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Keywords:

  • cell expansion;
  • eucalyptus;
  • lignification;
  • Rac-like ROP;
  • secondary cell wall;
  • secondary xylem;
  • wood quality

Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References
  • • 
    To better understand the genetic control of secondary xylem formation in trees we analysed genes expressed during Eucalyptus xylem development.
  • Using eucalyptus xylem cDNA libraries, we identified EgROP1, a member of the plant ROP family of Rho-like GTPases. These signalling proteins are central regulators of many important processes in plants, but information on their role in xylogenesis is scarce.
  • • 
    Quantitative real-time reverse-transcriptase polymerase chain reaction (qRT-PCR) confirmed that EgROP1 was preferentially expressed in the cambial zone and differentiating xylem in eucalyptus. Genetic mapping performed in a eucalyptus breeding population established a link between EgROP1 sequence polymorphisms and quantitative trait loci (QTLs) related to lignin profiles and fibre morphology. Overexpression of various forms of EgROP1 in Arabidopsis thaliana altered anisotropic cell growth in transgenic leaves, but most importantly affected vessel element and fibre growth in secondary xylem. Patches of fibre-like cells in the secondary xylem of transgenic plants showed changes in secondary cell wall thickness, lignin and xylan composition.
  • • 
    These results suggest a role for EgROP1 in fibre cell morphology and secondary cell wall formation making it a good candidate gene for marker-based selection of eucalyptus trees.

Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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, inline image 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.

Materials and Methods

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Cloning and sequencing of a full-length EgROP1 cDNA

Polymerase chain reaction was performed on an E. gunnii xylem full-length cDNA λZapII library (Poeydomenge et al., 1994) with the T3 universal primer and a specific primer (EgRAC1 up 5′-tgaggaccgggtcttgttag-3′) that hybridized the 3′-end of the 386 bp cDNA fragment corresponding to the 3′-end of EgROP1 (clone RTC(19H05); (Foucart et al., 2006)). A 914 bp PCR fragment containing the ORF and the 5′-UTR was cloned into the pGEMT-easy vector (Promega) to obtain pGEMT–EgROP1. Both strands of EgROP1 cDNA were sequenced at the Génopôle of Toulouse, France. Protein sequence comparisons were conducted with the blastx algorithm (Altschul et al., 1997) against the National Center for Biotechnology Information nonredundant database (http://blast.ncbi.nlm.nih.gov/Blast.cgi?CMD=Web&PAGE_TYPE=BlastHome). Phylogenetic classification was performed with the basic genebee clustalw1.83 software (http://www.genebee.msu.su/clustal/basic.html).

Tissue harvesting and RNA extraction

Shoot tips, petioles, vascular tissue samples and leaves were harvested in July 2007 from 7-yr-old field-grown E. gunnii trees (clone 0867; AFOCEL, Longages, France). They were immediately frozen in liquid nitrogen and stored at −80°C. The central vein of the leaves was removed to minimize the proportion of primary xylem. Secondary phloem was scraped from exposed vascular tissue after removal of the cork. Differentiating secondary xylem was scraped from the exposed xylogenic tissue after removal of the bark. The vascular cambium-enriched sample was obtained by smoothly scraping the inner side of the bark. Tips of growing roots were harvested from 2-yr-old glasshouse-grown E. gunnii trees of the same AFOCEL clone. RNA was extracted as described previously (Southerton et al., 1998; Foucart et al., 2006). Remaining traces of DNA were removed with RQ1-RNase free DNase (Promega). RNA quality and quantity was checked with an RNA 600 Nano kit on a BioAnalyser 2100 (Agilent, Santa Clara, CA, USA) and a Nanodrop ND-1000 Spectrophotometer (Labtech, Palaiseau, France).

Quantitative real time reverse-transcriptase polymerase chain reaction (qRT-PCR)

RNA was cleaned on an RNeasy column (Qiagen). First-strand cDNA synthesis was from 1 µg of total RNA using Superscript III RetroTranscriptase (Invitrogen). First-strand cDNAs were purified with the QIAquick purification kit for PCR products (Qiagen) and eluted in a final volume of 100 µl. Real-time PCR was performed on a Light Cycler (Roche Diagnostics) using the LightCycler FastStart DNA MasterPLUS SYBR Green I kit (Roche Diagnostics), as previously described (Foucart et al., 2006). Specific primer pairs were designed for EgROP1 (up, 5′-agcgcgtcgaggttcatcaa-3′; down, 5′-tggggaaagtgttgctggtata-3′) and E. globulus isocitrate dehydrogenase (EgIDH) (up, 5′-ctgctggaatctggtatgaaca-3′; down, 5′-tcactctggacatctccatctccatca-3′). EgIDH served as the reference gene as previously described (Paux et al., 2004; Goicoechea et al., 2005). The specificity of primer annealing was examined by monitoring the dissociation curve when real-time PCR reactions were completed, and the standard curve slope was determined for each PCR run and used to calculate PCR efficiency. Quantification of EgROP1 expression relative to EgIDH was determined using the 2−ΔCT method (Pfaffl, 2001).

Construction of expression vectors containing mutated EgROP1 cDNAs

Quick-change site-directed mutagenesis (Stratagene) was used to create mutant sequences encoding G15V-EgROP1 (EgROP1-CA) and T20N-EgROP1 (EgROP1-DN). Two pairs of oligonucleotides containing the desired changes (underlined) were used to mutate the EgROP1 sequence carried by the pGEMT-EgROP1 vector: EgRAC1M2b (5′-cggcgacgttgccgtcggcaaaacctgcc-3′) and EgRAC1M2bRev (5′-ggcaggttttgccgacggcaacgtcgccg-3′) for the G15V mutation; and EgRAC1M3b (5′-gcggtcggcaaaaactgcctgttgatttc-3′) and EgRAC1M3bRev (5′-gaaatcaacaggcagtttttgccgacggc-3′) for the T20N mutation. Mutated genes (EgROP1-CA and EgROP1-DN) were then cloned as BamHI–SalI digests into the BamHI and SalI sites of the binary pBLTi 121 vector (Pagny et al., 2000) downstream of the CaMV35S promoter. An EgROP1 (EgROP1-OX) overexpression construct was also made by cloning the EgROP1 wild-type cDNA downstream of the CaMV35S promoter.

Generation and molecular analysis of A. thaliana mutants

The pBLTi vectors carrying EgROP1 cDNAs were introduced into Agrobacterium tumefaciens strain GV3101 by using the freeze-thaw procedure (Holsters et al., 1978). Wild-type A. thaliana plants (ecotype Columbia, Col) were transformed by infiltration (Clough & Bent, 1998). Control lines were generated by transformation with the vector pBLTi 121 that did not contain the EgROP1 gene. Potential T1 transformants were selected by germination on sterile Murashige and Skoog (MS) medium containing kanamycin (50 µg ml−1). Transformation was confirmed by PCR on leaf disks (Klimyuk et al., 1993). Homozygous T3 transgenic plants with a single T-DNA insertion locus, as estimated by the Mendelian segregation of the kanamycin-resistant NptII gene, were used for phenotype characterization. Expression of the transgene was confirmed by RT-PCR using the SuperScript first-strand synthesis kit for RT-PCR (Invitrogen) performed on RNA extracted from 3-wk-old transgenic seedlings using the Extract-All kit (Eurobio, http://www.eurobio.fr).

Sample preparation for microscopy and imaging

Microscopic analyses of the secondary vascular system were performed on transgenic A. thaliana plants grown under short-day conditions (cycles of 9 h light and 15 h dark) at 22°C with 70% relative humidity for 2–4 months to induce substantial secondary thickening in the hypocotyl and epicotyl (Chaffey et al., 2002).

Semithin sections (120 µm) were prepared from fresh samples using a VT1000S vibratome (Leica). Lignin distribution in unstained samples was studied by using epifluorescence illumination (excitation filter, BP 340–380 nm, suppression filter LP 430 nm) and in sections stained with Maüle reagent (Meshitsuka & Nakano, 1979). Observations were made using an inverted microscope (DMIRBE; Leica) and images were acquired using a CCD camera (colour Coolview; Photonic Science, Robertsbridge, UK). Measurements and image treatments were performed using image pro plus software (Media Cybernetics, Bethesda, MD, USA).

Other sections were stained for confocal microscopy with an aqueous solution of Congo red (0.1% w : v) (Verbelen & Kerstens, 2000). Images were acquired with a SP2 SE spectral confocal laser scanning system (Leica) equipped with an upright DM 6000 microscope (Leica) and a ×40 (HCX PL APO, N.A. 0.8) water immersion objective lens. Samples were excited at 405 nm and 561 nm to reveal lignified (green) and nonlignified (red) cell walls, respectively. Pictures were computed by projection of 10–20 plan-confocal images acquired in the z dimension at 0.5–1 µm increments.

Immunolabelling was performed on samples fixed in 2.5% (v : v) glutaraldehyde in 50 mm cacodylate buffer (pH 7.0), then dehydrated in an ethanol series (20, 40, 60, 80, 95 and 100%) and embedded in LR White resin (Electron Microscopy Sciences, Hatfield, PA, USA). Cross-sections 1-µm thick were immunostained for (1-4)-β-d-xylans using a rat LM10 monoclonal antibody (Plant Probes, Leeds, UK) and a goat anti-rat IgG coupled to the fluorescent dye Alexa Fluor 633 (Molecular Probes, Eugene, OR, USA) as described previously (Peumans et al., 2007). Observations were carried out by confocal microscopy. Excitation and emission were at 633 nm and 640–680 nm, respectively. Cell walls were observed by their autofluorescence when irradiated with the 488 nm ray line of an argon laser, the emitted light being collected in the 500–550 nm range. Some sections of resin-embedded material were stained with 0.05% toluidine blue and examined by the use of bright-field microscopy.

For scanning electron microscopy, leaf samples were dehydrated in an ethanol series and dried using a CPD 750 EmScope critical point dryer. Tissue was then coated with gold–palladium (20 nm) and observed with a Hitachi S450 scanning electron microscope. Sizes of macerated secondary xylem cells were measured from approx. 15-mm long stem pieces corresponding to hypocotyls and basal parts of inflorescences prepared according to the method of Chaffey et al. (2002). Pulped material was mounted on a glass slide, images were acquired by using bright-field microscopy and measurements were performed using image pro plus software (Media Cybernetics).

Detection of EgROP1 polymorphisms

Genomic DNA from 201 full sibs of an interspecific hybrid F1 progeny of Eucalyptus urophylla and Eucalyptus grandis was extracted from dried leaves as described by Verhaegen & Plomion (1996). EgROP1 primer pairs were designed from the E. gunnii ROP1 sequence (EgRAC1 up, 5′-gatccgcatttccccatcgc-3′; EgRAC1 down, 5′-caagccacgccaattcaacc-3′) using oligo explorer 1.1.0 software (Gene Link, Hawthorne, NY, USA). The amplified EgROP1 promoter fragment contained a simple sequence repeat motif. Template DNA was amplified by PCR under the following conditions: preliminary denaturation for 4 min at 94°C, followed by 30 cycles of denaturation for 45 s at 92°C, annealing for 45 s at 58°C and extension for 90 s at 72°C, and a final extension for 10 min at 72°C. Single-stranded conformation polymorphism (SSCP) analysis was performed as previously described (Bodénès et al., 1996).

Wood chemical and fibre properties were estimated from several levels on each tree: a disk was taken for chemical analysis at half of the commercial height (1.3 m above ground) and at three-quarters of commercial height for fibre analysis. Lignins were characterized by thioacidolysis–solvolysis of 15 mg of extractive-free wood in a dioxane–ethanethiol mixture (9:1 v : v) containing 0.2 m boron trifluoride etherate, for 4 h in an oil bath at 100°C. The thioacidolysis-recovered monomers were quantified by GC-MS (Saturn 2000; Varian, Palo Alto, CA, USA) of their trimethylsilylated derivatives. Kraft pulping was done according to Gullichsen & Fogelholm (2000). Two grams of air-dry pulps were used for fibre morphology assessment with a PQM 1000 fibre analyser (Metso Paper, Helsinki, Finland). Fibre properties measured were: length, width, coarseness (a measure of mass per unit length of fibre) and curl index (measured as (real length/projected length – 1)100), an assessment of the straightness of the fibres).

The construction of the genetic maps of both E. urophylla and E. grandis parents was done following the double-pseudo-testcross mapping strategy (Grattapaglia & Sederoff, 1994) as previously described (Verhaegen & Plomion, 1996) using mapmaker/exp 3.0 software (Lander et al., 1987). Loci were assigned to linkage groups (LG) with a minimum LOD score of 3.0 and a maximum Kosambi distance of 40 cM. Graphs of linkage maps and QTLs were drawn using the mapchart software 2.2 (Voorrips, 2002). Detection of QTLs was carried out with the multiqtl V2.6 software (http://www.multiqtl.com). Interval mapping procedure was used to identify and position QTLs, as well as to estimate their percentage of explained variance (PEV). Empirical statistical significance thresholds were determined after 1000 permutations of the dataset (Doerge & Churchill, 1996). The empirical confidence interval at 95% for the QTL position and for the allelic effect were calculated using bootstrap analysis (Visscher et al., 1996) with 5000 resamplings.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

Cloning and sequence analysis of EgROP1, a RAC/ROP GTPase identified from a Eucalyptus xylem-subtracted cDNA library

We previously reported a protein with high homology (E-value = 6E-25) to the A. thaliana small GTPase AtROP1 that was cloned from two distinct suppressive subtractive hybridization libraries ((i.e. a secondary xylem versus leaves (Paux et al., 2004) and a xylem versus phloem library (Foucart et al., 2006)). These clones were used to obtain a full-length cDNA (914 bp) carrying the ORF with both 5′- and 3′-UTR extremities from an E. gunnii xylem cDNA library (Poeydomenge et al., 1994), that has been named EgROP1 (accession number: EF392836) (Fig. 1).

image

Figure 1. Phylogenetic tree and sequence comparison of Rac/ROP GTPases. (a) The phylogenetic relationship between EgROP1 and a set of Rac-like ROP GTPases. PtrROP is the putative product of the Populus trichocarpa estExt_fgenesh4_pg.C_1810025 gene. (b) Protein sequence alignment of EgROP1, PtrROP, PsROP1, BvRho1, AtROP1, AtROP3, AtROP5, NtRAC1 and MsRAC1. Residues identical to EgROP1 are indicated by dashes. The tinted boxes indicate conserved regions characteristic of ROP proteins: I and II, GTPase regions; III and V, GTP/GDP-binding regions; IV, RHO insert region; E, effector domain; VI, polybasic membrane localization domain; P, putative phosphorylation sites; CaaL, geranylgeranylation site. The SKK phosphorylation site specific to subgroup IV ROPs is indicated by ***. At, Arabidopsis thaliana; Bv, Beta vulgaris; Eg, Eucalyptus gunnii; Gh, Gossypium hirsutum; Ms, Medicago sativa; Nt, Nicotiana tabacum; Os, Oryza sativa; Ps, Pisum sativum; Ptr, P. trichocarpa; Ze, Zinnia elegans; Zm, Zea mays.

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The deduced protein sequence of EgROP1 contains 197 amino acid residues with a calculated molecular mass of 21.67 kDa. Sequence and phylogenetic comparisons with most of the ROP/RAC GTPases found in GenBank classified EgROP1 into the subgroup IV defined by Li et al. (1998) (Fig. 1a). This group is characterized by a unique phosphorylation site (SKK, 97–99), a C-terminal region containing the geranylgeranylation motif CaaL, the target for geranylgeranyl transferase II (GGTase II), and a CaaL-proximal polybasic domain involved in subcellular localization (Hancock et al., 1991). The EgROP1 protein sequence is more than 98% identical to a putative Populus trichocarpa ROP protein encoded by the estExt_fgenesh4_pg.C_1810025 gene model (http://genome.jgi-psf.org/Poptr1_1/Poptr1_1.home.html; Fig. 1b), which is preferentially expressed in stems (Gene Expression Omnibus series 6422; Id_ref 85853-55; http://www.ncbi.nlm.nih.gov/geo/). Also, EgROP1 shows up to 97% identity with PsROP1, BvRho1 and NtRAC1, 96% with AtROP1 and AtROP3, and more than 93% with MsRAC1 and AtROP5. As shown on the sequence alignment (Fig. 1b), all of the regions involved in GTP/GDP binding, GTPase activity, effector protein-binding domains and putative phosphorylation sites are highly conserved.

Quantitative RT-PCR analysis revealed preferential expression of EgROP1 in cambium-enriched fractions

We determined the expression levels of EgROP1 in nonvascular tissues (young and mature leaf blades), in meristematic zones (shoot and root tips, cambium enriched fraction), in petioles and in vascular tissues (secondary xylem and phloem) isolated from E. gunnii trees. The qRT-PCR results clearly indicated preferential expression of EgROP1 in the cambium-enriched fraction, with a ninefold higher level of transcripts than in young leaf blades, the calibrator tissue used in this study (Fig. 2). EgROP1 transcript levels were also two to three times higher in differentiating secondary xylem and shoot tips than in young leaves. This preferential expression in the cambial zone and developing secondary xylem (i.e. wood-forming tissues) suggests a role for EgROP1 in signalling processes occurring during xylogenesis.

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Figure 2. Expression levels of EgROP1 in eucalyptus tissues. The relative expression of EgROP1 was monitored by qRT-PCR on various tissues of Eucalyptus gunnii: MLB, mature leaf blades; ST, shoot tips; RT, root tips; PT, petioles; CEF, cambium enriched fraction; SP, secondary phloem; DSX, differentiating secondary xylem. Relative expression levels were calculated using EgIDH as the reference gene and young leaves as the calibrator tissue. Data were obtained from three RT-PCR replicates each of three different trees. Means and standard deviations are shown. Significant differences were statistically assessed using one-way anova and Tukey's HSD test which provided three groups of tissues expressing EgROP1 differentially (a, b and c).

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EgROP1 sequence polymorphism is linked to variations in the chemical properties of wood and in fibre morphology in E. urophylla × E. grandis progeny

To assess whether EgROP1 may indeed be involved in xylem formation, we sought a link between possible effect of EgROP1 polymorphisms and phenotypic variations of wood properties in eucalyptus. The EgROP1 polymorphism was assayed in a full sib family of 201 interspecific E. urophylla × E. grandis hybrids using the SSCP technique (Orita et al., 1989). EgROP1 SSCP patterns indicated two alternative ROP alleles in the E. urophylla parent (not shown). Segregation of these alleles was observed in the progeny, which were in two classes corresponding to the allele inherited from the E. urophylla female parent. No significant departure from Mendelian segregation ratios was observed in a χ2 test of fit. EgROP1 was mapped to linkage group 6 of the E. urophylla genetic map previously established by Gion et al. (2000) (Fig. 3).

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Figure 3. Genetic mapping of EgROP1 and QTLs related to lignin profiles and wood quality on Eucalyptus urophylla map. Single-stranded conformation polymorphism (SSCP) located EgROP1 on linkage group 6 (LG6). Distances along the linkage group are Kosambi centimorgans (cM). Quantitative trait loci (QTLs) are indicated by bars adjacent to the linkage group region where a locus had a significant threshold by permutation test. Lines represent the empirical confidence interval at 95% for the QTL position that was calculated using bootstrap analysis with 5000 resamplings. [GC], G content; [S/G], S : G ratio; [FW], fibre width; [CI], curl index.

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Quantitative traits related to wood chemical properties (extractive content, S content, G content and S : G ratio) as well as to fibre properties (weighted fibre length, fibre width, coarseness and curl index) were variable in the hybrid progeny (see Coefficient of Variation, Table 1). Coefficients of variation were higher for chemical traits (from 12 to 17%) than for fibre properties (from 3 to 7%). A total of four QTLs were detected on linkage group 6 of E. urophylla for the wood quality-related traits studied (Table 2). The two major QTLs concerned the G content and S : G ratio, explaining 14% and 28% of the phenotypic variation, respectively. The two other QTLs concerned fibre width (7.6%) and curl index (6.2%). All QTLs were closely linked to EgROP1 gene (Fig. 3). ‘S : G’ and ‘G content’ QTLs colocalized, which is logical as these two traits are both related to the G content of secondary cell walls. Furthermore, the allelic substitution effect is positive for the S : G ratio and negative for G content and fibre width (Table 2). This observation indicates that the less favourable allele for the S : G trait corresponds to the favourable allele for the two other traits.

Table 1.  Descriptive statistics for quantitative traits observed in Eucalyptus urophylla × Eucalyptus grandis hybrid progeny
 Trait definition (unit)MeanSDCV (%)
  1. SD, standard deviation; CV, coefficient of variation of the traits studied.

Chemical propertiesExtractive content (%)3.700.6217
G content (% extractive content)6.420.7912
S content (% extractive content)27.554.1815
H content (µmol g−1 lignin) 9557
S : G ratio4.030.5313
Fibre propertiesWeighted fibre length (mm)1.280.05 4
Fibre width (µm)331 3
Fibre coarseness (µg mm−1)0.0970.003 3
Fibre curl index (%)15.81.1 7
Table 2.  Quantitative trait loci (QTLs) detected on linkage group 6 by interval mapping in Eucalyptus urophylla and Eucalyptus grandis for wood quality traits and fibre morphology
 TraitnLOD scoreP-valuePosition (cM)PEV (%)d
  1. The QTL position is given in cM from the top marker of the linkage group. Values of the maximum LOD score and probability associated to the 1 QTL (H1 vs H0) or 2 QTLs (H2 vs H0 and H2 vs H1) models are given for each QTL after 1000 permutations. n, number of genotypes; PEV, percentage of phenotypic variation; d, allelic substitution effect.

Wood chemical compositionG content1895.0< 10−4 8514.0−0.59
S : G ratio18912.4< 10−4 8728.0+0.56
Fibre propertiesFibre width1783.00.0041027.6−0.56
Curl index1782.40.0071026.2−0.53

Overexpression of mutated EgROP1 in A. thaliana affects leaf cell morphology

Due to the inherent difficulties of carrying out functional studies in Eucalyptus, a tree genus that is difficult to transform, we expressed EgROP1 heterologously in A. thaliana, which has previously been used to study secondary xylem formation (Chaffey et al., 2002; Nieminen et al., 2004). Transgenic A. thaliana plants overexpressing wild-type (EgROP1-OX), constitutively active (EgROP1-CA) and dominant negative (EgROP1-DN) forms of the EgROP1 protein were generated. The Gly-15 to Val substitution in EgROP1-CA was expected to inactivate the intrinsic GTPase activity, keeping the mutant protein in its active GTP-bound state (Li et al., 2001). In addition, such ‘constitutively active’ ROPs are insensitive to the action of ROPGAPs and can constitutively activate effectors (Zheng & Yang, 2000). EgROP1-DN was produced by replacing Thr-20 with Asn, a mutation that locks the protein in the GDP-bound or nucleotide-free inactive form (Farnsworth & Feig, 1991; Li et al., 2001). ‘Dominant-negative’ ROP overexpression may have the ability to block endogenous ROP activation by sequestering activators (GEFs). Finally, plants transformed with the empty vector were used as control. Five to 25 independent transgenic lines were obtained for each construct. Transgene expression was confirmed by RT-PCR in all EgROP1 transgenics (not shown). Further phenotypic analyses were carried out on homozygous T3 plants recovered from three independent transformants that expressed high levels of EgROP1 transgenes. Two independent phenotypic analyses were carried out on two sets of five plants for each of the 12 transgenic lines (three control lines and nine EgROP1 lines) that were placed in a fully randomized experimental design. No difference in plant developmental stages (growth rate, flowering time, seed production and life span) that could have had introduced a difference in age and/or developmental stage of the xylem tissues was observed between the different lines (not shown). Results described in the next sections were consistent between independent transformed lines.

The rosette and inflorescence leaves of the transgenic plants overexpressing both wild-type and mutant forms of EgROP1 were dramatically different from the leaves of the control plants (Fig. 4a). Leaf blades were curled downward and were either cup- or sickle-shaped. To investigate further the alterations of leaf morphology in arabidopsis plants overexpressing EgROP1, we examined epidermal cells lying in the central zone of the adaxial side of fully expanded mature leaves (fifth rosette leaves) using transmission and scanning electron microscopy (SEM) (Fig. 4b,c). In contrast to mature pavement cells (Fig. 4b), the shape of the epidermal cells of transgenic lines overexpressing EgROP1-CA and EgROP1-OX was very regular. However the EgROP1-OX phenotype was less marked than that of the EgROP1-CA. In lines overexpressing EgROP1-DN, the cell morphology was also altered, with alternating rectangular and spindle-shaped cells and smaller circular or triangular-shaped cells. These shapes were notably different from those observed in the EgROP1-OX and EgROP1-CA lines.

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Figure 4. Rosette leaf morphology and leaf epidermal cell morphology of Arabidopsis thaliana plants overexpressing wild-type and dominant mutant EgROP1 proteins. (a) Control and transgenic plants overexpressing EgROP1-CA, EgROP1-OX or EgROP1-DN. (b) Scanning electron microscopy of the adaxial side of rosette leaves from control and transgenic plants, as in (a). (c) Micrographs of transverse sections of rosette leaves from control and transgenic plants, as in (a), stained with toluidine blue. Arrows point to swollen epidermal cells. Bars, (b) 100 µm, (c) 30 µm.

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The SEM analysis further revealed that the epidermal cells of the transgenic lines were particularly swollen compared with controls (Fig. 4b). Similar observations were made with bright-field microscopy (data not shown) thus eliminating a possible technical effect of the critical-point drying protocol for SEM. Consistent with this swollen shape, leaf pavement cells appeared more triangular and sometimes ‘pointed’ in transgenic lines (Fig. 4c), as if they expanded more in the longitudinal dimension than the control pavement cells. Furthermore, both palisade and spongy mesophyll cells were also smaller and rounder in plants overexpressing EgROP1-CA and EgROP1-DN. These data indicate general alterations in anisotropic growth in several cell types of the leaves of transgenic plants.

As A. thaliana trichomes serve as a system for studying the control of form during single cell morphogenesis (Mathur, 2004), we investigated the effect of EgROP1 expression on the development of trichomes on leaves of transgenic plants. In control leaves most of the trichomes formed three (72%), two (24%) or four branches (4%) (Fig. 5). Although three-branched trichomes remained the most prevalent in the transgenic plants, the proportions of two- and four-branched trichomes were inverted in plants expressing EgROP1-CA (3.2% and 12.5%) or EgROP1-OX (3.1% and 21%). No significant difference was observed in plants overexpressing EgROP1-DN when compared with the control, indicating that this dominant negative protein does not interfere with trichome branching mechanisms. These data confirm that anisotropic cell expansion is altered in leaves of plants overexpressing EgROP1 proteins. The cell shape defects observed in leaf cells suggest that EgROP1 plays a role in the morphogenesis of cells undergoing axial growth.

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Figure 5. Mature leaf trichome morphology in Arabidopsis thaliana overexpressing wild-type and dominant mutant EgROP1 proteins. (a) Relative proportions of two-branched (light-tinted bars), three-branched (dark-tinted bars) and four-branched (closed bars) trichomes as a percentage of the number (n) of trichomes analysed in each transgenic line. Values shown are means ± SD, n = 120–800. *, Statistically significant differences (χ2 test; P < 0.001) between control and mutant trichomes. (b) Scanning electron microscopy of three-branched and four-branched leaf trichomes. Bars, 50 µm.

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Overexpression of EgROP1 increases the length and diameter of secondary xylem elements

As EgROP1 was preferentially expressed in wood-forming tissues, we investigated the effect of overexpression of EgROP1 wild-type and mutant forms on the vascular system. We took advantage of the ability of arabidopsis to form secondary vascular tissues that closely resemble angiosperm wood (Chaffey et al., 2002). Sections were taken from the basal portion of inflorescence stems from two independent sets of 2-month-old plants from three independent lines for each EgROP1 transgene and control lines, which exhibited comparable developmental patterns. Fluorescence microscopy showed a blue ring of lignified tissues in EgROP1-CA secondary xylem and, to a lesser extent in EgROP1-DN secondary xylem, that was narrower than control and EgROP1-OX lines (Fig. 6a). Congo red-stained sections revealed that the primary xylem bundles were similar in all transgenic lines whereas the secondary xylem in the interfascicular region was reduced to a few large cells in EgROP1-CA plants when compared with the other lines (Fig. 6b).

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Figure 6. Secondary xylem cell morphology in Arabidopsis thaliana inflorescence stems overexpressing wild-type and dominant mutant EgROP1 proteins. (a) Epifluorescence microscopy of unstained tissues in 2-month old inflorescences. Lignified tissues appear in blue. (b) Laser confocal scanning microscopy of sections from the interfascicular regions of 2-month-old inflorescences stained with Congo red. The white arrows indicate newly formed cells in which the cell wall is not yet lignified (red), in contrast to the green-coloured components of lignified cell walls. Red arrows point to large cells characteristic of the secondary xylem of EgROP1-CA transgenic plants. (c) Bright-field microscopy of phloroglucinol stained transverse sections. Red arrows point to large cells characteristic of the secondary xylem of EgROP1-CA transgenic plants. (d) Bright-field microscopy of Maüle-stained transverse sections from 2-month-old and 4-month-old (4 m) inflorescences. Brown arrows point to large cells with G-rich lignins in their secondary cell walls. Black arrows show unlignified cells within secondary xylem. (e) Bright-field microscopy of toluidine blue-stained longitudinal sections from 2-month-old inflorescences. Arrows show vessel-like elements. Ep, epidermis; Co, cortex; Ph, secondary phloem; CZ, cambial zone; Fi, secondary xylem fibres; V, vessel-like elements; IF, interfascicular fibres; Pi, pith; SX, secondary xylem. Bars, (a) 500 µm, (b) 80 µm, (c) 80 µm, (d) 80 µm, (e) 50 µm.

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Measurements of cell size confirmed that the newly formed, unlignified cells were significantly larger in EgROP1-CA plants (Student test, P < 10−10), with an average lumen cell area of c. 150 ± 3.1 µm2 (n = 570) compared with 108 ± 2.1 µm2 (n = 710) in control plants (not shown). This phenotype effect must be caused by the constitutively active EgROP1 protein as no measurable change in the lumen cell area was observed in mutants overexpressing EgROP1-OX or EgROP1-DN.

As these observations suggested a possible effect of EgROP1 overexpression on xylem cell size, we macerated A. thaliana stems that had undergone secondary xylem formation in order to isolate and characterize vessel elements and fibres from each transgenic line (Table 3). Overexpression of wild-type and constitutively active EgROP1 genes increased dramatically both the length (by c. 30%) and diameter (by c. 15%) of xylem fibres when compared with the control. EgROP1-CA had a more marked effect on diameter but a lesser effect on length than EgROP1-OX. EgROP1-DN lines had the opposite phenotype with a significant decrease in diameter (by c. 20%) of the secondary xylem elements in comparison with control lines, a phenotype that is consistent with the opposing activities of the CA and DN forms of EgROP1. Interestingly, although the size of the secondary xylem elements was affected, no change in shape and morphology was detected (not shown).

Table 3.  Sizes of xylem cell elements in Arabidopsis thaliana inflorescence stems overexpressing wild type and dominant mutant EgROP1 proteins
 LineLength (µm)Length variation (%)Diameter (µm)Diameter variation (%)n
  1. Data are given as the mean ± standard deviation of n cells, as indicated, obtained from two sets of five plants each of three independent transgenic lines for each construct. Length and diameter variations are expressed as percentage of the control. *Statistically significant difference (Dunnett test; P-value < 0.01) between control and mutant lines.

FibresControl269 ± 10 19.9 ± 0.5 200
EgROP1-OX358 ± 10 33*22.6 ± 0.3 14*300
EgROP1-CA343 ± 8 28*22.4 ± 0.4 19*300
EgROP1-DN243 ± 9−1015.3 ± 0.4−19*200
Vessel elementsControl149 ± 3 28.7 ± 0.5 200
EgROP1-OX175 ± 4 18*31.3 ± 0.5 13*300
EgROP1-CA167 ± 3 13*32.3 ± 0.5 16*300
EgROP1-DN147 ± 4 −122.1 ± 0.3−21*200

Overexpression of EgROP1 alters secondary cell wall composition

To visualize lignin distribution in secondary xylem, we stained sections with phloroglucinol and Maüle reagent. The intensity of phloroglucinol staining was similar between transgenic lines, suggesting no change in lignin distribution (Fig. 6c). As described earlier, the EgROP1-CA plants showed only a small number of enlarged cells in their secondary xylem (Fig. 6c,d). Moreover, these enlarged cells, newly formed from the cambium, stained brown with the Maüle reagent, a colour characteristic of guaiacyl-rich (G-rich) lignins found in vessel cell walls in A. thaliana wild-type secondary xylem (Chaffey et al., 2002). To clarify whether these cells were vessel elements or other enlarged cell types with modified lignin composition in their secondary cell walls, we performed longitudinal sections on the basal part of 2-month-old inflorescences from wild-type and EgROP1-CA plants (Fig. 6e). In sharp contrast to the secondary xylem of control plants, which consisted of several layers of fibres alternating with a few vessels, this zone in EgROP1-CA transgenic plants was reduced to only a few layers of newly formed cells, most being vessels or vessel-like elements as shown on Fig. 6e.

In 4-month-old EgROP1-CA transgenic lines striking patches of unlignified cells were seen in contact with vessel elements enriched in guaiacyl units (indicated with black arrows in Fig. 6d). This lack of lignins seemed to affect only fibre-like cells. Similar defects in lignification were also observed in the EgROP1-DN lines, but in a smaller proportion of cells. As these lignification defects could occur either through a modified developmental pattern or an alteration of the lignification process, we stained sections immunochemically to assess whether these unlignified patches contained xylem cells with secondary cell walls (Fig. 7). Immunostaining with the LM10 monoclonal antibody specific for (1-4)-β-d-xylan showed that the cell walls in unlignified patches did not contain xylans specific to secondary cell walls, contrary to wild-type-like zones with lignified large cell walls (Fig. 7b,c) (McCartney et al., 2005), suggesting that the formation of the secondary cell wall was impaired in EgROP1-CA transgenics. Moreover, a closer view of the same zone stained with toluidine blue confirmed that the cells in these unlignified patches had thinner cell walls than cells in lignified regions (Fig. 7d).

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Figure 7. Lignin and (1-4)-β-d-xylan distribution in secondary xylem cell walls of 4-month-old Arabidopsis thaliana inflorescence stems overexpressing EgROP1-CA protein. (a) A semithin transverse section stained with Maüle reagent and observed by using bright-field microscopy. (b–d) Successive 1-µm thick sections obtained from the same inflorescence stem shown in (a) were immunostained with LM10 monoclonal antibody against (1-4)-β-d-xylan (b,c) and observed by using laser confocal microscopy. Antibody staining revealed secondary cell walls (red) whereas autofluorescence at 488 nm showed cell walls in green. White and black arrows point to unlignified secondary xylem cells that did not react with the antibody. Dashed arrows point to wild-type lignified secondary xylem cells. The white box delineates the region shown in (d) at ×40 magnification and stained with toluidine blue. Bars, 50 µm.

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Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

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.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References

We thank Carol Featherstone, Carl Douglas and Jill Harrison for their critical comments on the manuscript. We are grateful to Christian Brière for statistical analyses. C.F. was supported by a grant from the French Ministère de l’Education Nationale, de l’Enseignement Supérieur et de la Recherche. This work was supported by the French Centre National de la Recherche Scientifique and the Université Toulouse III.

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Acknowledgements
  8. References