Rab, Ran, Arf and Rho GTPases (Hall, 1998; Etienne-Manneville & Hall, 2002). The Rho family itself is divided into three subfamilies: Rho, Rac and Cdc42. Interestingly, there is no evidence of Ras family proteins in plants although many small GTPases of the Rho family have been identified from different plant species (Winge et al., 1997; Yang, 2002; Christensen et al., 2003), especially Rac proteins (Winge et al., 1997). Plant RACs seem to constitute a unique subfamily of the Rho GTPases, called ROP (Rho-related GTPase from plants) (Yang & Watson, 1993; Li et al., 1998; Vernoud et al., 2003). Both RAC and ROP are currently used synonymously in the literature to identify the same type of small plant GTPases (Yang, 2002).
During the past decade, ROP GTPases have emerged as key regulators of a number of cellular processes in plants (Zheng & Yang, 2000; Yang, 2002; Gu et al., 2004; Nibau et al., 2006; Yang & Fu, 2007; Yalovsky et al., 2008), including actin cytoskeleton organization, cell polarity, cell growth and differentiation, cell wall formation, cell death, regulation of cytosolic Ca2+ concentration, and H2O2 production, ubiquitin-dependent proteasome-mediated proteolysis and stress-induced or hormone signal transduction, all of which are known to play roles in xylogenesis (Ye, 2002).
Here, we report the cloning and functional analysis of EgROP1, a Rac-like ROP gene preferentially expressed in the differentiating xylem zone of eucalyptus. Association studies in a eucalyptus breeding population colocalized EgROP1 with QTLs (quantitative trait loci) related to wood fibre morphology and lignin content. We tested the hypothesis that EgROP1 is involved in xylogenesis by heterologous functional characterization of EgROP1 activity in transgenic Arabidopsis thaliana plants. This suggested that EgROP1 plays a role in xylem cell differentiation during the early events of secondary xylem formation. Our results suggest that EgROP1 is a vascular-expressed Rac-like gene that likely affects secondary xylem differentiation, making it a reasonable candidate for marker-based selection of eucalyptus for altered wood property traits.
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As part of a functional genomic approach aimed at cloning candidate regulatory genes involved in xylogenesis in Eucalyptus, we identified an expressed sequences tag (EST) encoding EgROP1, a putative Rac-like ROP protein, from two distinct subtractive cDNA libraries enriched in xylem-expressed sequences (Paux et al., 2004; Foucart et al., 2006). Because ROPs play key roles as molecular switches in signalling and are involved in cellular processes known to occur during xylogenesis, we chose this candidate gene for further functional studies. The work we describe in this paper suggests that EgROP1 may indeed be involved in secondary xylem differentiation, a developmental process of particular importance in vascular plants.
Phylogenetic comparison of EgROP1 with other ROPs placed it in subgroup IV (Li et al., 1998). Several reports suggest that ROPs within a phylogenetic group may be involved in similar cellular processes (Yang, 2002; Gu et al., 2004). Group IV proteins, such as AtROP1 and the closely related AtROP3 and AtROP5, are expected to have overlapping functions in the maintenance of tip growth of root hairs or pollen tips by regulating cell polarity and cell expansion through actin organization (Lin & Yang, 1997; Kost et al., 1999; Li et al., 1999; Fu et al., 2001, 2002; Molendijk et al., 2001). The high protein sequence similarity between EgROP1 and other group IV ROPs indicate that it may play a similar role to AtROP1, AtROP3 and AtROP5, particularly in anisotropic growth through cytoskeleton organization.
We assessed the function of EgROP1 in A. thaliana plants overexpressing wild-type, constitutively active and dominant negative forms of the protein. Overexpression of EgROP1 genes affects cell morphology in various cell types in A. thaliana leaves. The leaf pavement cell phenotypes of these plants resemble those observed in plants expressing AtROP2-CA or AtROP7-CA, two other subgroup IV ROPs expressed in leaves (Fu et al., 2002, 2005; Brembu et al., 2005). Recently, Fu et al. (2005) proposed a model for ROP/RAC control of leaf pavement cell morphogenesis. Briefly, ROP2 activates the RIC4 pathway to promote lobe outgrowth by creating a fine cortical microfilament network, while it inhibits the RIC1 pathway, which suppresses outgrowth by promoting the formation of well-organized cortical microtubules. Although EgROP1 is unlikely to be involved in controlling leaf epidermal cell morphology, as it is not expressed preferentially in leaves, its function is similar enough to other subgroup IV arabidopsis ROPs to affect related mechanisms when expressed ectopically in A. thaliana.
Overexpression of either EgROP1-CA or EgROP1-OX also affected trichome morphology by increasing the proportion of trichomes with four branches. Trichome formation is an extreme example of anisotropic growth (Mathur et al., 1999; Mathur, 2004). Trichome branching in arabidopsis seems to be mediated by transiently stabilized microtubule structures (Mathur et al., 1999; Mathur & Chua, 2000). A previous case of altered trichome morphology was reported for transgenic plants overexpressing AtROP2-CA (Fu et al., 2002). Interestingly, phenotypes similar to those presented in EgROP1 transgenic plants (i.e. tubular-shaped and swollen epidermal cells as well as a reduction in trichome branch number) have also been described in A. thaliana plants defective in AtKTN1, a protein involved in regulating microtubule disassembly (Burk et al., 2001). Consistent with this idea, numerous cytoskeletal-associated proteins have now been shown to be involved in regulating cell morphogenesis, some of them being regulated through ROP activation (Mathur & Hulskamp, 2002; Wasteneys & Yang, 2004; Yalovsky et al., 2008). This data, along with the leaf cell morphology phenotypes we observed in plants overexpressing EgROP1, suggest that EgROP1 may interfere with the normal function of ROPs that control the direction of cell elongation through microtubule polymerization and organization.
Interestingly, EgROP1-DN-induced phenotypes in leaves were not the opposite of those observed with EgROP1-OX and EgROP1-CA. Our hypothesis is that EgROP1 may fail to fully interact with arabidopsis GAP and GEF proteins in the leaf, an organ in which it may not normally function in eucalyptus. In this case, EgROP1-CA and EgROP1-DN expression would not have complete dominant effects in A. thaliana, although they might induce some similar perturbing effects. Nevertheless, real-time RT-PCR confirmed that transgenes were expressed, and the EgROP1-OX, CA and DN secondary xylem cell morphologies were distinguishable, suggesting that all three kinds of EgROP1 proteins had different protein activities.
Consistent with previous transcriptomic studies that showed that an SSH clone from the EgROP1 gene was classified as preferentially expressed in Eucalyptus secondary xylem in comparison with reaction wood or leaves (Paux et al., 2004, 2005; Foucart et al., 2006), EgROP1 is expressed in the vasculature, particularly in the cambial and differentiating secondary xylem zones. Two other ROP genes, AtROP7 and ZeRAC2, are also expressed preferentially in vascular cells (Nakanomyo et al., 2002; Brembu et al., 2005). This specific EgROP1 expression pattern is consistent with studies on other ROP genes, which described distinct patterns of expression and intracellular sites of protein activation (Delmer et al., 1995; Li et al., 1998; Molendijk et al., 2001; Nakanomyo et al., 2002; Brembu et al., 2005). This pattern may allow for functional diversity between proteins that share a very high degree of identity (Valster et al., 2000). Consequently, the action of a given ROP protein may be determined more by its cellular and subcellular localization than by its functional domains, which could be more or less identical to other ROPs. If this is the case, the pattern of EgROP1 expression would indicate that EgROP1 is likely involved in the signalling processes involved in xylogenesis. This conclusion is strengthened by our studies of the effects of EgROP1 expression in the vascular system of A. thaliana.
Cell size in the secondary xylem of EgROP1 transgenic plants was significantly affected, especially in EgROP1-CA lines, with an increase of up to 30% in cell size when compared with the control. By contrast, secondary xylem elements were smaller in EgROP1-DN lines than in the control. Interestingly, a Rac GTPase-activating protein was identified within a short list of arabidopsis genes coexpressed with IRX3 and potentially involved in secondary cell wall formation in arabidopsis (Brown et al., 2005), which suggests that such a small GTPase-mediated signalling pathway likely exists in this developmental process. Moreover, the opposite effects of EgROP1-CA and EgROP1-DN on secondary xylem cell size indicate that EgROP1-DN may effectively block the molecular mechanisms that control cell expansion during xylogenesis in arabidopsis, which may involve similar proteins in arabidopsis and eucalyptus, thus suggesting a role for EgROP1 in xylem cell expansion in eucalyptus too.
In plants overexpressing EgROP1-CA, the interfascicular secondary xylem cells were not only larger than in control plants but were also stained brown by Maüle reagent, similar to arabidopsis vessel elements, which contain only G units. Longitudinal sections confirmed that these cells were vessel-like elements. Moreover, patches of unlignified cells with thin cell walls and no secondary cell wall-specific xylans were observed in EgROP1-CA overexpressors, suggesting that EgROP1 function may influence later stages of xylogenesis such as secondary cell wall formation. Interestingly, this secondary cell wall defect seemed to affect only fibre cells, a phenotype that has already been described for plant-specific transcription factors of the NAC family that regulate xylary fibre development (Zhong et al., 2006; Ko et al., 2007). As already proposed for ZeRAC2 in Zinnia elegans (Nakanomyo et al., 2002), we hypothesize that EgROP1 might influence xylem cell composition by favouring the formation of tracheary elements. As a consequence of this increased vessel to fibre ratio, the lignin profiles will be modified because arabidopsis vessels only contain G units whereas fibres also contain S units. A decrease of the S : G ratio is therefore expected since more vessels are formed and a large number of the newly formed fibres in secondary xylem do not synthesize lignified secondary cell walls.
The QTLs for fibre cell morphology and lignin monomer composition colocalized with the EgROP1 locus on the linkage group 6 of E. urophylla. EgROP1 may underlie these QTLs, as the gene has significant effect on fibre width and G content (and therefore on the S : G ratio) in eucalyptus wood. This idea is consistent with the observations made in the secondary xylem of arabidopsis plants overexpressing EgROP1, which exhibit larger vessel-like cells containing G units. Therefore, QTL analysis supports the hypothesis that EgROP1 plays a role in secondary xylem cell formation in eucalyptus and may affect secondary cell wall formation.
This work shows that EgROP1 may play a role in xylogenesis, a process of fundamental importance in woody species. Importantly, the results we obtained from both functional characterization in transgenic A. thaliana plants and linkage analysis on field-grown eucalyptus trees demonstrate that functional characterization of further eucalyptus genes in this model species will be a valuable approach towards identifying candidate genes for molecular marker-assisted selection of wood quality traits.