JXB Advance Access originally published online on February 27, 2008
Journal of Experimental Botany 2008 59(4):939-950; doi:10.1093/jxb/ern017
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© 2008 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
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RESEARCH PAPER |
Modulation of eIF5A1 expression alters xylem abundance in Arabidopsis thaliana
Department of Biology, University of Waterloo, 200 University Avenue West, Waterloo, Ontario N2L 3G1, Canada
* To whom correspondence should be addressed. E-mail: jet{at}uwaterloo.ca
Received 5 November 2007; Revised 26 December 2007 Accepted 10 January 2008
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Abstract |
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Eukaryotic translation initiation factor 5A (eIF5A) is thought to facilitate protein synthesis by participating in the nuclear export of specific mRNAs. In Arabidopsis, there are three isoforms of eIF5A. One of them, AteIF5A1, has been shown to be expressed in vascular tissue, specifically developing vessel members, using GUS as a reporter. In order to determine whether AteIF5A1 plays a role in xylem formation, its full-length cDNA was constitutively over-expressed in transgenic Arabidopsis plants. Microscopic analysis revealed that the cross-sectional area of the xylem in the main inflorescence stems of transgenic plants was 1.9-fold higher than those of corresponding inflorescence stems of wild-type plants. In wild-type stems, the primary xylem typically comprised six cell layers and was
105 µm thick, but increased to 9–11 cell layers, 140–155 µm thick, in transgenic stems. Similarly, the secondary xylem increased from six cell layers, 70 µm thick, in control stems to 9 cell layers, 95–105 µm thick, in transgenic stems. Moreover, constitutive down-regulation of AteIF5A1 using antisense technology resulted in the major suppression of xylem formation compared with control plants, and the antisense transgenic plants were also stunted. These data collectively indicate that eIF5A1 plays a role in xylogenesis. Key words: Arabidopsis thaliana, eukaryotic translation initiation factor 5A, inflorescence stem, xylem
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Introduction |
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Xylem plays an essential role in plant life by reason of being a tissue designed to carry out long-distance transport of water and solutes as well as to provide mechanical support. It is composed of tracheary elements (vessel members or tracheids), parenchyma cells, and often fibres. The tracheary elements and fibres are dead at maturity. Indeed, programmed cell death (PCD) is known to be a key feature of xylogenesis (Fukuda, 1996; Ye, 2002). Moreover, there is evidence that the death of tracheary elements is characterized by fragmentation of DNA, which is a characteristic feature of apoptosis (Mittler and Lam, 1995).
Xylem is ontogenetically classified as primary or secondary. Primary xylem is formed by the procambium, a meristem tissue that also gives rise to primary phloem. Eudicotyledonous species also develop a vascular cambium, which is derived from certain procambium and interfascicular parenchyma cells, and gives rise to secondary xylem and secondary phloem (Esau, 1977). The maturation of tracheary elements entails a sequence of cell specification, cell expansion, secondary cell wall deposition and lignification, and cell death. Useful insights into the regulation of these processes have been obtained by in vitro culture of Zinnia elegans mesophyll cells into tracheary elements (Fukuda, 1996). Vascular development is under the control of a network of gene expression cascades that, in turn, are regulated by hormones (Fukuda, 2004). Auxin is believed to be the major signal involved in the ontogeny of vascular tissues (Aloni, 1987; Sachs, 2000). In particular, there is evidence that, during the primary phase of plant development, high auxin levels in cell files promote their differentiation into vascular bundles (Sachs, 1991). Indeed, it has been proposed that auxin may initiate a signal transduction pathway regulating the development of vascular tissue from the procambium (Nieminen et al., 2004). This contention is consistent with the finding that auxin transporter genes are expressed along the cambial zone in hybrid aspen (Schrader et al., 2003). Several studies in which IAA has either been exogenously applied or, in some cases, constitutively over-produced have shown that it promotes xylem production (Klee et al., 1987; Tuominen et al., 1995, 2000; Zhong and He, 2001; Little et al., 2002; Ko et al., 2004).
The promotion of vascular tissue development by IAA during both the primary and secondary phases of plant growth is probably achieved through an effect on gene expression. For example, Arabidopsis ATHB-8, -9, -14, -15, and REV, members of the homeodomain-leucine zipper III (HD-Zip III) family, are all expressed in developing vascular tissues (Sessa et al., 1998; Zhong and He, 1999; Baima et al., 2001; Ohashi-Ito and Fukuda, 2003), and ATHB-8 expression is positively regulated by auxin (Baima et al., 1995; Mattsson et al., 2003). Moreover, constitutive over-expression of ATHB-8 has been shown to result in increased xylem formation (Baima et al., 2001). Analyses of ATHB-8 expression using GUS as a reporter are consistent with its involvement in cell proliferation during vascular development (Baima et al., 1995; Kang and Dengler, 2002). Studies of leaf vein formation and primary vascular patterning suggest that ATHB-8 may translate the auxin signal into early events of procambium development and function in defining xylem identity (Kang and Dengler, 2002; Kang et al., 2003). By contrast, ATHB-15 appears to be a negative regulator of vascular development, playing an antagonistic role to ATHB-8 during vascular morphogenesis (Kim et al., 2005).
In the present study, evidence is described to show that the eukaryotic translation initiation factor 5A (eIF5A) is involved in xylogenesis. Experiments with yeast and mammalian cells have indicated that eIF5A is not a conventional translation initiation factor. Rather, it is thought to facilitate translation by shuttling specific mRNAs from the nucleus to the cytoplasm for translation (Bevec et al., 1996; Bevec and Hauber, 1997; Schatz et al., 1998; Rosorius et al., 1999). In yeast and mammalian cells, eIF5A is synthesized as an inactive protein that is post-translationally activated by the sequential actions of two enzymes, deoxyhypusine synthase (DHS, EC 2.5.1.4 [EC] 6) and deoxyhypusine hydroxylase (DHH, EC 1.14.99.29 [EC] ). DHS catalyses the transfer of a butylamine residue from the polyamine, spermidine, to a conserved lysine in the N-terminus of the inactive eIF5A, resulting in the formation of deoxyhypusine. Deoxyhypusine hydroxylase then converts deoxyhypusine to hypusine giving rise to hypusinated eIF5A, which is the active form of the protein (Park et al., 1993, 1997; Park, 2006). The finding that recombinant plant DHS, like yeast and mammalian cell DHS, is capable of catalysing the formation of deoxyhypusine on plant eIF5A indicates that plant eIF5A is also synthesized as an inactive protein and post-translationally hypusinated (Ober and Hartmann, 1999; Wang et al., 2001). That hypusinated eIF5A selectively translocates mRNAs from the nucleus into the cytosol is supported by the finding that inhibitors of eIF5A hypusination cause the disappearance of only certain mRNAs from polysomes (Hanauske-Abel et al., 1995).
Genes encoding eIF5A are thought to be present in all eukaryotic cells (Jenkins et al., 2001). Full-length cDNA clones encoding eIF5A have been isolated from several plant species including tobacco, tomato, Arabidopsis, and rice (Chamot and Kuhlemeier, 1992; Wang et al., 2001, 2003; Chou et al., 2004), and in each case there is evidence for a multi-gene family. Herein, evidence is described indicating that AteIF5A1, one of the isoforms of eIF5A in Arabidopsis (Wang et al., 2003), is involved in xylogenesis.
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Materials and methods |
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Plant material
Arabidopsis thaliana (L.) Heynh. plants, ecotype Columbia, were grown in PRO-MIX® BX (Premier Horticulture, Ltd., Rivière-du-Loup, Québec). Seeds were sown in 32-well flats covered with plastic wrap and maintained at 4 °C for 2 d. The covers were then removed, and the flats were transferred to a growth chamber operating at 22 °C, 80% RH and daily 16 h light (150 µmol m–2 s–1 photosynthetically active radiation)/8 h dark cycles where the plants were grown to maturity.
Modulation of eIF5A expression
Two constructs designed to modify the expression of eIF5A were expressed in transgenic Arabidopsis plants. In one set of transgenic plants, AteIF5A1 (GenBankTM accession number AF296082; Gene locus tag AT1G13950) was up-regulated by over-expressing the coding region of the gene. The coding region was amplified from a full-length cDNA isolated previously (Wang et al., 2001) using the upstream primer, 5'-GAAGCTCGAGGCTGCAACCATGTCC-3' containing an XhoI site (underlined) and downstream primer, 5'-GGGGAGCTCTTGTTAGTCTCACTTGG-3' containing a Sac1 site (underlined). The resultant PCR fragment was subcloned into the binary vector, pKYLX71 (Schardl et al., 1987), in the sense orientation under the control of two copies of the constitutive cauliflower mosaic virus promoter (CaMV-35S) giving rise to the construct Pro35S:sense AteIF5A1. The pKYLX71 vector contains a tetracycline-resistance gene in the bacterial replication region as well as a kanamycin-resistance gene (nptII) between the right- and left-border regions. In a second set of transgenic plants, AteIF5A1 was suppressed by constitutively expressing the 3'-UTR of the gene in its antisense orientation. For this purpose, the 3'-UTR was amplified from AteIF5A1 full-length cDNA (Wang et al., 2001) using the upstream primer, 5'-CTCGAGTAGTGGTGAGTGTGATGTCAGCTATGG-3' containing an XhoI site (underlined) and downstream primer, 5'-AAGCTTAGAAGAAGTATAAAAACCATC-3', containing a HindIII site (underlined). The resultant PCR fragment was then subcloned into pKYLX71 in the antisense orientation under the control of two copies of the CaMV 35S promoter giving rise to the construct Pro35S:antisense 3'-UTR AteIF5A1.
Each of the recombinant binary vectors as well as the empty binary vector were introduced individually into Agrobacterium tumefaciens, strain GV3101 by electroporation, and 4-week-old Arabidopsis plants were infected with the transformed agrobacteria by vacuum infiltration (Bechtold et al., 1993). The treated plants were grown to maturity, and seeds were collected. Transgenic plants were selected by germinating these seeds on media containing kanamycin. The seeds were surface-sterilized in a solution of 1% sodium hypochlorite, rinsed three times in sterile water, and plated on 0.7% agar containing 
MS salt (Sigma-Aldrech, Oakville, Ontario, Canada) and 50 µg ml–1 kanamycin. The plates were maintained at 4 °C for 2 d and then transferred to a tissue culture chamber operating at 22±3 °C with 16 h light (150 µmol m–2 s–1 photosynthetically active radiation)/8 h dark cycles. After 10 d, the surviving seedlings were transferred to PRO-MIX® BX and grown to maturity in a growth chamber under the conditions specified above. For Pro35S:sense AteIF5A1 and Pro35S:antisense 3'-UTR AteIF5A1 transgenic plants, seed was harvested and the selection process repeated until homozygous lines were obtained. The presence of the transgene in the transgenic plants was confirmed by PCR using genomic DNA as a template. The DNA was isolated from the rosette leaves of 3-week-old plants using the Wizard® genomic DNA extraction kit (Promega). Genomic DNA from wild-type plants and those carrying empty binary vector was used as a control.
Analysis of AteIF5A1 promoter activity
To localize AteIF5A1 expression in Arabidopsis, a β-glucuronidase (GUS) transgene regulated by the promoter for AteIF5A1 was expressed in transgenic plants. The 5' region 2.1 kb upstream of the ATG start codon in the genomic sequence for AteIF5A1 (GenBankTM accession number AC007576) was amplified by PCR using the upstream primer, 5'-GACAAGCTTCCTAGATTCGTGGACACAGATCG-3', containing a HindIII site (underlined) and the downstream primer, 5'-TTAGTCGACGAGCTTCGACGAGGATTTTGC-3', containing a SalI site and genomic DNA isolated from 4.5-week-old wild-type plants as the template. The PCR product was subcloned into pGEM T-Easy® vector, amplified in E. coli, sequenced and subcloned into the binary vector, pB1101, upstream of the β-glucuronidase (GUS) gene, giving rise to the construct ProAteIF5A1:GUS. The recombinant binary vector was introduced into A. tumefaciens strain GV3101 by electroporation, and Arabidopsis transformation and progeny selection were carried out as above. Transgenic plant material was stained for histochemical visualization of GUS activity according to Jefferson et al. (1987). For microscopic analyses, free-hand sections were cut just below the first internode on the main inflorescence stems of 4-week-old Arabidopsis plants. The sections were examined using a Zeiss Axiophot® microscope (Carl Zeiss Canada, Don Mills, ON) equipped with a Q-Imaging digital camera system (Quorum Technologies, Inc., Guelph, ON).
Antibody production and western analysis
Polyclonal antibodies against the coding region of eIF5A were raised in rabbits using Arabidopsis recombinant AteIF5A1 protein eluted from SDS-polyacrylamide gels as antigen (Wang et al., 2001). Isoform-specific antibodies against AteIF5A1 (GenBankTM accession number AF296082; Gene locus tag AT1G13950), AteIF5A2 (GenBankTM accession number BE039424; Gene locus tag AT1G26630), and AteIF5A3 (GenBankTM accession number AV526594; Gene locus tag AT1G69410) were raised in rabbits using synthetic peptides (Fig. 1) as antigens. Uniqueness of the peptides was confirmed by interrogation using protein BLAST. The carrier protein, KLH, was conjugated to the N-terminal cysteine of the peptides using m-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) as described by Kaup et al. (2002). Briefly, dissolved KLH (10 mg ml–1 in water) was mixed drop-wise with dissolved MBS (10 mg ml–1 in dimethylformamide) and stirred at room temperature for 30 min. The MBS–KLH complex was purified using a Sephadex 25 column and mixed with the synthetic peptide (1:1, w:w) for the production of primary antibodies in rabbits.
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For western analysis, total protein was extracted from 2-week-old seedlings or from rosettes of plants at specified ages. To this end, 200 mg of tissue was homogenized on ice with a mortar and pestle in 500 µl of extraction buffer (50 mM HEPES, pH 7.4). The protein was quantified according to Bradford (1976), fractionated on 12% SDS-polyacrylamide gels and the separated proteins transferred to Hybond®-P membranes (Amersham-Pharmacia). Immunoblotting was performed according to Wang et al. (2001) using the rabbit polycolonal antiserum (above) and alkaline phosphatase-conjugated goat anti-rabbit IgG (Roche Molecular Biochemicals, Basel).
Anatomical measurements
Fully mature, 8-week-old plants were chosen for xylem measurements. Free-hand cross-sections were cut 10 mm from the base of the main inflorescence stems, which is well below the first internode. For both transgenic and wild-type plants, the stem region from which the sections were cut was 30-d-old, i.e. the number of days after the initiation of bolting. The sections were treated with phloroglucinol-HCl, which selectively stains lignified cell walls such as those of mature tracheary elements and fibres, and examined microscopically. Primary and secondary xylem tissues were distinguished on the basis of two criteria. First, the vessel members of secondary xylem are of similar diameter and, along with the fibres, are arrayed in radial files, whereas for primary xylem there is a gradient of vessel member size ranging from small (protoxylem) to larger (metaxylem). Secondly, Arabidopsis secondary xylem contains fibres, which are not present in primary xylem. Xylem was quantified by three methods. (i) Cell layers along the radial axis of both primary and secondary xylem tissues were counted. Interfascicular sclerified cell layers (including fibres) were also counted. (ii) The thickness of xylem tissues was measured in the radial direction using a calibrated ocular scale on the sections. The thickness of the interfascicular sclerified cells (including fibres) was also measured. (The regions used for these measurements are identified in Fig. 7A.) (iii) Cross-sectional areas of the stem and selected stem tissues were measured. To do so, micrographs were digitized using Adobe® Photoshop® 7.0 and printed at suitable factors of enlargement. Regions corresponding to the entire stem, the xylem, and the phloem were cut out of the prints. The areas of the cut-outs were measured using an LI-3000A area meter coupled to a LI-3050A conveyer (John Morris Scientific, USA).
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Results |
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Expression of AteIF5A1 in developing xylem
AteIF5A1 (GenBankTM accession number AF296082 and Gene locus tag AT1G13950) is one of three isoforms of eIF5A in Arabidopsis. The expression of AteIF5A1 was examined in transgenic Arabidopsis plants expressing a GUS reporter gene under the regulation of the promoter for AteIF5A1. GUS staining proved to be prominent in the leaf vasculature (Fig. 1A) and in developing main inflorescence stems (Fig. 1B, C) of ProAteIF5A1:GUS plants. Microscopic examination of the vasculature in leaf and stem sections from ProAteIF5A1:GUS plants revealed that the GUS staining was present in xylem tissue but not in phloem (Fig. 2A, B), and in developing vessel members, but not in mature vessel members (Fig. 2C, D). As expected, no GUS staining was observed in vessel members of stems of wild-type plants (Fig. 2D).
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Effect of AteIF5A1 up-regulation on xylem development
AteIF5A1 was constitutively over-expressed in transgenic plants in order to determine whether it plays a role in xylem development. Three separate over-expressing transgenic lines designated S-AteIF5A1 line 1, S-AteIF5A1 line 2, and S-AteIF5A1 line 3 were examined. The presence of the transgene in each of these lines was confirmed by PCR. It was also clear from Western blots probed with antibody raised against the coding region of AteIF5A that levels of AteIF5A1 protein were up-regulated in each of the lines in comparison with wild-type plants and plants transformed with an empty binary vector (Fig. 3A). The coding region of eIF5A1 is highly conserved in the three Arabidopsis isoforms of this protein (Fig. 4), and hence this antibody detects endogenous protein corresponding to AteIF5A1, AteIF5A2, and AteIF5A3 as well as trans AteIF5A1 protein. For one of the transgenic lines, S-AteIF5A1 line 2, which exhibited the highest level of over-expression, an additional band that cross-reacted with antibody against the AteIF5A coding region was evident in the western blot (Fig. 3A). eIF5A is synthesized as an inactive protein that is post-translationally modified by two enzymatic reactions that convert a conserved lysine in the N-terminus to hypusine (Wang et al., 2001). Thus, it is possible that the additional band in the western blot for S-AteIF5A1 line 2 corresponds to the inactive, unhypusinated form of eIF5A1 protein. That this additional band was detectable for the S-AteIF5A1 line 2 and not for the other two lines examined may be related to the higher level of over-expression of the protein in the S-AteIF5A1 line 2.
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Over-expression of AteIF5A1 resulted in larger rosette leaves but had no effect on the timing of the transition from vegetative to reproductive growth (Fig. 5). Specifically, the transgenic and empty vector plants bolted and flowered at comparable times after planting (Fig. 5). However, inasmuch as the over-expression lines produce more and larger rosette leaves, bolting may actually occur later in terms of number of plastochrons/plant age. This would, in turn, mean that, since bolting occurs at the same chronological age in over-expressing and wild-type plants, development is accelerated in the over-expressing lines. Of particular interest is the finding that over-expression of AteIF5A1 resulted in thicker inflorescence stems and an increased abundance of xylem therein (Fig. 6A, C, D, E). These comparisons were made by examining cross-sections of the main inflorescence stems of 8-week-old control and transgenic plants taken 10 mm from the base, which corresponded to stem tissue that was 30-d-old. By this stage of development, the inflorescence stems were completely developed and had reached their maximum sizes. For control (Figs 6A, 7A, B
) and transgenic (Figs 6C–E; 7C, D) stems, the epidermis, cortex, vascular tissues, and interfascicular regions, and a parenchymatous pith were clearly evident in cross-sections. The primary xylem was separated by regions of interfascicular sclerified cells (Fig. 6A, B). The secondary xylem consisted of both fascicular and interfascicular regions derived from the fascicular cambium and the interfascicular cambium, respectively (Altamura et al., 2001). The increased thickness of the transgenic inflorescence stems correlated with enhanced amounts of primary and secondary xylem tissues (Figs 6A, C–E, 7A–D, 8, 9, 10) and also with enhanced levels of parenchymatous pith (Fig. 6A, C–E). Transgenic plants also exhibited wider bands of primary interfascicular sclerified cells (Figs 8, 9). By contrast, there was little change in levels of phloem in the transgenic inflorescence stems (Figs 6, 8–10
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0.05) relative to corresponding empty binary vector control values determined using the paired t test.
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Xylem was quantified by measuring the radial thickness of xylem tissues in stem sections. Cell layers in the radial direction were also counted. In control stems, the primary xylem typically comprised six cell layers and was
105 µm thick; in addition there were three layers of interfascicular sclerified cells that were collectively 75 µm thick (Figs 8, 9). By contrast, the primary xylem of transgenic stems typically comprised 9–11 cell layers and was 140–155 µm thick (Figs 8, 9). In addition, there were 4–5 layers of interfascicular sclerified cells that were collectively 95–120 µm thick (Figs 8, 9). The secondary xylem in control stems typically consisted of six cell layers in 70 µm thick radial files in fascicular regions (Figs 8, 9). By contrast, the secondary xylem of transgenic stems typically consisted of 9 cell layers in 95–105 µm thick radial files in the fascicular regions and 6 cell layers 65–75 µm thick in the interfascicular regions (Figs 8, 9). Moreover, the cells of the secondary xylem in the transgenic stems were of a smaller diameter than their counterparts in control stems, indicating accelerated cell maturation (Fig. 7B, D). Overall, there was more enhancement of secondary xylem than of primary xylem in the transgenic stems relative to control stems (Figs 8, 9). Based on measurements of cross-sectional area, the transgenic inflorescence stems proved to be 1.7-fold larger than those for the corresponding control plants, and this was accompanied by an 1.9-fold enhancement of xylem (including primary xylem, secondary xylem, and primary fibres) (Fig. 10). However, there was no significant increase in the cross-sectional area of the phloem in the stems of transgenic plants (Fig. 10).
Effects of AteIF5A1 down-regulation on xylem development and rosette leaf senescence
AteIF5A1 was suppressed in transgenic Arabidopsis plants by constitutive expression of the corresponding 3' UTR cDNA in its antisense orientation. The presence of the 3' UTR cDNA in three separate lines, namely, A-AteIF5A1 line 2, A-AteIF5A1 line 4, and A-AteIF5A1 line 6, was confirmed by PCR. Western blots of rosette leaf protein probed with AteIF5A1-specific antibody indicated that levels of AteIF5A1 protein were reduced by 47% in A-AteIF5A1 line 2, 5% in line 4, and 46% in line 6 relative to levels in corresponding empty binary vector control rosettes (Fig. 3B, C). Isoform- specific antibodies for eIF5A proteins were raised against synthetic peptides corresponding to unique regions in their carboxy termini (Fig. 4). Xylem development was reduced in the AteIF5A1-suppressed plants, particularly lines 2 and 6 in which AteIF5A1 was more strongly suppressed (Figs 6A, F–H, 7A, E). Within the vascular bundles of the AteIF5A1-suppressed plants, there were only four primary xylem cell layers in 58 µm thick radial files for lines 2 and 6 compared with six primary xylem cell layers in 105 µm thick radial files for control stems (Figs 8, 9). In keeping with the lower level of AteIF5A1 suppression in line 4 (Fig. 3B, C), the number of primary xylem cell layers in the vascular bundles was about the same as that for control stems, but the thickness of the radial file was 80 µm compared to 105 µm for the control stems (Figs 8, 9). The inflorescence stems of the transgenic plants were also thinner than those of control plants, particularly for A-AteIF5A1 lines 2 and 6 (Fig. 6A, F, H).
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Discussion |
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eIF5A is thought to be ubiquitously present in eukaryotic cells and is highly conserved across the plant and animal kingdoms (Thompson et al., 2004). Studies with mammalian cells have indicated that eIF5A functions as a shuttle protein, translocating mRNAs from the nucleus to cytosolic ribosomes for translation (Rosorius et al., 1999). The structure of eIF5A is consistent with the contention that it binds to nucleic acids. Specifically, the C-terminal domain, which is thought to contain RNA-binding domains, resembles the oligonucleotide-binding domain found in prokaryotic cold-shock protein, prokaryotic translation initiation factor IF1, staphylococcal nuclease and the N-terminal domain of yeast Asp-tRNA synthase (Kim et al., 1998). There are two isoforms of eIF5A in the human genome designated eIF5A1 (GenBankTM accession number NM_001970) and eIF5A2 (GenBankTM accession number NM_020390), and there is evidence that one of these is required for cell division and that the other plays a role in apoptosis (Jakus et al., 1993; Taylor et al., 2004). If this is so, it seems likely that different isoforms of eIF5A translocate different species of mRNA from the nucleus into the cytosol. The selective nature of eIF5A involvement in mRNA translation is supported by the finding that inhibitors of the enzymes mediating its post-translational activation cause the disappearance of only certain mRNAs from polysomes (Hanauske-Abel et al., 1995).
In Arabidopsis, there are three isoforms of eIF5A, and it is apparent from the present study that one of these, AteIF5A1, is expressed in developing xylem tissue. This was evident from an analysis of GUS activity in ProAteIF5A1:GUS-expressing plants. This expression pattern has also been observed using GFP as a reporter (Z Liu, unpublished data). Moreover, xylem development was enhanced in transgenic plants over-expressing AteIF5A1, and curtailed in AteIF5A1-suppressed transgenic plants. Phloem development, however, was not significantly affected by changed expression of AteIF5A1. These observations are consistent with the notion that AteIF5A1 facilates xylem development.
It is clear from xylem-related EST collections and cDNA libraries for several species including pine (Pinus taeda L.; Allona et al., 1998), poplar (Sterky et al., 1998; Hertzberg et al., 2001; Milioni et al., 2002; Dejardin et al., 2004), Zinnia elegans (Demura et al., 2002), and Arabidopsis (Zhao et al., 2000) that numerous genes are expressed during xylem development. Among these is ATHB-8, a member of the homeodomain-leucine zipper (HD-Zip) family. The expression of ATHB-8 is positively regulated by auxin (Baima et al., 1995; Mattsson et al., 2003), which is in keeping with the fact that exogenously applied auxin and, in some cases constitutively over-produced auxin, have been shown to enhance vascular development (Tuominen et al., 1995, 2000, and literature therein; Zhong and He, 2001; Little et al., 2002; van der Graaff et al., 2003; Ko et al., 2004). Moreover, constitutive over-expression of ATHB-8 has been shown to result in increased xylem formation (Baima et al., 2001).
The findings in the present study that AteIF5A1 is expressed in xylem tissue and that its over-expression results in increased xylem formation suggest that this protein also plays a pivotal role in xylogenesis. Of particular note is the fact that GUS activity reflecting AteIF5A1 expression was detectable in developing vessel members of stem xylem tissue, but not in mature vessel members. The latter observation is to be expected inasmuch as mature vessel members are dead. Nonetheless, these findings are consistent with a prospective involvement of eIF5A1 in the maturation of vessel members. However, the effect of over-expressed eIF5A on xylem formation appears to go well beyond simply impacting vessel member development. This is evident from the finding that there was an increase in both primary and secondary xylem tissue in the stems of AteIF5A1-over-expressing plants. That there was more primary xylem tissue can be interpreted as indicating that constitutive AteIF5A1 over-expression results in an enlargement of procambial strands involving more cell divisions. That primary xylem, but not phloem, is affected reflects a selective effect on the capacity of the cambium and the procambium to produce xylem. The increase in secondary xylem, on the other hand, reflects more cell division within the vascular cambium of AteIF5A1-over-expressing plants, division that preferentially forms derivatives destined to become vessel members. These are over-arching effects reminiscent of the established regulatory role of auxin in determining vascular pattern formation (Torrey, 1957) and, therefore, raise the prospect that AteIF5A1 may impact IAA formation and transport. This possibility is currently being examined.
Although less is known about the function of plant eIF5A than of animal and yeast eIF5A, it has been shown that plant eIF5A, like its mammalian and yeast counterparts, is post-translationally hypusinated by the addition of a butylamine residue from spermidine to a conserved lysine (Ober and Hartmann, 1999; Wang et al., 2001). Hypusinated eIF5A is thought to be the active form of eIF5A for mammalian cells and yeast (Jakus et al., 1993; Chen et al., 1996). In Arabidopsis, suppression of deoxyhypusine synthase (DHS), the first of two enzymes required for the hypusination of AteF5A1, has been shown to delay leaf senescence (Wang et al., 2003). This was interpreted as indicating that hypusinated eIF5A plays a role in senescence. In the present study, up-regulation of AteIF5A1 was shown to enhance xylem formation. Previous studies with mammalian cells have shown that over-expressed eIF5A accumulates as unmodified (unhypusinated) eIF5A, presumably because the enzymes mediating hypusination are overwhelmed by the large amount of trans eIF5A protein being expressed (Wohl et al., 1995; Clement et al., 2006). This raises the possibility that unhypusinated eIF5A is the form of the protein that is involved in xylogenesis. A number of xylem-related genes have been identified in Arabidopsis (Ko et al., 2006), and it is possible that unhypusinated AteIF5A1 recruits mRNAs formed from one or more genes responsible for xylem development.
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Acknowledgements |
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The authors are grateful to Dr Carol Peterson and Daryl Enstone for a critical review of the manuscript. Financial support from the Natural Sciences and Engineering Research Council of Canada is gratefully acknowledged.
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Abbreviations |
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ATHB-8, Arabidopsis thaliana homeobox gene 8; DHS, deoxyhupusine synthase; eIF5A, eukaryotic translation initiation factor 5A; GUS, β-glucuronidase; IAA, indoleacetic acid; PCD, programmed cell death.
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References |
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