JXB Advance Access originally published online on July 4, 2005
Journal of Experimental Botany 2005 56(418):2211-2227; doi:10.1093/jxb/eri221
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RESEARCH PAPER |
Molecular changes associated with the setting up of secondary growth in aspen
1Laboratory of Plant Biotechnology, Université Libre de Bruxelles, Chaussée de Wavre 1850, B-1160 Brussels, Belgium
2UMR CNRS/UPS 5546, Surfaces Cellulaires et Signalization chez les Végétaux, Pôle de Biotechnologie Végétale, Chemin de Borde Rouge 24, F-31326 Castanet Tolosan, France
3Laboratory for Wood Biology and Xylarium, Royal Museum for Central Africa, Tervuren, Leuvense Steenweg 13, B-3080 Tervuren, Belgium
4Department of Plant Systems Biology, Flanders Interuniversity Institute for Biotechnology (VIB), Ghent University, Technologiepark 927, B-9052 Gent, Belgium
* To whom correspondence should be addressed. Fax: +32 2 6509175. E-mail: mbaucher{at}ulb.ac.be
Received 7 December 2004; Accepted 9 May 2005
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Abstract |
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Vascular secondary growth results from the activity of the vascular cambium, which produces secondary phloem and secondary xylem. By means of cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis along aspen stems, several potential regulatory genes involved in the progressive transition from primary to secondary growth were identified. A total of 83 unique transcript-derived fragments (TDFs) was found to be differentiated between the top and the bottom of the stem. An independent RT-PCR expression analysis validated the cDNA-AFLP profiles for 19 of the TDFs. Among these, seven correspond to new genes encoding putative regulatory proteins. Emphasis was laid upon two genes encoding, respectively, an AP2/ERF-like transcription factor (PtaERF1) and a RING finger protein (PtaRHE1); their differential expression was further confirmed by reverse northern analysis. In situ RT-PCR revealed that PtaERF1 was expressed in phloem tissue and that PtaRHE1 had a pronounced expression in ray initials and their derivatives within the cambial zone. These results suggest that these genes have a potential role in vascular tissue development and/or functioning.
Key words: AP2/ERF, aspen, cDNA-AFLP, in situ RT-PCR, RING-H2, secondary growth, vascular cambium
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary materialReferences |
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Plant growth originates from meristems, localized tissues with stem cell features that are at the origin of all organs of the plant (Bäurle and Laux, 2003
). Apical meristems, contained in shoot and root tips, are involved in the extension of the components of the primary plant body. During this primary growth stage, water and sap conduction are ensured, respectively, by the primary xylem and the primary phloem that originate from procambial initials (Esau, 1965). In plants undergoing secondary growth (gymnosperms and dicotyledonous angiosperms), secondary vascular tissues that originate from cambium provide mechanical strength and allow long-distance transport of water and sap. Secondary growth, which results in the increase in the girth of stems, branches and roots, may have originated in the Middle Devonian, and cambium is present in several unrelated groups of vascular plants from the Upper Devonian (Kenrick and Crane, 1997; Lachaud et al., 1999).
The cambium has typically two morphologically distinct cell types, or initials: the axillary elongated fusiform initials leading to the axial system (including tracheids, vessel elements, fibres, axial parenchyma cells, sieve elements, and companion cells) and the smaller isodiametrical ray initials giving origin to the radially orientated parenchymatous rays (Iqbal and Ghouse, 1990). Anticlinal divisions of the cambial initials cause enlargement of the circumference of the cambial cylinder whereas periclinal divisions produce either a phloem or a xylem mother cell (also called derivatives), leaving initial cells in the meristem. Fusiform and ray derivatives may divide several times before differentiating into vascular tissues (reviewed by Lachaud et al., 1999; Dengler, 2001; Mellerowicz et al., 2001). Because most anatomical criteria are often not sufficient to discriminate between cambial initials and derivatives, the term cambial zone is used to denote these cell types. However, differences in cell wall composition (Catesson et al., 1994) or ultrastructural characteristics (Arend and Fromm, 2003) between cells of the cambial zone suggest that differentiation occurs early in cambial derivatives.
The vascular cambium has to maintain a balance between cell proliferation and cell differentiation, and, as for the apical meristem, this process is most likely under strict developmental control (reviewed by Dengler, 2001). For instance, in the shoot apical meristem, it has been demonstrated that WUSCHEL (WUS) promotes stem cell division whereas CLAVATA 1–3 (CLV1–3) repress mitotic activity and promote the commitment to leaf primordia formation (Brand et al., 2000; Schoof et al., 2000). The expression of CLV1 is up-regulated in Arabidopsis stems undergoing secondary growth, but no expression was monitored for CLV3 and WUS in the cambium (Ko and Han, 2004). Accordingly, the putative orthologue PttCLV1 has been shown to be expressed in the cambial zone of Populus, but not PttWUS or PttCLV3, suggesting that similar but not identical mechanisms and/or genes are involved in the identity and maintenance of the vascular meristem, compared with the apical meristem (Schrader et al., 2004; Ko and Han, 2004).
In Arabidopsis, the onset of body weight-induced secondary growth has been investigated by microarray analysis. Several up-regulated genes in wood-forming stems were shown to possess auxin responsive cis-acting elements in their promoter region, indicating auxin-mediated regulation of secondary growth (Ko et al., 2004). However, although the Arabidopsis system has been suggested to be a good model to study wood formation, an apparent major structural difference in secondary xylem between Arabidopsis and poplar is the lack of rays in Arabidopsis (Chaffey et al., 2002). Throughout the life of woody plants, the transition from primary to secondary growth is progressive and may require a specific regulatory network of molecular interactions.
The aim of the present study was to investigate the spatio-temporal transition from primary to secondary vascular growth in hybrid aspen. A cDNA-AFLP approach, which permits distinction between highly homologous genes among families, allowed the identification of novel putative regulatory genes associated with secondary growth in aspen. Particular attention was paid to two genes, the expression of which was localized in vascular tissues, as assayed by in situ RT-PCR.
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Materials and methods |
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Plant growth conditions and sampling
Populus tremulaxP. alba (clone INRA 717 1B-4) was micropropagated in vitro on half-strength MS medium (Duchefa Biochemie, Haarlem, The Netherlands). Six-week-old plants were acclimatized in the greenhouse. For cDNA-AFLP analysis, stem samples were harvested in May 2000 from three 6-month-old trees (approximate height of 150 cm). Top samples (T) consisted of the 15 cm of the stem below the shoot apical meristem and the bottom samples (B) were harvested at a height of between 30 and 50 cm above the soil. The bark was peeled away and the samples were immediately frozen in liquid nitrogen. For RT-PCR analysis stem samples were harvested in March 2003 from a 6-month-old greenhouse-grown aspen tree. The sampling was the same as for the cDNA-AFLP analysis (B, T) with an additional two middle samples (M1 and M2) in between. Leaf samples consisted of mature leaves from which main veins were removed (L) and of entire unexpanded young leaves (YL).
Safranin-stained cross-sections
Top and bottom stem samples were soaked in polyethyleneglycol (PEG) 1500 for 7 d at 65 °C. Thin cross-sections (15–30 µm) were made using a sledge microtome armed with a C-profile solid knife (Reichert, Vienna, Austria) and soaked in 75% (v/v) ethanol. Cross-sections were treated successively with 8% (v/v) commercial bleach, 10% (v/v) acetic acid, 50% (v/v) ethanol, and 0.1% (w/v) safranin. Progressive dehydration (from 50% to 100% ethanol) was performed, before washing thin sections in parasolve overnight and mounting on glass-slides. Pictures were taken with an Olympus PM-20 camera system coupled to an Olympus BX60 microscope (Olympus, Tokyo, Japan).
cDNA-AFLP analysis
Total RNA was extracted from the plant samples according to Verwoerd et al. (1989). cDNA-AFLP was performed according to Bachem et al. (1996) and Durrant et al. (2000), with some minor modifications. Double-strand (ds) cDNA was synthesized using a cDNA Synthesis Module (Amersham Biosciences, Uppsala, Sweden). Ds cDNA was digested with EcoRI (Promega, Madison, WI, USA) and MseI (New England Biolabs, Beverly, MA, USA) and specific adaptors were ligated with T4-DNA ligase (Amersham Biosciences). Adaptors were made from oligos synthesized by Proligo (Paris, France): 5'-GACGATGAGTCCTGAG-3' and 3'-TACTCAGGACTCAT-5' for MseI sites and 5'-CTCGTAGACTGCGTACC-3' and 3'-CTGACGCATGGTTAA-5' for EcoRI sites. Pre-amplification was performed at an annealing temperature of 60 °C for 28 cycles with the following primers: 5'-GACTGCGTACCAATTC-3' (EcoRI-adaptor primer) and 5'-GATGAGTCCTGAGTAA-3' (MseI-adaptor primer). Selective amplification was performed with 176 combinations of EcoRI-adaptor(+NN) and MseI-adaptor(+NN) primers. Differential bands were cut out of the gels and DNA was eluted either in TE or by electro-elution on 1% low-melt agarose (Bio-Rad, Hercules, CA, USA) gels. The eluted DNA was then re-amplified, using the same primer set used for the selective amplification. cDNA fragments were cloned in the PCR4®-TOPO vector (InvitrogenTM, Carlsbad, CA, USA) and sequenced. Similarity searches were carried out with WU-BLASTX+BEAUTY (http://searchlauncher.bcm.tmc.edu/seq-search/nucleic_acid-search.html). NCBI BLAST 2.0 analyses were also performed against the EST databases PopulusDB (http://poppel.fysbot.umu.se/), PoplarDB (http://mycor.nancy.inra.fr/BLAST/blast.html), and AspenDB (http://aspendb.mtu.edu/), against the P. trichocarpa draft genome sequence database (http://genome.jgi-psf.org/Poptr1/Poptr1.home.html) and against the TAIR database (http://www.arabidopsis.org/Blast/)(Altschul et al., 1997).
RT-PCR analysis
Total RNA was prepared using an RNeasy Plant Mini Kit (Qiagen, Valencia, CA, USA) then treated with DNase I (DNAfreeTM from Ambion, Austin, TX, USA). RNA concentrations were estimated spectrophotometrically at 260 nm and were further adjusted using the RiboGreen® reagent (Molecular Probes, Eugene, OR, USA). Single-stranded cDNA was synthesized using a Reverse Transcription System (Promega). PCR reactions were performed using a Master Mix from Promega, with various PCR conditions depending on the sequences and the primer pairs (Table S1: supplementary data that can be found at JXB online). 18S rRNA was used as internal control. Primer pairs used for RT-PCR analysis were designed either on cDNA-AFLP TDFs, or on highly similar ESTs (identity 
93%) when those were available (Table S1: supplementary data that can be found at JXB online).
Reverse northern analysis
cDNA was amplified from each cDNA-AFLP clone using M13 Forward (F) 5'-TGACCGGCAGCAAAAT-3' and M13 Reverse (R) 5'-GGAAACAGCTATGACCATG-3' primers. Genomic DNA and EF1-
cDNA were used as internal controls, and PM43 (a plasmid containing a set of cDNA-AFLP adaptors) and TE buffer were used as negative controls to monitor background. PCR products were arrayed on nitrocellulose filters using a MicroGrid II robot (Biorobotics, Cambridge, UK). Each cDNA-AFLP TDF was spotted 12 times. Genomic DNA, EF1-, PM43, and TE buffer controls were spotted 70, 70, 94, and 114 times, respectively. Total RNA was prepared as described above from samples corresponding to those used for cDNA-AFLP. For each sample, 5 µg total RNA was reverse-transcribed with ABgene Reverse-iT RTase Blend (Epsom, UK) and labelled with dCTP [33P] (Amersham Biosciences). Membranes were prehybridized for 3 h and hybridized overnight in 3x SSC, 0.5% SDS, 10% PEG 6000, and 0.2% skimmed milk at 60 °C. Membranes were washed twice with 3x SSC, 0.5% SDS and once with 1x SSC, 0.5% SDS and exposed in a PhosphorImager cassette (Molecular Dynamics) for 72 h. PhosphorImager screens were then scanned with a Storm 850 scanner (Amersham Biosciences). Detection and quantification of the signals were performed using the imaging software ImageQuant 5.0 (Amersham Biosciences). The mean background of each membrane (estimated as the mean pixel intensity of TE buffer spots) was subtracted from each spot. A Wilcoxon paired-sample test was then performed on the 12 repetitions to identify TDFs that showed a significant differential pattern (P <0.01).
In situ RT-PCR analysis
Transverse sections were hand-cut on stems of 1-month-old acclimatized aspens. Liquid phase in situ RT-PCR was performed according to Pesquet et al. (2004) with modifications for nucleotide-based epitopic detection. Genomic DNA restriction was performed with one or several of the following enzymes, depending on the sequence of the cDNA to be amplified: TaqI, MspI, HinfI, HaeIII, or RsaI (Promega). For RT-PCR, the following pairs of primers were used: F 5'-TCAACACAGCATTCGAGAGG-3' and R 5'-ATGCAGCGCATTCAATTACA-3' for CAD cDNA (accession no. AF217957), F 5'-CTTCTGGAAGGGATGCATTT-3' and R 5'-CGAACCCTAATTCTCCGTCA-3' for 18S rRNA gene (accession no. AJ236016), F 5'-TCTCCACCTTTACGCTCGTT-3' and R 5'-CACTCGGTGTGGAAACTGTG–3' for PtaRHE1, and F 5'-TTCTATCCCGGTCAATGGAG-3' and R 5'-GCTTCCACAAAGACGGATTC-3' for PtaERF1. During PCR, products were labelled with Dig-11-dUTP (Roche, Mannheim, Germany) at 10 pmoles per reaction (for a total content of 1 µmole dNTP per reaction). After PCR, cross-sections were washed twice in 100 mM TRIS–HCl pH 7.5, 150 mM NaCl, then incubated for 1 h in 100 mM TRIS–HCl pH 7.5, 150 mM NaCl, 5% (w/v) BSA. Anti-digoxigenin-alkaline phosphatase Fab fragments (Roche) were then added and the reaction was allowed to proceed for 30 min. Cross-sections were then washed three times in 100 mM TRIS–HCl pH 9.5, 150 mM NaCl, and 0.2% (v/v) Tween. Subsequently, cross-sections were incubated for 15–30 min in alkaline phosphatase substrate solution prepared from NBT/BCIP tablets (Roche). The staining reaction was stopped by washing the cross-sections in TE buffer pH 8. Pictures were taken with a light-microscope (Nikon Eclipse E800M, Tokyo, Japan) equipped with a digital still camera-based system (DXM1200 Nikon).
Cloning of PtaRHE1 and PtaERF1 coding sequences
Poly(A) mRNA was isolated from aerial parts of 3-month-old aspen using a PolyATtract®mRNA Isolation System III (Promega). The 5' end of the PtaRHE1 cDNA sequence was isolated using a Marathon cDNA Amplification Kit (Clontech Laboratories, Palo Alto, CA, USA) with the following gene-specific primer: 5'-AAGCCGATCCCAAATGATCCTCCTC-3'. The 3' end of PtaRHE1 was recovered from a 96% identical overlapping homologous sequence (scaffold_77: 1287648:1288442) of the P. trichocarpa draft genome sequence. The 987 bp sequence of PtaRHE1 was amplified by PCR using gene-specific primers F 5'-TGCTAAACCAAACCCATTATCC-3' and R 5'-CTAACCGGGTCAAGAATTGC-3'. The 5' and the 3' ends of the PtaERF1 cDNA sequence were isolated using a Marathon cDNA Amplification Kit with the following gene-specific primers 5'-GGATGCCTTGCTGAAGCACATGGAG-3' and 5'-CGTGGCAAGAAAGCGAAGGTGAACT-3', respectively. The 1409 bp sequence of PtaERF1 was amplified by PCR using the gene-specific primers F 5'-ATAATCACCATCAATCA-3' and R 5'-CTAAATAAGTCACACCACATT-3'. Both cDNAs were subsequently cloned in a PCR4®-TOPO vector and sequenced.
The sequences of PtaRHE1 and PtaERF1 were analysed with the BLAST programs on the NCBI and PopulusDB databases, as well as on the TAIR database (http://www.arabidopsis.org/Blast/) (Altschul et al., 1997). Protein sequences alignment was made with Clustal W (http://www.ebi.ac.uk/clustalw) (Thompson et al., 1994), and page setup was made with Boxshade (http://www.ch.embnet.org/software/BOX_form.html). Molecular weight was determined with the software Pepstats (http://bioweb.pasteur.fr/seqanal/interfaces/pepstats.html).
Nucleotide sequence accession numbers
Sequences of PtaRHE1 and PtaERF1 are deposited in the GenBank nucleotide sequence database under Accession numbers AY780430 and AY780431, respectively. Sequences of the 83 TDFs are registered under the Accession numbers CV555406-CV555488.
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Results |
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Anatomical features of aspen stem tissues used for cDNA-AFLP analysis
The goal was to identify genes encoding regulatory proteins involved in the progressive transition from primary to secondary growth in aspen. Towards this end, cDNA-AFLP analysis was performed on stem tissues corresponding to two different developmental stages along a stem of a 6-month-old aspen tree (150 cm) (Fig. 1A). The first sample (top sample) consisted of the 15 cm of stem located immediately beneath the apical meristem. In the upper part of this sample (Fig. 1B, section 1), the vascular system consists of primary xylem and phloem, whereas the lower part of this stem sample is characterized by the presence of a cambium producing secondary vascular tissues (Fig. 1C, section 2). This stem sample represents therefore a spatio-temporal progression of vascular tissue differentiation, from primary xylem to young secondary xylem. The second sample (bottom sample), located at the bottom of the stem (30–50 cm above soil level), comprises a well-structured secondary xylem separated from the bark by the vascular cambium (Fig. 1D, section 3).
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For the cDNA-AFLP analysis, the bark region was removed and the total remaining stem tissue was used for RNA extraction. An exception was made for the upper part of the top sample (corresponding to Fig. 1B), where the cortex was gently peeled off from the stem sample. Compared with the bottom sample that is mainly constituted of secondary xylem, the top sample is enriched in primary vascular tissues, and contains a significant proportion of pith.
Isolation and identification of genes differentially expressed during the transition from primary to secondary growth by cDNA-AFLP analysis
In order to ensure the reproducibility of the gene expression profiles, cDNA-AFLP was performed independently on three individual trees (T1–3). cDNA fragments were amplified using 176 selective primer combinations out of the 256 possible. Figure 2A shows the reproducibility of the expression pattern among the three individual trees. Figure 2B shows an example of differential gene expression between the top and the bottom samples. An estimated 26 000 TDFs were screened. In order to avoid artefacts due to the variability of the biological material, only the bands that were differential in all three individual trees were selected for further investigation. A total of 106 TDFs, with a length between 51 bp and 456 bp, were differentiated between the top and the bottom samples. The TDFs were excised from the gels, re-amplified with the corresponding cDNA-AFLP primer combinations, cloned, and sequenced. After removal of 17 chimeric sequences (clones containing two or more ligated TDFs) and 6 redundant TDFs, a set of 83 unique TDFs was retained for further analysis. Most of the TDFs (55 out of 83) showed a higher expression in the top sample (‘top TDFs’) rather than in the bottom sample (‘bottom TDFs’). Table 1 displays the sequence similarities for the 83 TDFs. The poplar origin was confirmed for all TDFs after BLASTN analysis with the draft poplar genome sequence. Subsequently, a BLASTX search against GenBank database and a BLASTN search against poplar EST databases were performed and, according to their similarities, TDFs were classified into four different groups (Group 1, Regulation and signalling; Group 2, Cell wall biogenesis; Group 3, Various metabolic processes; and Group 4, Hypothetical/unknown proteins). Of the top TDFs (Table 1A), 11 were classified in Group 1, including five putative transcription factors (Pta011, Pta070, Pta075, Pta086, and Pta091), two translation-related factors (Pta004 and Pta097), two receptor-like protein kinases (Pta043 and Pta050), one ankyrin-like protein (Pta041), and one RING (Really Interesting New Gene) zinc finger protein (Pta099). Group 2 comprises a xyloglucan endotransglycosylase/hydrolase (XTH) precursor (Pta026) and a pectinesterase-like (PME) gene (Pta101). Group 3 consists of 19 TDFs similar to genes involved in various metabolic processes. Group 4 is composed of 23 TDFs similar to hypothetical/unknown proteins or for which no significant similarity (E values above 0.2) could be found, probably due to their small size or to their possible location within the non-coding regions in the cDNAs. The same classification was made for the bottom TDFs (Table 1B). Table 2 shows the relative distribution of the 83 TDFs within the four different groups. The TDFs classified in Group 1 are clearly more abundant in the top than in the bottom (11 out of 13 TDFs). Only four genes (two top and two bottom TDFs) related to cell wall biogenesis were identified in this study, indicating that the comparative approach used (top versus bottom) allowed the subtraction of main cell wall-related genes. Among them, the two top TDFs are similar to enzymes modulating cell wall expansion, cellular adhesion, and cell wall cross-linking (PME and XTH). The two bottom TDFs, encoding cinnamyl alcohol dehydrogenase (CAD) and 4-coumarate coA:ligase (4CL), are associated with lignin biosynthesis, a characteristic of secondary cell wall formation (reviewed by Boerjan et al., 2003). Almost 50% of the TDFs correspond to hypothetical or unknown proteins and, for about half of the TDFs, no close ESTs/contigs (identity >90%) were found. The availability of the poplar genome sequence will help to annotate these TDFs further.
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Expression analysis of top TDFs along a poplar stem and in leaf tissues
To validate the cDNA-AFLP results, an independent expression study was performed for 25 TDFs by RT-PCR (Table 1). The expression analysis included four xylem samples harvested along the stem of 6-month-old aspen trees [one base sample (B), two intermediate samples (M1 and M2), and one top sample (T)] and two leaf samples [one consisting of dissected interveinal tissue of mature leaves (L), the other consisting of entire young unexpanded leaves, mainly constituted of vein tissues (YL)]. As the objective of this study was to identify genes potentially involved in the setting up of secondary growth, a focus was made on the top TDFs. Figure 3 shows RT-PCR profiles of 19 top TDFs for which the cDNA-AFLP profile was confirmed. For the six remaining TDFs, the differential expression was not validated by RT-PCR (data not shown). As shown in Figure 3A, the RT-PCR profiles are in agreement with the cDNA-AFLP results. Globally, a progressive decrease of the expression was observed from the top to the base samples. In the leaf samples, the RT-PCR expression profiles were variable. As shown in Fig. 3B, the expression levels for most of the TDFs were either similar in both leaf samples or higher in young leaf samples, except for Pta041 that was not detected in either of the leaf samples. Pta050 and Pta058 had a pronounced expression in interveinal tissue compared with that in the young leaf samples and are, therefore, not likely to be associated with vascular tissue.
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To assess the differential expression with a non PCR-based method, an independent reverse northern analysis was done. The 106 TDFs were spotted on two nitrocellulose membranes. These membranes were hybridized with labelled cDNA synthesized from total RNA, extracted either from the top or from the bottom part of a bark-free stem of a 6-month-old aspen. The TDFs from Group 1 that revealed significantly different hybridization signals (P <0.01) were Pta075 and Pta099 (Fig. 4). The signals of the other top TDFs from Group 1 were either too low or not different. These results may be due to the fact that homologous cDNAs hybridize and average the signal.
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In situ RT-PCR localization of gene expression in aspen stem sections
To investigate the localization of gene expression at the tissular and cellular levels, in situ RT-PCR was performed on hand-made stem transversal sections of 1-month-old acclimatized aspen plants. Young stem tissues were best suited for in situ RT-PCR analysis since experiments with older tissues were hampered by phenolics that inhibit enzyme activities (data not shown). Figure 5A shows the negative control in which all the steps of the experimental protocol were carried out with a mock RT reaction (without reverse transcriptase) for PtaRHE1. While no cytoplasmic signal was observed in mock RT reactions, a nuclear staining was often observed, presumably due to incomplete DNA digestion. In order to validate the in situ RT-PCR protocol, the expression of two control genes, CAD and 18S, was investigated. CAD expression was localized in the primary xylem cells and in ray parenchyma cells surrounding vessels (Fig. 5B), a localization that is in agreement with the expression pattern conferred by a Eucalyptus gunnii EgCAD2 promoter-GUS fusion in transgenic poplar (Hawkins et al., 1997
; Lauvergeat et al., 2002). The in situ expression analysis for the 18S ribosomal gene, regarded as constitutive (Pesquet et al., 2004), shows that this gene is expressed in almost all cell types including cortex parenchyma cells and in both differentiating phloem and xylem (Fig. 5C), with a more pronounced and homogenous expression in the cambial zone (Fig. 5D).
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Subsequently the localization of gene expression corresponding to the two selected top TDFs from Group 1 (Pta099 and Pta075), was examined. Their expression profile was associated with the vascular system (Fig. 5E, H). Pta099 expression was associated with the cambial zone and to a lesser extent with differentiating phloem, primary xylem, and ray parenchyma cells of secondary xylem (Fig. 5E). A closer observation (Fig. 5F) revealed that within the cambial zone, Pta099 gene expression was localized in the ray initials and derivatives and not in their fusiform counterparts when compared to the 18S gene expression pattern (Fig. 5D). Pta075 expression was confined to the phloem tissue (Fig. 5G, H).
PtaERF1 (Pta075) cDNA cloning and sequence analysis
A PtaERF1 cDNA of 1409 bp, including a 19 bp 5' untranslated region (UTR) and a 250 bp 3' UTR, was cloned. The 1140 bp open reading frame (ORF) predicts a 380 amino acid polypeptide with a calculated molecular weight of 42 kDa. This ORF shows a 97% identity with the scaffold LG_III at the coordinates 6664108:6666668 in the poplar draft genome sequence, and a 99% identity with the contig POPLAR.406.C2 in the PopulusDB. This polypeptide is homologous to AP2/ERF (Apetala2/Ethylene responsive factor) transcription factors. Figure 6 shows the alignment of the deduced amino acid sequence of PtaERF1 with the four most similar deduced proteins, including that of Nicotiana tabacum NtDRF1 (AY286010
[GenBank]
) (52% identity, 62% similarity over 380 amino acids), Lycopersicon esculentum LeJERF1 (AY044235
[GenBank]
) (47% identity, 61% similarity over 380 amino acids) and LeJERF3 (AY383630
[GenBank]
) (48% identity, 59% similarity over 311 amino acids), and Fagus sylvatica ERF1 (FsER1— AJ420195
[GenBank]
) (55% identity, 68% similarity over 370 amino acids). The most similar polypeptide of A. thaliana is At1g53910 (RAP2-12) (43% identity, 52% similarity over 380 amino acids). As shown in Fig. 6, sequence comparison revealed a conserved N-terminal region (domain I), a putative nuclear localization signal (NLS) KRKRK (domain II), and a conserved ERF DNA binding domain (domain III). Domain I, of unknown function, typifies a new class of ERF proteins, named class IV (Tournier et al., 2003).
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PtaRHE1 (Pta099) cDNA cloning and sequence analysis
A PtaRHE1 cDNA of 987 bp, including a 74 bp 5' UTR and a 34 bp 3' UTR, was cloned. The 879 bp ORF predicts a 293 amino acid polypeptide with a calculated molecular weight of 33 kDa. This ORF shows a 98% identity with the scaffold_77 at the coordinates 1287428:1288306 in the poplar draft genome sequence, and a 99% identity with the contig POPLAR.9452.C1 in the PopulusDB. Homology throughout the sequence indicates that PtaRHE1 belongs to the RHE subgroup of RING-H2 proteins described by Jensen et al. (1998)
. Figure 7 shows the alignment of the deduced amino acid sequence of PtaRHE1 with the four most similar deduced proteins including the Arabidopsis At3g16720 (ATL2) (54% identity, 65% similarity over 280 amino acids), At3g62690 (ATL5) (41% identity, 64% similarity over 234 amino acids), At2g47560 (49% identity, 69% similarity over 144 amino acids), and the N. tabacum AF211532
[GenBank]
(Avr9/Cf-9 rapidly elicited-132, ACRE-132) (39% identity, 58% similarity over 228 amino acids). The predicted polypeptides have four conserved regions, including a putative transmembrane hydrophobic region at the N terminus (region I), a region rich in basic amino acids (region II), a region with highly conserved amino acid sequences (region III), and a RING-H2 zinc finger domain (region IV) (Salinas-Mondragón et al., 1999) (Fig. 7). The latter conforms to the stringent RING-H2 consensus sequence of the RING-H2 domain (CX2CX14-15CXHX2HX2CX3WX6-8CPXC (X means other amino acid) (Jensen et al., 1998).
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary materialReferences |
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During the last decade, the analysis of secondary growth by various new molecular tools has allowed the identification of a large number of differentially expressed genes in both woody (Whetten et al., 2001
; Hertzberg et al., 2001; Yang et al., 2003; Sterky et al., 2004) and non-woody species (Zhao et al., 2000; Fukuda, 2004; Ko et al., 2004). Until now, few of these genes have been functionally characterized and data on cell-specific expression is lacking for most of them.
In this study, genes encoding putative regulatory proteins that could be involved in the transition from primary to secondary growth in aspen were identified. Poplar is the model tree species in molecular biology, for which there is a large poplar EST resource (Sterky et al., 2004) and for which the genome sequence has been released (http://genome.jgi-psf.org/Poptr1/Poptr1.home.html).
The experimental strategy consisted of gene expression comparison, by cDNA-AFLP, between the top and bottom stem samples from 6-month-old aspen trees. The top sample corresponds to tissues in which secondary growth is being set up whereas in the bottom sample, the secondary growth is well established (Fig. 1). In this context, the bottom sample was used as a subtractive control for typical secondary growth gene expression (e.g. genes involved in cell wall biosynthesis and modifications).
In order to limit false positives due to biological variation, only TDFs that showed an identical expression pattern for three independent trees were considered (Fig. 2). Consequently, a restricted number of 83 differential TDFs was selected (Table 1). A focus was made on TDFs that showed a higher expression level in the top than in the bottom sample. RT-PCR expression analysis validated the cDNA-AFLP profiles for 19 top TDFs and revealed a clear decreasing expression level along the aspen stem from the top to the base (Fig. 3). At the biological level and considering the vascular tissue development, three hypotheses can be considered to explain this decreasing gene expression pattern: (i) the TDFs correspond to genes associated with primary xylem but not with the transition to secondary growth, (ii) the TDFs correspond to genes specific for the transition from primary to secondary growth, a process that is linked to vascular cambial activity, and (iii) the TDFs correspond to genes associated with both situations. To document these hypotheses further, particular attention was given to six TDFs (Pta011, Pta041, Pta070, Pta075, Pta086, and Pta099) classified in the ‘Regulation and signalling’ group (Figs 3, 4). To the authors' knowledge, the function of the corresponding genes has not been revealed yet. For Pta041 and Pta070, only sequence data are available. Pta041 is homologous to At3g12360, encoding an ankyrin-like protein and Pta070 is homologous to At5g05610, a gene coding for a plant homeodomain (PHD) finger protein. For the four remaining TDFs, a possible involvement of their corresponding genes in plant developmental processes can be suggested. One TDF (Pta011) is similar to CPRF4b (Common Plant Regulatory Factors 4b), a parsley basic region/leucine zipper (bZIP) DNA-binding protein. CPRF4b is 86% identical to CPRF4a, which has been suggested to be involved in light signal transduction (Kircher et al., 1998). Light is known to control several central plant developmental processes, such as germination and transition from the vegetative to the reproductive stage. The role of light in the transition from primary to secondary growth is worth further investigation.
Another TDF (Pta086) is similar to At1g61140, a gene coding for a SWI2/SNF2 (Switch 2/Sucrose Non Fermenting 2)-like protein. SNF2-like proteins are ATPases involved in ATP-dependent chromatin remodelling machines that are multisubunit complexes altering DNA-histone interactions via ATP hydrolysis. Loss of the SNF2-like function in plants affects cytosine methylation of genomic DNA, transcriptional activity and development, such as in the Arabidopsis pickle mutant in which embryonic traits are expressed after germination (reviewed by Reyes et al., 2002).
Pta075 (PtaERF1) belongs to the subfamily of the AP2/ERF domain plant-specific transcription factors, characterized by an ERF DNA binding domain, and represented by 124 members in the Arabidopsis genome (Riechmann, 2002). ERF genes are induced by abiotic (Sakuma et al., 2002) and biotic (Berrocal-Lobo et al., 2002) stresses, but are also reported to play a role in plant growth and development (Kirch et al., 2003). PtaERF1 is homologous to the tomato JERF3 (Fig. 6), a transcriptional activator that binds to the GCC box, an element responsive to ethylene and jasmonic acid as well as to dehydration responsive element (DRE) (Wang et al., 2004). These authors showed that transgenic tobacco plants expressing JERF3 ectopically have an enhanced salt tolerance and an increased expression of several pathogenesis-related genes. In situ RT-PCR showed that PtaERF1 is expressed in phloem tissue (Fig. 5G, H). Further functional studies of PtaERF1 should reveal its potential role in phloem functioning and/or development.
The last TDF, Pta099 (PtaRHE1), shows homology to RING-finger proteins. These proteins are involved in diverse regulatory processes such as protein–protein interactions (Borden, 2000), proteolysis as ubiquitin E3 ligases (Joazeiro and Weissman, 2000), and signal transduction (Saurin et al., 1996). The RING finger motif is a zinc binding domain defined by a conserved pattern of cysteine and histidine residues capable of co-ordinating two atoms of zinc within a characteristic cross-brace structure (reviewed by Saurin et al., 1996). PtaRHE1 is most homologous to the Arabidopsis ATL2 (Fig. 7), a gene involved in the early response to elicitors such as chitin and inactivated cellulase preparations (Martínez-García et al., 1996). Promoter–GUS fusion expression studies in Arabidopsis have shown that ATL2 is expressed in the root of etiolated seedlings and in the shoot apical meristem of light grown-seedlings, with the strongest expression in the leaf primordia and in the stipules (Salinas-Mondragón et al., 1999). The function of ATL2 has still to be elucidated. However, for other RING-H2 proteins, a role in various plant developmental processes has been reported. For instance, the expression of the alfalfa MsRH2-1, which is related to the ATL family, is correlated with the differentiation of vascular tissues in lateral roots and symbiotic nodules (Karlowski and Hirsch, 2003). In situ RT-PCR localization studies in stem cross-sections indicated that PtaRHE1 is expressed in primary xylem, in ray parenchyma cells, and exhibits a differential pattern in vascular cambium (Fig. 5E, F). Expression of several aspen genes has also been localized in the cambial zone, including the MADS-box gene PtM5, (Cseke et al., 2003), the homeobox genes PttHB1 and PttHB2 (Hertzberg and Olsson, 1998), and PttRPS18 encoding the S18 ribosomal protein (Johansson et al., 2003). By contrast, PtaRHE1 exhibits a sharp differential pattern of expression between the two initial cell types within the cambial zone (Fig. 5E, F). Although caution should be taken when interpreting these in situ RT-PCR signals in secondary vascular tissues due to differences in cell size (Gray-Mitsumune et al., 2004), the PtaRHE1 signal in the cambial zone (Fig. 5F) seems to be located only in ray initials and their derivatives, when compared to the ubiquitous 18S signal (Fig. 5D). A potential role for this gene in the determination and/or the maintenance of cambial cell identity can therefore be suggested. Reverse genetics should confirm or refute this hypothesis.
In the cambial zone, at least three patterns of cell differentiation can be distinguished. The first pattern is related to the differentiation of the cambial derivatives to either xylem or phloem through periclinal segmentations. The second differentiation pattern is related to the specification of the different components of either xylem or phloem tissue within the cambial zone. Little is known about the genetic control of cell type identity leading to the differentiation of vessels, fibres, axial parenchyma cells, sieve elements, or companion cells. Nevertheless, comprehensive genomic approaches have resulted in the identification of genes, the expression of which is associated with particular tissues or cell types in phloem (Vilaine et al., 2003; Ivashikina et al., 2003) and xylem (Hertzberg et al., 2001; Demura et al., 2002; Milioni et al., 2002). PtaERF1 is apparently expressed mainly in phloem tissue. A suggestion for the functional significance of PtaERF1 as a transcription factor in phloem is its involvement in cellular processes related to phloem identity, functioning (e.g. defence signalling) and/or development.
The third differentiation pattern concerns the identity of the two cambial initials, either fusiform or ray initials. Rays are essential determinants for secondary growth in plants because they ensure the translocation of nutrients between phloem and xylem and the transmission of messenger molecules (Lachaud et al., 1999). Until now, the induction and the regulation of vascular ray differentiation have received only limited attention. As PtaRHE1 expression is localized in ray initials and derivatives within the cambial zone, a role for this gene in the identity of this cell type and/or in the communication between xylem and phloem can be postulated.
In conclusion, the approach used to analyse the setting up of secondary growth in aspen has allowed the identification of several regulatory genes. The expression of two of them was localized in particular cell types within the vascular system. PtaRHE1, encoding a RING-H2 finger protein, is expressed in ray initials within the cambial zone and PtaERF1, encoding an AP2/ERF transcription factor is expressed in phloem tissue. Further work will focus on the determination of the role of these genes in vascular tissue differentiation by transgenic approach.
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary materialReferences |
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Supplementary information (Table S1: Primer pairs used for RT-PCR analysis) can be found at JXB online.
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Acknowledgements |
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The authors acknowledge Björn Sundberg for giving PopulusDB EST data, Antje Rohde, Jan Van Doorsselaere, and Jan Zethof for their assistance with the cDNA-AFLP technique, and Nathalie Verbruggen for access to image acquiring facilities. DVR was the recipient of a pre-doctoral grant from the ‘Fonds pour la Formation à la Recherche dans l'Industrie et l'Agriculture (FRIA)’ and of a short-term Marie Curie Fellowship. MB is a Research Associate from the Belgian National Funds for the Scientific Research (FNRS). This work received partial financial support from the ‘Fonds de la Recherche Fondamentale Collective’ (no. 2.4524.02) and from the France–Belgium Tournesol program (no. 03/003).
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