JXB Advance Access originally published online on November 16, 2005
Journal of Experimental Botany 2006 57(1):101-111; doi:10.1093/jxb/erj009
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RESEARCH PAPER |
A root- and hypocotyl-specific gene coding for copper-containing amine oxidase is related to cell expansion in soybean seedlings
1Laboratory of Molecular Biology, Department of Agricultural Biotechnology, Agricultural University of Athens, Iera Odos 75, 11855 Botanikos, Athens, Greece
2Laboratory of Plant Physiology and Morphology, Department of Agricultural Biotechnology, Agricultural University of Athens, Iera Odos 75, 11855 Botanikos, Athens, Greece
* To whom correspondence should be addressed. Fax: +30 210 5294314. E-mail: bmbi2kap{at}aua.gr
Received 6 May 2005; Accepted 29 September 2005
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Abstract |
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Polyamines are considered to participate in various processes of plant development. In this study, the possible implication of putrescine catabolism by the copper-containing amine oxidases (CuAOs, EC 1.4.3.6 [EC] ) in the development of roots and hypocotyls was examined. For this purpose, two cDNA clones of Glycine max (L.) Merr. cv. Williams, designated as GmCuAO1 and GmCuAO2, exhibiting extensive similarity with previously characterized CuAO clones from other plants, have been isolated and characterized. The expression of the GmCuAO1 gene is root- and hypocotyl-specific, while GmCuAO2 seems not to be expressed in a tissue-specific manner. Moreover, the GmCuAO1 gene is predominantly expressed in tissues which are characterized by rapid extension growth, such as the apical segments of etiolated hypocotyls. Using convex and concave segments of the etiolated hypocotyl apical hook it has been demonstrated that GmCuAO1 is strongly expressed in expanding cells of the concave part when exposed to light, while the same pattern is also followed by the activity of enzymes involved in putrescine catabolism. In dark and photoperiodically grown hypocotyls, activity measurements of the enzymes involved in putrescine catabolism have shown that the activity of these enzymes is several-fold higher in rapidly growing tissues. Furthermore, the cellular and tissue distribution of GmCuAO1 gene transcripts in the root axis and in hypocotyls confirmed their abundance in developing tissues and expanding cells. The results provide evidence suggesting that a tissue-specific gene coding for CuAO is correlated with cell expansion in fast-growing tissues of root and hypocotyls.
Key words: Copper-containing amine oxidase, Glycine max, hypocotyl development, in situ hybridization, polyamines, root development
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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The hypocotyl of dicotyledonous plants is widely used as a model for the study of cell elongation mechanisms (Cosgrove and Durachko, 1994
). Lack of light causes seedling hypocotyls to elongate, a phenomenon that is characteristic of etiolation (skotomorphogenesis). As soon as the dark-grown seedlings are exposed to light, a significant reduction of the elongation rate occurs and the apical hook straightens. Cell extension growth in turgid organs such as soybean hypocotyls is brought about by the chemical loosening of growth-limiting cell walls, resulting in the relaxation of wall tension and concomitant osmotic water uptake (Cosgrove, 1999, 2000).
The biochemical mechanism that regulates the wall-loosening reaction has not yet been elucidated, although numerous potential wall-loosening proteins have been investigated. These proteins are thought either to catalyse the enzymatic degradation of particular cell-wall polysaccharides in vitro or to belong to the family of expansins which cause stretch-dependent extension in acidified cell walls by breaking non-covalent bonds (Cosgrove, 1999, 2000). A novel wall-loosening mechanism, in auxin-induced growth, involving the controlled production of hydroxyl radicals (.OH) in the cell walls has been put forward (Fry, 1998; Chen and Schopfer, 1999). Biological production of .OH is believed to be mediated by the reduction of hydrogen peroxide (H2O2) with superoxide anion (
) (Haber-Weiss reaction), catalysed either by Fe or Cu ions or by peroxidases (Chen and Schopfer, 1999; Schopfer 2001). Besides the participation in wall-loosening events, it has been hypothesized that peroxidase activity is also implicated in H2O2-dependent wall stiffening that causes mechanical fortification and confers extension irreversibility during cell growth (Fry, 1986; Hohl et al., 1995; Schopfer, 1996).
Furthermore, H2O2 in the apoplast can be produced by the action of amine oxidases including copper-containing amine oxidases (CuAO: EC 1.4.3.6
[EC]
) oxidizing the diamines putrescine and cadaverine (Angelini et al., 1993; Møller and McPherson, 1998; Laurenzi et al., 2001; 
ebela et al., 2001) and flavin-containing polyamino oxidases, which oxidize spermidine and spermine at the secondary amino groups (PAO: EC 1.5.3.3
[EC]
) (Bagni and Tassoni, 2001; Cona et al., 2003). It has been suggested that H2O2, generated by diamines and polyamines, is important in lignin biosynthesis and cell-wall cross-linking reactions that may regulate growth as well as defence, wounding, and cell death responses (Angelini et al., 1993; Møller and McPherson, 1998; Laurenzi et al., 2001; ebela et al., 2001; Cona et al., 2003; Walters, 2003; Rea et al., 2004).
A considerable body of evidence indicates that CuAO and PAO are predominantly located in the cell wall (Angelini et al., 1993; ebela et al., 2001; Cona et al., 2003) and that CuAO can be released to the apoplast (Møller and McPherson, 1998). As a result of CuAO activity, putrescine is converted into 
1-pyrroline with the simultaneously release of H2O2 and ammonia as by-products. Then 1-pyrroline can be further catabolized to 
-aminobutyric acid (GABA) by the action of pyrroline dehydrogenase. GABA is subsequently transaminated and oxidized to succinic acid, which is incorporated into Kreb's cycle (Bhatnagar et al., 2002). Thus, polyamine catabolism is not simply a degradable process but is also an important link between amino acid and carbon metabolism in plants, resulting in the recycling of nitrogen as well as the carbon skeleton of putrescine (Bhatnagar et al., 2002).
The level of CuAO in etiolated legume seedlings was found to be higher than in light-grown ones (Federico and Angelini, 1988). These data may suggest that CuAO activity has a role in emerging plants development. In addition, comparative immuno- and histochemical localization of CuAO in Pisum sativum (L.), Lens culinaris (Medik.), and chickpea plants (Cicer arietinum (L.)) treated under different light conditions has been reported (Laurenzi et al., 2001). In that report, CuAO activity in etiolated seedlings of pea and lentil was present in all root cortical cell walls, whereas in sections taken from the basal internode of older seedlings CuAO also appeared in central cylinder parenchyma. It is worth noticing that in sections taken from roots and epicotyls of de-etiolated plants the CuAO activity almost disappeared from the cortical cells.
The expression pattern of genes correlated with putrescine degradation in plants has not yet been elucidated although, in Arabidopsis thaliana, the Atao1 gene coding for CuAO is expressed in the lateral root cap, in differentiating regions of the root vascular tissue, and in the tracheary elements of hypocotyls. It has been suggested that the Atao1 expression pattern is correlated either with cell-wall cross linking/lignification or with developmental programmed cell death (Møller and McPherson, 1998).
In the present study, the involvement of CuAO in the process of tissue development and cell expansion was investigated. Two Glycine max (L.) cDNA clones coding for CuAO, one of which is organ-specific, have been identified. Using semi-quantitative reverse transcription-PCR (RT-PCR), in situ hybridization, CuAO activity measurements, and histochemical localization techniques it is concluded that the organ-specific gene coding for CuAO is involved in cell-wall expansion processes during soybean root and hypocotyl development.
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Materials and methods |
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Plant material and growth conditions
Soybean (Glycine max (L.) Merr cv. Williams) seeds were pregerminated between two sheets of moist paper, in Petri dishes, in the dark at 26 °C, for 2 d. Germinated seeds were left in the Petri dishes in the dark at 26 °C for a further 6 d and total RNA was isolated from hypocotyls, cotyledons, and root segments representing the region of cell division and elongation, the root hair region, and the lateral root emergence region. For in situ hybridization, roots and hypocotyls from the 6–8-d-old seedlings grown in the dark were used. Plants were also developed in a modified 0.5x Hoagland solution, under a 16/8 h light/dark cycle, at 150 µmol photons m–2 s–1 and 26 °C for 1 month. Young leaves, mature leaves, and stems of 30-d-old seedlings were used to isolate total RNA. For the histochemical analysis, seeds of soybean were soaked in tap water for 1 h and germinated in a growth chamber on wet sterilized perlite at 26 °C. The seedlings were grown in these conditions for 7 d under continuous dark or in a 16/8 h light/dark cycle at 150 µmol photons m–2 s–1. Uniformly grown seedlings were selected with an average hypocotyl length of about 10 cm for the dark and about 4 cm for the photoperiodically grown seedlings. The sampling for the dark-grown hypocotyls took place under a green safe light. The hypocotyls were separated in three equal parts (apical, middle and basal) and were used for total RNA isolation, histochemical and assay analysis (Fig. 1). Moreover, the apical hooks of 7-d-old seedlings growing under continuous dark were exposed to 150 µmol photons m–2 s–1 for 0, 30, 90, and 150 min to light and were split longitudinally to give the convex and the concave halves of hook curvature. These segments were also used for total RNA isolation and assay analysis.
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Growth measurements
Ten 5-d-old etiolated seedlings were selected with a hypocotyl length of about 3 cm and were marked at 1 cm intervals with Indian ink. After 24, 48, and 72 h the distance between marks was measured using a micrometer under safe green light and the growth rate of each segment was calculated. The angle between the middle point of the cotyledons and the hypocotyl axis in ten dark-grown seedlings was measured from pictures taken at 0, 30, 90, 150, and 210 min after the exposure to light and the rate of curvature angle change was calculated.
Semi-quantitative RT-PCR analyses
Semi-quantitative RT-PCR analysis was performed using total RNA, which was isolated according to Brusslan and Tobin (1992) and extracted from the tissues of interest as described above. Total RNA was quantified by spectrophotometry and agarose gel electrophoresis. All RNA samples were treated for 20 min at 37 °C with DNase I (Promega, Madison, WI) to eliminate DNA contamination. First strand cDNA was reverse transcribed from 1 µg of DNase-treated total RNA. All the DNase-treated total RNA samples were denatured at 65 °C for 5 min followed by a quick chill on ice in a 12 µl reaction mixture containing 500 ng oligo(dT)12–18mer and 1 µl of 10 mM dNTPs. After the addition of 4 µl of 5x First-strand buffer (Invitrogen, Paisley, UK), 1 µl (40 U) RNaseOUT (Invitrogen) ribonuclease inhibitor and 2 µl of 0.1 M dithiothreitol (DTT), the reaction was preheated at 42 °C for 2 min before the addition of 1 µl (200 U) of SuperScript II reverse transcriptase (Invitrogen). The reaction mixture was incubated at 42 °C for 50 min, followed by heat inactivation at 70 °C for 15 min. For normalization of the different RNA preparations, a 260 bp fragment of G. max ubiquitin was amplified, using two gene-specific primers designated as GmUBQ-F (5'-GGGTTTTAAGCTCGTTGT-3') and GmUBQ-R (5'-GGACACATTGAGTTCAAC-3'). For each gene under study, a pair of gene-specific primers was designed. These were designated as GmCuAO1-F (5'-CAGCATTGGTGTTTACCACG-3'), GmCuAO1-R (5'-ATGCAGTGCTCAATAATGGC-3'), GmCuAO2-F (5'-AATGGGTAGGAGGGTCATAC-3'), and GmCuAO2-R (5'-TTTTCAGTCCAATGCGCACC-3'). PCR amplification was performed for each gene under study from 22 to 30 cycles at 94 °C for 1 min, 55 °C for 1 min, and 72 °C for 1 min. A complete final extension for the PCR products was performed at 72 °C for 20 min. Amplified products were separated on 1.5% agarose gels. The PCR products were then extracted and ligated into the poly-T site of pGEM-T Easy vector (Promega, Madison, WI) and the complete sequence of products was determined.
In situ mRNA hybridization
Root and hypocotyls segments were fixed in 4% (w/v) paraformaldehyde supplemented with 0.25% glutaraldehyde in 10 mM sodium phosphate buffer pH 7.4, for 1 h, in a vacuum aspirator. Fixed tissues were dehydrated in ethanol series, and then exchanged with xylene before embedding in paraffin. For the GmCuAO1 gene a pair of gene-specific primers was designed and a 551 bp fragment, exhibiting 65% similarity to the GmCuAO2 clone, was ligated into the pGEM-T Easy vector (Promega, Madison, WI) and the complete sequence of the product was determined. Antisense and sense RNA probes were labelled with digoxigenin (DIG)-11-rUTP (ROCHE) by in vitro transcription of a PCR product derived from the GmCuAO1 clone using the SP6 and T7 promoters of the pGEM-T Easy vector. Sections (10 µm) were placed on poly-L-lysine slides, digested with proteinase K for 30 min at 37 °C, treated with acetic anhydride, dried in ethanol, and then hybridized with the appropriate gene-specific DIG-labelled probes overnight, at 42 °C. After washing with 4x SSC containing 5 mM DTT, the slides were treated with RNase A for 30 min at 37 °C, washed with RNase A buffer 500 mM NaCl, 1 mM EDTA (ethylenediaminetetraacetic acid), 10 mM TRIS–HCl, pH 7.5, containing 5 mM DTT, and then processed for revealing the DIG antigen. This involved blocking with DIG-blocking reagent and bovine serum albumin, followed by incubation with an anti-DIG antibody conjugated to alkaline phosphatase, and washing with blocking reagent. The colour revealed by incubation in 5-bromo-3-chloro-3-indolyphosphate nitroblue tetrazolium and the reactions were stopped with water, the slides dehydrated, air-dried, and then mounted in DPX before viewing. The sections were examined using a Zeiss Axiolab microscope (Carl Zeiss, Jena, Germany) and pictures were taken with a SONY DSC-S75 38P/45 (SONY Corporation, Japan) digital camera system.
Preparation of plant extracts for enzyme activity measurements
Hypocotyl segments from 7-d-old seedlings were immediately transferred in liquid nitrogen and homogenized at a ratio of 1:1 (w/v) with 0.1 M potassium phosphate buffer, pH 7.0. Extracts were filtered through nylon cloth and centrifuged at 12 000 g, for 20 min at 4 °C. In each assay, three replicates of ten hypocotyl segments (apical, middle, or basal) were used. Similarly, three replicates of ten convex or concave halves of hook curvature were also analysed.
Measurements of enzymatic activity involved in putrescine catabolism
The activity of enzymes involved in putrescine catabolism was determined by using a coupled reaction with horseradish peroxidase and guaiacol (Luhová et al., 2003), with putrescine as a substrate. The reaction mixture (final volume 1 ml) contained 0.1 M potassium phosphate buffer pH 7.0, 0.5 mM guaiacol, 1 U of peroxidase and plant extract. The reaction was started by the addition of 5 µl of putrescine (final concentration 2.5 mM). The time-dependent increase in absorption at 436 nm was recorded for a period of 3 min. Enzyme activity was assayed using a Hitachi U-2800 spectrometer. No enzyme activity was observed in the hypocotyls without the addition of putrescine in the reaction mixture.
Histochemical detection of enzyme activity involved in putrescine degradation
Root and hypocotyl segments were washed with sterile water and were then fixed in 2% (v/v) formalin pH 7.0, 2% (w/v) polyvinylpyrrolidone-40 and 1 mM DTT for 30 min at 4 °C according to Sergeeva and Vreugdenhil (2002). Fixed tissues were washed with sterile water twice more and placed in Triton 2% (v/v) at –20 °C overnight. The tissues were then washed several times with sterile water before being assayed. For enzyme visualization a modification of the Luhová et al. (2003) method was used. The reaction mixture contained 50 mM phosphate buffer pH 7.0, 5 mM putrescine as substrate, 2.5 mM 4-chloronaphthol, and 5 U ml–1 horseradish peroxidase. The colour revealed by incubation of segments for 30 min at room temperature and the reactions were stopped with water. No distinct differences in the intensity of the colour were observed after the addition of water in the samples. As a control, segments of roots and hypocotyls were treated with a reaction mixture without the addition of the putrescine.
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Results |
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Growth measurements
The highest growth rate of etiolated hypocotyls was found to be restricted to the apical segment of the hypocotyls (Fig. 2). Calculations of changes in the degree of etiolated hypocotyl apical hook curvatures 0, 30, 90, 150, and 210 min after the exposure to light (Fig. 2, insert) showed that significant changes were observed only after 150 min.
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Isolation and characterization of cDNA clones coding for GmCuAO
CuAO genes generally consist of a multigene family in various plant species (Tipping and McPherson, 1995
; Møller and McPherson, 1998). Digital northern searches among public soybean EST databases revealed the presence of a number ESTs in G. max coding for enzymes involved in putrescine degradation. The corresponding ESTs were obtained and the complete nucleotide sequence of the larger cDNA clones was determined. The deduced amino acid sequence revealed the presence of CuAO cDNAs representing a full-length clone designated as GmCuAO1 (GenBank accession no CAE47488), coding for a polypeptide of 673 amino acids and a partial cDNA clone of 821 bp designated as GmCuAO2. The deduced amino acids sequence of CuAO1 of G. max was interrogated with other known plant CuAOs and revealed a 45%, 78%, and 80% similarity to A. thaliana, L. culinaris, and P. sativum, respectively. Similarly, comparison of the partial GmCuAO2 and other previously characterized plant CuAOs amino acid sequences showed a high level of sequence conservation exhibiting a 58%, 66% and 67% similarity to A. thaliana, L. culinaris, and P. sativum, respectively. Amino acid residues that have been shown to be important for the catalytic activity of plant CuAO are apparently conserved in both GmCuAOs polypeptides (Fig. 3).
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The amino acid sequence between 409 and 419 of PsCuAO, that has previously been characterized to comprise the cofactor TPQ (2,4,5-trihydroxyphenylalanine quinone)-containing peptide (Janes et al., 1992
), is conserved in all plant CuAOs published so far, including GmCuAO1. Furthermore, the N-terminal sequence of plant CuAOs that was predicted to be a secretion signal with the cleavage site between residues 25 and 26 of PsCuAO are conserved and in GmCuAO1 (Tipping and McPherson, 1995). This is in good agreement with previously obtained evidence that all plant copper amine oxidases so far examined are extracellular enzymes (Tipping an McPherson, 1995; Møller and McPherson, 1998). Thus, GmCuAO1 is also probably an extracellular enzyme. In addition, the conserved consensus in many amine oxidases N-Y-D (Asn-Tyr-Asp) (residues 411 to 413 of GmCuAO1), within which the Tyr has been shown to become modified to the TPQ cofactor (Janes et al., 1992; Mu et al., 1992; Tipping and McPherson, 1995) are also conserved in GmCuAO1. Four histidines, three of which correspond to copper ligands (Parson et al., 1995; Kumar et al., 1996; Møller and McPherson, 1998) (residues 467, 469, and 628 of GmCuAO1) are conserved in both GmCuAO1 and GmCuAO2.
GmCuAO1 is a root and hypocotyl-specific gene in soybean
As observed in Fig. 4 the highest accumulation of GmCuAO1 transcripts was found in hypocotyls, root tip, the root hair region, and the lateral root emergence region, while no signal was detected in mature and young leaves, cotyledons, and stems. The accumulation of GmCuAO2 gene transcripts was found in all tissues examined except the cotyledons and root tips, while its transcripts were most abundant in mature leaves and the lateral root emergence region (Fig. 4). These data indicate that the GmCuAO1 gene is root- and hypocotyl-specific, while GmCuAO2 is not expressed in a tissue-specific manner, suggesting that the GmCuAO1 and GmCuAO2 genes are differentially regulated in soybean.
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The accumulation of GmCuAO1 gene transcripts in hypoctyl segments treated under different light conditions
The highest accumulation of GmCuAO1 gene transcripts was found in the apical part of dark-growing hypocotyls, which corresponds to the zone of elongation (Li et al., 1997
), while lower levels of gene transcripts were detected in apical part of hypocotyls growing under 16/8 h light/dark photoperiod. In the other hypocotyl segments, regardless of light conditions, the accumulation of GmCuAO1 gene transcripts was significantly lower (Fig. 5).
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GmCuAO1 expression is correlated with the straightening of the etiolated hypocotyl apical hook after light treatment
To extend the experiments described in the previous section, the hypocotyl hook straightening system was used which depends on asymmetric cell expansion (Silk and Erickson, 1978
). The accumulation of GmCuAO1 gene transcripts exhibited an asymmetric distribution that was most pronounced 90 min after exposure to light. Indeed, the transcript levels of the GmCuAO1 gene were more abundant in the concave half of the apical hook, than in the convex half. Upon transfer to light GmCuAO1 transcript levels were also increased in the convex part of the hook and remained constant throughout the experiment. The presence of a relatively high expression of the GmCuAO1 gene in the convex half 30 min after the illumination of hypocotyls hook could be explained by a quicker response of the convex part to light, but finally to a lesser extent (Fig. 6). However, the levels of GmCuAO2 gene transcripts are more or less constant in all tissues examined, indicating that GmCuAO2 expression remains unaffected by light treatment (Fig. 6). Therefore, it is concluded that the GmCuAO1 transcript levels correlate with the light stimulated straightening of the hypocotyls apical hook in soybean.
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Distribution of putrescine catabolism in hypocotyls and the hypocotyl apical hook of soybean seedlings
The highest activity of enzymes involved in putrescine catabolism was measured in the apical part of dark-growing hypocotyls, which showed a several-fold higher rate of putrescine catabolism compared with the other two-thirds of dark-grown or photoperiodically grown hypocotyls. Although, the dark-grown hypocotyls possess higher activity of these enzymes with respect to photoperiodically grown hypocotyls, in both treatments the highest activity was measured in the apical part of hypocotyls, which corresponds to the fast-growing part of hypocotyls (Fig. 7). Consequently, putrescine catabolism was higher in fast-growing parts of hypocotyls and the dark-grown hypocotyls possessed the highest activity of enzymes involved in putrescine catabolism. It was noticeable that the distribution of putrescine catabolism and the distribution of GmCuAO1 transcripts in soybean hypocotyls followed the same pattern, which indicates that putrescine catabolism and the expression of GmCuAO1 in certain tissues are probably related.
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Although in all the samples taken 0, 30, and 90 min after exposure to light, putrescine catabolism was slightly higher in the convex half of the hypocotyl apical hook, in samples taken 150 min after the exposure to light putrescine catabolism was approximately 2-fold higher in the concave half compared with all the other samples, including the convex half from the hypocotyl apical hook 150 min after the exposure to light (Fig. 7). This is in good agreement with results taken from RT-PCR, where the expression of the GmCuAO1 gene are asymmetrically distributed in the convex and the concave halves 90 min after exposure to light. Furthermore, these data strongly suggest that the activity of enzymes involved in putrescine catabolism is correlated with the rapid extension cell growth that occurs in the concave half of the hypocotyl apical hook, as can be conferred from the rate of changes in curvature 150 min after exposure to light (Fig. 2, insert).
Localization of GmCuAO1 gene transcripts in main, lateral root and hypocotyls
A pair of gene-specific primers was designed for GmCuAO1 and a 551 bp fragment, exhibiting 65% similarity to the GmCuAO2 clone, was used as a probe. Sections of various regions of roots and hypocotyls from 6–8-d-old etiolated seedlings were hybridized with DIG-11-rUTP labelled RNA. Both antisense and sense-labelled RNA transcripts were used as probes (Fig. 8).
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When median longitudinal sections of main roots and hypocotyls were hybridized with the GmCuAO1 antisense probe, hybridization signals were largely detected in the root tip region and hypocotyls (Fig. 8A, E, F, G). On the basis of main root and hypocotyl anatomical features, transcripts were highly abundant in the root cap, cortex parenchyma, and central root cylinder, as well as in hypocotyl vascular bundles and cortex parenchyma, while the hybridization signal was barely detected in meristematic tissues of the root axis. In addition, a strong signal was detected in cortex parenchyma of the apical part of etiolated hypocotyls (Fig. 8E), corresponding to the zone of elongation, while the cortex parenchyma of the basal and middle part of the hypocotyls was barely stained (Fig. 8F), which is in good agreement with previous results obtained from RT-PCR analysis. Furthermore, in hypocotyl curvatures, the signal was abundant on the concave side where the cells were relatively unexpanded, compared with a hardly detected signal in the expanded cells of convex side (Fig. 8G).
To identify the cell layers in which GmCuAO1 is expressed, in situ hybridization was conducted with cross-sections of main roots and hypocotyls (Fig. 8B, C, H). On the basis of developmental stages, sections were chosen representing the root zones of cell division (Fig. 8B) and elongation (Fig. 8C) as well as the hypocotyl elongation zone (Fig. 8H), where the CmCuAO1 gene transcripts were abundant, and hybridized with the antisense probe. A strong hybridization signal at a distance of 180 µm from the root tip was detected in root cap cells and expanding cells immediately surrounding the meristem, while no significant signal was detected in the meristematic tissues (Fig. 8B). As shown in Fig. 8C, which corresponds to a cross-section of the root elongation zone (900 µm from the root apex), very heavy stain was found in the expanding cells of the cortex parenchyma and elongating cells of the central cylinder, but not in root cap and procambium layers. Taken together, the available data suggest that the expression of the GmCuAO1 gene is strongly correlated with cell expansion events in developing roots.
Cross-sections from the elongation zone of hypocotyls have been used for the in situ hybridization approach to identify cell layers in which the GmCuAO1 gene was preferentially expressed. Although the hybridization signal was detected in all the cells in the hypocotyl elongation zone, strong signal was detected in epidermal cells, cortex parenchyma, central cylinder, and the vascular bundles of elongating hypocotyls (Fig. 8H). These data support the suggestion that GmCuAO1 is preferentially expressed in elongating tissues.
The RT-PCR analysis revealed that high levels of GmCuAO1 gene transcripts were also detected in the lateral root emergence region. To investigate the tissue and cellular distribution of this gene in lateral roots, in situ hybridization analysis was performed in sections taken from primary roots of 6–8-d-old seedlings (about 3 cm from the root tip), where the lateral root-initiating region was longitudinally sectioned. As observed in Fig. 8D, the cells in the lateral root meristem were weakly stained, whereas in the cells of pericycle, sieve tubes, xylem, and cortex parenchyma of the main root the signal was higher. In addition, hybridization signal was detected in cortex parenchyma of emerging lateral root primordia. The results presented here suggest that GmCuAO1 gene expression is possibly involved in the development of lateral roots.
Histochemical detection of enzyme activity involved in putrescine catabolism
Etiolated hypocotyl and root segments were fixed and subjected to a histochemical localization method of enzymes involved in putrescine catabolism. The H2O2 produced by putrescine catabolism formed a blue precipitate with chloronapthol by the action of peroxidase. In the root axis, the tissue distribution of the enzymes involved in putrescine degradation was similar to the localization pattern of GmCuAO1 gene transcripts (Fig. 8I, J). These enzymes are localized mainly in the root cap, the cortex parenchyma of the elongation zone, and in vascular bundles of the root axis. In hypocotyls, the histochemical detection revealed that these enzymes were present in all the tissues of the hypocotyls although a strong signal was detected in the cortex parenchyma (Fig. 8K). Not surprisingly, in all the tissues examined, the precipitate was abundant in cell walls, indicating that enzymes involved in putrescine catabolism are probably localized predominantly in cell walls.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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In the present report, the possible implication of putrescine catabolism in tissue development and cell expansion was examined. For this purpose, two G. max genes coding for CuAO, designated as GmCuAO1 and GmCuAO2, and which exhibited high similarity to other previously characterized plant CuAO genes were isolated and characterized. GmCuAO1 gene transcripts were detected only in roots and hypocotyls, while the GmCuAO2 gene was not expressed in a tissue-specific manner. Although, transcripts of genes coding for CuAO have been found in all organs examined except cotyledons, these genes have a different regulation in soybean seedlings, suggesting that CuAO, besides regulating the putrescine levels in various plant organs, may have a more specific physiological role to accomplish. Tissue-specific expression of CuAO genes was also found in A. thaliana, where the Atao1 gene is expressed in the lateral root cap, in differentiating regions of the root vascular tissue, and in tracheary elements of hypocotyls (Møller and McPherson, 1998
).
In soybean hypocotyls treated under different light conditions, GmCuAO1 gene transcripts accumulated in the apical part of etiolated hypocotyls, whereas in the other segments the expression of GmCuAO1 was significantly lower. In etiolated soybean hypocotyls, it has been shown that the rate of growth is predominantly distributed in the apical third (Fig. 2), which corresponds to more than 90% of the total growth with respect to the mature and the basal zones (Li et al., 1997). The high expression of genes involved in putrescine biosynthesis in soybean hypocotyls (Delis et al., 2005) as well as the high expression levels of the GmCuAO1 gene in the fast-growing tissues of the hypocotyl, suggest that CuAO could be involved either in the regulation of putrescine levels, which is important for tissue development, or that putrescine catabolism is involved in another mechanism in the tissue developmental processes.
In transgenic plants, over-expression of heterologous or homologous genes involved in polyamine biosynthesis (either arginine or ornithine decarboxylases) causes elevated levels of putrescine, while the levels of spermine and spermidine remains unaffected (Bastola and Minocha, 1995; Bhatnagar et al., 2001). In addition, an increase in putrescine levels in most of the transgenic plants does not lead to an abnormal phenotype and/or tissue development and cell expansion. These data suggest that the plants are able to tolerate high concentration of putrescine, which is not directly involved in plant development. Furthermore, in a previous work, Scoccianti et al. (1990) have found that the putrescine levels in soybean hypocotyls were several-fold higher in growing than in grown hypocotyls. Similarly, the levels of a diamine oxidase followed the same pattern and decreased sharply in grown hypocotyls. The data suggest that, in growing hypocotyls, high putrescine biosynthesis is accompanied by high putrescine degradation, whereas in grown hypocotyls both biosynthesis and catabolism are sharply decreased.
During germination of the seedling, a hook-like structure is formed at the apical part of the hypocotyl. The hook is maintained until the seedling emerges and upon exposure to light, the hook opens. During opening of the apical hook, cells at the concave edge elongate throughout the straightening process (Raz and Ecker, 1999). According to the results of this study, the expression level of GmCuAO1 was asymmetrically distributed in the convex and concave parts of the soybean apical hook and was most pronounced 90 min after exposure to light. Moreover, the accumulation of GmCuAO1 gene transcripts was higher in the concave part of the hypocotyl apical hook where the cells are growing faster to allow the hypocotyl to straighten. On the other hand, the accumulation levels of GmCuAO2 in apical hook edges were unaffected during light stimulation. These data suggested that GmCuAO1 is strongly correlated with the rapid extension growth of the cells during light stimulation.
The implication of polyamine catabolism in fast-growing tissue development is further supported by activity measurements of the enzymes involved in putrescine catabolism in soybean hypocotyls and the hypocotyl apical hook after exposure to light. These measurements revealed that the apical fast-growing parts of etiolated hypocotyls (Fig. 2) and the expanding cells of the concave half of the hypocotyl apical hook (Fig. 2, insert) 150 min after the exposure to light possess the highest activity. Furthermore, the localization of enzymes involved in putrescine catabolism in the cortex parenchyma of the main root elongation zone, in vascular bundles of the root axis, and in the cortex parenchyma of hypocotyls, could provide evidence that expression of GmCuAO1 is probably involved in cell expansion.
Vianello et al. (1993) reported that the activity of amine oxidase, measured with a method similar to ours and isolated from soybean etiolated seedlings, was completely lost a few minutes after incubation with Cu-binding inhibitors (cyanide, diethyldithiocarbamate). Moreover, incubation with pargyline, an inhibitor of FAD-containing amine oxidase has no effect on the activity of amino oxidase in soybean seedlings. With regard to this, the measured activity of the enzymes involved in putrescine degradation in soybean hypocotyls can probably be ascribed to copper-containing amine oxidase.
It has previously been shown that genes involved in putrescine biosynthesis are mainly localized in regions where root and hypocotyl cells are expanding (Delis et al., 2005). Not surprisingly, in situ hybridization revealed that the GmCuAO1 gene was also mainly expressed in regions of cell elongation of primary, secondary roots, and hypocotyls. The hypothesis that putrescine degradation may participate in cell expansion processes is further supported by recent evidence, where it was shown that a root-specific expansin is correlated with root cell elongation, exhibiting an almost identical pattern of expression to that seen for soybean GmCuAO1 (Lee et al., 2003). Moreover, the high accumulation of GmCuAO1 transcripts in the apical segments of hypocotyls compared with the basal and middle segments, further supports the above suggestion. Similarly, in hypocotyl curvatures, the hybridization signal was detected in the concave part, where the cells are expanding, while the signal in the convex part, where the cells are already expanded, was hardly detected. These results indicated that polyamine degradation could participate in cell expansion during growth in soybean roots and hypocotyls.
Recently, it has been proposed that cell-wall loosening during auxin-induced growth is controlled by an apoplastically produced hydroxyl radical (.OH) (Chen and Schopfer, 1999; Schopfer, 2001). It is believed that .OH production is mediated by the reduction of hydrogen peroxide (H2O2) with superoxide anion (O2–) catalysed either by Fe or Cu ions or by peroxidases. Furthermore, it has been found that the formation of O2– is induced by auxin, in the growth-controlling epidermis of the coleoptiles, while .OH scavengers inhibited the auxin-induced elongation growth of maize coleoptiles (Chen and Schofer, 1999; Schopfer, 2001). In addition, more recently it has been shown that ROS (Reactive Oxygen Species) production is required for root growth in A. thaliana (Foreman et al., 2003).
To gain a further insight into the biochemical mechanism and the contribution of the particular ROS in cell-wall loosening events, it is important to examine their derivation. In higher plants, several sources are known to exist for the generation of the ROS (Bolwell, 1999). These include a membrane-bound NADPH oxidase, a plant analogue of gp91phox, the core polypeptide of mammalian enzyme (Keller et al., 1998; Torres et al., 1998), lipoxigenase (Croft et al., 1990), and apoplastic peroxidase (Bolwell et al., 1995). In addition, H2O2 is produced in the cell wall either by dismutation (either spontaneously or catalysed by cell wall superoxide dismutases), or from the oxidative cycle of peroxidases in the presence of a reductant (Bolwell, 1999; Cona et al., 2003, Passardi et al., 2004).
The derivation of apoplastic H2O2 a as by-product of putrescine catabolism via the cell-wall located CuAO activity (Angelini et al., 1993; ebela et al., 2001) may be incorporated in the production of hydroxyl radicals that participate in the breaking of cell-wall polysaccharides, such as pectin and hemicelluloses (xyloglucan) (Fry, 1998). These suggestions are in agreement with evidence in onion roots, where it has been shown that H2O2 is mainly located in cell walls, while the amount of H2O2 inside the cells was very low (Cordoba-Pedregosa et al., 2003). This hypothesis is further supported by the fact that these extremely reactive molecules (H2O2, O2–, .OH), which are capable of splitting covalent bonds in all kinds of organic molecules, are likely to derive it from a tightly regulated pathway such as polyamine metabolism, where most enzymes have short half-lives, indicating that they are an important metabolic control point in the cell (Malmberg et al., 1998; Bhatnagar et al., 2002). This scenario is further supported by the abundance of polyamines in the plant cell, due to the fact that if .OH is able to act stoichiometrically against the covalent bonds of cell-wall hemicelluloses, then it will probably be produced by molecules with high concentration.
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Footnotes |
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Abbreviations: CuAO, Copper-containing amine oxidase; DIG, digoxigenin; DTT, dithiothreitol; GABA,
-aminobutyric acid; PAO, flavin-containing polyamine oxidase; RT-PCR, reverse transcription PCR; ROS, reactive oxygen species; TPQ, 2,4,5-trihydroxyphenylalanine quinone. |
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