Blackwell Science, LtdOxford, UK PCEPlant, Cell and Environment0016-8025Blackwell Science Ltd 2001 24 757 Expression of oxalate oxidase in ryegrass stubble C. Davoine et al. Original ArticleBEES SGML
Plant, Cell and Environment (2001) 24, 1033–1043
Specific and constitutive expression of oxalate oxidase during the ageing of leaf sheaths of ryegrass stubble C. DAVOINE,1 E. LE DEUNFF,1 N. LEDGER,1 J.-C. AVICE,1 J.-P. BILLARD,1 B. DUMAS2 & C. HUAULT1 1Laboratoire
de Physiologie et Biochimie Végétales, UMR. INRA 950/UCBN, Institut de Recherche en Biologie Appliquée, Université de Caen 14032 Caen Cedex, France and 2Signaux et Messages Cellulaires chez les végétaux, UMR CNRS-UPS 5546, Pôle de Biotechnologie Végétale, 31326 Castanet-Tolosan, France
ABSTRACT Changes in the activity of oxalate oxidase (OxO) and of the concentrations of oxalate and H2O2 were investigated during the ageing of leaf sheaths of ryegrass (Lolium perenne L.) stubble. The accumulation of H2O2 during ageing coincides with the increases of both oxalate level and OxO activity. Western and Northern blot analyses using protein and RNA extracts of the different categories of leaf sheaths suggested that OxO gene expression, as well as Ca-oxalate synthesis, are crucial events of ageing for leaf sheaths. Immunocytochemistry experiments have revealed that OxO, which is an extracellular enzyme, is nearly always present in the parenchymatous cells surrounding the vascular bundles and in the cells of the lower epidermis. Overall, results suggest that in ryegrass that synthesizes both Caoxalate and OxO, the production of H2O2 and Ca2+ during ageing of stubble might be involved in the constitutive defences against pathogens, thus allowing the phloem mobilization of nutrient reserves from the leaf sheaths towards elongating leaf bases of ryegrass. Key-words: Lolium perenne; Ageing of leaf sheaths; Caoxalate oxidase; H2O2; oxalate.
INTRODUCTION Oxalate oxidase (oxalate oxygen oxidoreductase, EC 1·2·3·4) was first reported by Zaleski & Reinhard (1912) from studies of powdered wheat grains. Remarkably, it was more than 80 years before Lane et al. (1993) and Dumas et al. (1993) determined in wheat the similarity between oxalate oxidase (OxO) and germin, the synthesis of which coincides with the onset of germination of embryos. It has been shown recently that OxO belongs to the superfamily of cupins (Dunwell, Khuri & Gane 2000), which includes germin-like polypeptides, seed storage globulin and also sucrose-binding proteins. OxO is a manganese containing homohexameric glycoprotein (Woo et al. 2000), showing a great resistance to degradation by proteases and to dissociation by heat, extreme pHs and SDS (Grzelczak & Lane 1984). These unusual properties have made it possible to
Correspondence: Erwan Le Deunff. Fax: +33 2 31 56 53 60; e-mail:
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assay for OxO activity on nitrocellulose membranes after sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE)-Western blot and to confirm the identity between germin and OxO. OxO converts Ca-oxalate to H2O2 and Ca2+ in the presence of O2. For many years, calcium oxalate was considered as a terminal waste product which forms in a large variety of plants, shape-specific crystals (Webb 1999). In Poacea, oxalate crystals have been found in many higher plant species in both reproductive and vegetative organs. Genetic analysis has suggested that Ca-oxalate crystal formation is a process involving a set of gene loci (Nakata & Mc Conn, 2000). In Poacea, Ca-oxalate accumulates in cereal seeds (Webb & Arnott 1982) and also in hay, straw and leaf sheaths (Yoshihara, Sogawa & Villareal 1979; Libert & Franceschi 1987). Ca-oxalate seems to be involved in calcium sequestration and accumulates mainly in vacuoles and in cell walls. However, some other studies suggest that Caoxalate formation is not an end process, but is a cell-mediated process that can be rapidly reversed in order to ensure calcium regulation (Franceschi 1989). At the physiological level, Ca-oxalate appears to control stomatal functioning (De Silva, Hetherington & Mansfield 1998), anther dehiscence (Horner & Wagner 1980) and growth (Lane 1994). Studies on cereal germination (Dumas, Freyssinet & Pallett 1993) have also suggested that oxalate degradation by OxO (and correlatively oxalate synthesis) should be developmentally or environmentally regulated. During the first days of barley germination of cereal embryos, OxO is detected mainly in coleorhyza (Dumas et al. 1993, 1995; Caliskan & Cuming 1998). In these tissues, OxO is located in the extracellular matrix zones, especially in the epidermis and vascular bundles of proliferating and elongating cells. These facts led to the suggestion that at least during cereal germination, the H2O2 produced by OxO is required for peroxidative cross-linking involved in cell-wall restructuring. In another connection, Ca-oxalate and OxO synthesis seems to be an essential determinant of pathogenicity or to the contrary, plant resistance. Although oxalate has been considered in a few cases, and paradoxically, as an inducer of plant defences (Davis et al. 1992; Jung et al. 1995), oxalic acid secreted by some micro-organisms is known to be both a toxic compound against predators (Patschovsky 1920) and a pathogenicity factor (Dunwell et al. 2000). The main effects of oxalic acid are to chelate calcium and to suppress 1033
1034 C. Davoine et al. the oxidative burst induced by pathogens (Cessna et al. 2000) and therefore to cancel the induction of plant defences. On another hand, induction of OxO expression in plant cells in response to oxalate-secreting pathogens might participate in the defence against pathogens and lead to broad resistance (Dunwell et al. 2000). The involvement of OxO in the general defence against pathogens was first proposed by Dumas et al. (1995) and Zhang, Collinge & Thordal-Christensen (1995). More recently, Schweizer, Christoffel & Dudler (1999) demonstrated that the transient expression of the germin gene in wheat epidermal cells confers disease resistance and that germin might also be involved in cell-wall cross-linking by the pathogeninduced oxidative microburst. However, H2O2 generated by OxO might either directly inhibit pathogen infection (Bolwell & Wojtaszek 1997) or act as a signal leading to the synthesis of defence proteins, such as pathogenesis-related (PR) proteins (Chamnongpol et al. 1998). It is interesting to note that PR proteins are often expressed in the lower epidermis and vascular strands of leaves (Mauch, Meehl & Staehelin 1992). Defence genes (Buchanan-Wollaston 1997) and for instance those coding for PR proteins are also expressed during senescence of Brassicus napus L. (Hanfrey, Fife & Buchanan-Wollaston 1996). Although H2O2 plays a general part in senescence leading to the overproduction of reactive oxygen species and to lipid peroxidation (Leshem 1988), H2O2 might also play a crucial role as a redox signal involved in the establishment of constitutive disease resistance at this late stage of development. This assumption is supported by the well-established fact that H2O2, in connection with nitric oxide and salicylic acid, is involved in the induction of defences against pathogens (Van Camp, Van Montague & Inzé 1998). Perennial ryegrass is a cool forage plant that accumulates storage compounds in leaf sheath bases (Ourry, Boucard & Salette 1988; Morvan et al. 1997). These carbon and nitrogen compounds are then mobilized from the stubble and used for regeneration of new shoots, especially after defoliation by herbivores or cutting. Separate analyses of leaf sheaths and elongating leaf bases (ELB) provides a convenient model to characterize the different stages of leaf senescence from the inner sheath to the outer leaf sheath. Indeed, senescence of stubble is an active process, which allows redistribution of nutrients to the growing ELB and therefore needs a very efficient protection against pathogens and predators. In connection with this, we have recently shown that the accumulation of H2O2 during ageing of leaf sheaths of ryegrass coincided with a drop of glutathione content and a decrease of ascorbate-recycling activity (Piquery et al. 2000). These results also have been obtained in naturally senescent cotyledons of Cucumis sativus (Kanazawa et al. 2000). Paradoxically, a high ascorbate level and a high ascorbate peroxidase activity were maintained during ageing of leaf sheaths (Piquery et al. 2000). Apart from the fact that ascorbate might ensure the link between sugar mobilization, which is very active in ryegrass leaf sheaths (Morvan et al. 1997), and ascorbate/oxalate synthesis, as also suggested by Yoneyama et al. (1997), Hor-
ner, Kausch & Wagner (2000) and Kostman et al. (2001), OxO might play a role in H2O2 production during ageing of leaf sheaths. In this study, we present results supporting the involvement of OxO in the process of senescence of ryegrass and demonstrate that the oxalate content as well as the OxO activity increase from the inner to the outer leaf sheaths and that OxO mRNA expression increases similarly in these tissues. Moreover we demonstrate that the OxO activity is located principally around the vascular bundles and also in the lower epidermis. These overall facts suggest that the OxO/oxalate system should be involved in the protection of stubble against pathogen infections allowing the remobilization of nutrients towards the regrowing tissues.
MATERIALS AND METHODS Plant material Seeds of ryegrass (Lolium perenne L. var Bravo) were germinated and grown for 8 weeks under a controlled environment on a nutrient solution as described previously by Morvan et al. (1997). After 2 months, plants were defoliated at 4 cm above ground level. The residual material (stubble) was composed of leaf sheaths tightly folded one inside the other and of ELB. Four categories of leaf sheaths (I, inner leaf sheath; MI, internal medium leaf sheath; ME, external medium leaf sheath; O, outer leaf sheath) were separated before analysis (Fig. 1).
Figure 1. Schematic representation of a longitudinal section of the different categories of leaf sheaths in a tiller of ryegrass (Lolium perenne L. var Bravo). ELB: elongating leaf base; I: inner leaf sheath; MI: internal medium leaf sheath; ME: external medium leaf sheath; 0: outer leaf sheath.
© 2001 Blackwell Science Ltd, Plant, Cell and Environment, 24, 1033–1043
Expression of oxalate oxidase in ryegrass stubble 1035
Enzyme assays For detection of the OxO activity, 0·5 g of fresh tissue was ground in 4 mL distilled water containing 20% polyvinylpolypyrrolidone (PVPP), then centrifuged at 15000 ¥ g for 10 min. The supernatant was precipitated between 30 and 70% of (NH4)2SO4 saturation. The pellet was resuspended in water, desalted on Sephadex G-25 and then assayed according to Dumas et al. (1993).
Determination of H2O2 and oxalic acid concentration H2O2 was extracted by homogenizing plant tissues (0·5 g) with 4 mL of 200 mM perchloric acid. The homogenate was centrifuged at 12000 ¥ g for 10 min and the resulting supernatant purified on Dowex AG1-X2 (Bio-Rad, Yvry sur Seine, France) before H2O2 measurement following the method of Okuda et al. (1991). Oxalate concentration was determined with the diagnostic Oxalate kit (Sigma, St Quentin, France) and represents the sum of soluble and insoluble oxalate. Fresh tissue (0·5 g) was ground in 4 mL water, centrifuged at 1500 ¥ g and the supernatant was used for the quantification of the oxalate level in the different categories of leaf sheaths.
Protein extraction and sample preparation One gram leaf tissue was extracted at 4 ∞C in 7 mL of 25 mM Tris-HCl buffer (pH 7·5) containing 2 mM phenylmethylsulfonylfluoride (PMSF), 0·1% (v/v) b-mercaptoethanol, 1 mM EDTA and 10 mM leupeptin. After centrifugation (3200 ¥ g, 4 ∞C for 10 min), the supernatant was mixed with 7 mg protamin and centrifuged at 18 000 ¥ g, 4 ∞C for 10 min. The proteins in the resulting supernatant were precipitated using the sodium deoxycholate–trichloroacetic acid procedure described by Peterson (1983). The resulting pellet was used for determination of soluble proteins according to the procedure of Lowry et al. (1951). The pellet was also resuspended in 65 mM Tris-HCl, 20% (v/v) glycerol, 2·3% (w/v) SDS, pH 6·8, containing 100 mM dithiothreitol (DTT) and 0·04% (w/v) bromophenol blue, boiled for 5 min to denature the proteins and then cooled until examined using electrophoresis.
Electrophoresis and Western blotting SDS-PAGE was performed as described by Laemmli (1970), using a 5·5% stacking gel and a 15% acrylamide separation gel. Equal mass of samples (5 mg of soluble proteins) was loaded per lane, according to the Lowry assay. Low and broad pre-stained molecular weight markers were obtained from Bio-Rad. Following electrophoresis, the gels were stained using silver nitrate or analysed using the Western blot technique. Electrophoretic transfer of polypeptides from SDS-PAGE gels on to polyvinylidene difluoride (PVDF) membrane (Immobilon-P; Proteigene, St Marcel, France) was conducted by semi-dry electroblotting (2·5 mA
for 30 min, Milli Blot System; Proteigene), according to the protocol described by Towbin, Staehelin & Gordon (1979). After blotting, PVDF membranes were treated with affinity-purified polyclonal anti-OxO antibodies at 1 : 20 000 dilution (Dumas et al. 1993). The antigen–antibody complex was visualized with alkaline phosphatase linked to goat (Ovis L.) anti-rabbit (Oryctolagus cuniculus L.) IgG as described by Blake et al. (1984). Gels were then quantitatively analysed using the computerized image analysis system (Bioimage, Vilbert Lourmat, Marne la Vallée, France).
Immunostaining for light microscopy Stubble was fixed in freshly prepared 2% (w/v) paraformaldehyde and 0·1% (v/v) glutaraldehyde in 0·1 M sodium phosphate buffer (pH 7·2) at 4 ∞C for 2 h. Following an overnight incubation in 0·1 M sodium phosphate buffer (pH 7·2), the tissues were dehydrated through a graded ethanol series and placed in several changes of hydrophilic Unicryl (British Biocell International, Cardiff, UK) resin polymerized for 72 h at 4 ∞C by illumination with UV light (360 nm) as previously described by Avice et al. (1996). Sections (2 mm thick) of stubble were cut with a LKB ultramicrotome and transferred to drops of water on clean, microscope slides and were allowed to dry for 30 min at 37 ∞C. Immunogold labelling was carried out by first incubating sections at room temperature in a blocking solution consisting of 40 mL of 0·1% bovine serum albumin/10% normal goat serum (v/v) in 20 mM TBS (Tris-HCl buffer pH 7·8) for 30 min. Excess solution was then drained from the sections and replaced with 40 mL of primary antibody solution (anti-OxO IgG at 1 : 100 dilution) prepared in TBS0·1% (v/v) bovine serum albumin containing 0·05% (v/v) Tween 20 overnight at 4 ∞C. Sections were then washed thoroughly and repeatedly for 5 min in TBS-0·1% bovin serum albumin (pH 7·8), and the excess buffer removed without allowing the sections to dry. Sections were immersed in secondary antibody consisting of 40 mL goat anti-rabbit IgG conjugated to colloidal gold (Biocell; 5 nm particle size 1 : 200 working solution in TBS-0·1% bovine serum albumin) for 60 min at room temperature and then washed thoroughly in TBS-0·1% bovine serum albumin followed by a final rinse in distilled water. Silver enhancement was carried out for 15 min at room temperature in accordance with the manufacturer’s instructions (Biocell SE kit; British Biocell International). Slides were rinsed in distilled water and directly mounted with Biomont (mounting medium; British Biocell International). Negative controls were performed to confirm the specific labelling of the sections by substitution of primary antibody with TBS buffer.
RNA and genomic DNA extraction Total RNA was extracted from 1 g of fresh leaf sheaths placed in a frozen mortar containing liquid nitrogen and ground together with 0·25 g of PVPP with a pestle. Total RNA was purified from the resulting powder with the aid of
© 2001 Blackwell Science Ltd, Plant, Cell and Environment, 24, 1033–1043
1036 C. Davoine et al. 8 mL of Tri Reagent according to the manufacturers modified instructions (MRC Euromedex, Mundolsheim, France). Poly (A)+ mRNA was extracted directly from total RNA by using oligotex mRNA midi Kit (Quiagen, Courtaboeuf, France). Genomic DNA was extracted from 1 g of fresh ELB tissue which was first ground in liquid nitrogen and the gDNA was obtained using the protocol of the genomic DNA isolation kit (Sigma).
Cloning of oxalate oxidase The OxO cDNA was obtained by the conjunction of RTPCR, 3¢ and 5¢ rapid amplification of cDNA ends (RACE) with the aid of the Marathon cDNA amplification kit (Clonetech, Montigny le Bretonneux, France). Two degenerate oligonucleotide primers were first designed from the conserved sequence domains of a number of aligned OxO sequences. These two oligonucleotide primers were used in a reverse transcriptase (RT)-polymerase chain reaction (PCR) reaction (as described later) to amplify a 412 bp fragment which was gel purified (Quiagen) and cloned in a pGEM-T vector (Promega, Charbonnieres, France). The plasm id DNA was extracted and sequenced. Analysis of the sequenced fragment by the Blast Program (http:// www.Infobiogen.fr) showed the sequence had a strong similarity (83–86%) to the other OxO gene sequences. To obtain the full length sequence the method of 3¢ and 5¢ RACE was used. One microgram of Poly (A)+ mRNA was used for the first and second-strands cDNA synthesis before the ligation of double strand cDNA adaptators according to the manufacturer’s instructions (Clonetech). First strand cDNAs were first synthesized using a polydT in a reverse transcription reaction as described below. 5¢ and 3¢ RACE cloning of the OxO cDNA were conducted by using two newly designed primers (OxOGSP2: 5¢-CAGAG TCTGGATGTGGCAGAGTGGCCCGG-3¢; OxOGSP1 : 5¢-GGCCACGGGAGA TGAGGAACGTCTCCCC-3¢) that produce overlapping of 5¢ and 3¢-RACE products. Touchdown PCR was performed with advantage 2 polymerase mix for 35 cycles: 5 cycles at 94 ∞C for 30 s, 72 ∞C for 4 min; 5 cycles at 94 ∞C for 30 s, 70 ∞C for 4 min and 25 cycles at 94 ∞C for 30 s, 68 ∞C for 4 min. Amplified products were then gel-purified with QIAquick Gel Extraction Kit (Quiagen) and cloned directly into the pGEM-Teasy cloning vector following the manufacturer’s protocol (Promega). For nucleotide sequence analysis, plasmid DNA was isolated with the flexiprep kit (Amersham, Orsay, France), and sequenced with ABI PRISM dRhodamine terminator of Perkin Elmer and run on an ABI PRISM 377 automated sequencer (Perkin Elmer Applied Biosystem, Orsay, France). Nucleotide sequences were compared with EMBL and Genebank databases using the Blast algorithm.
Northern blot analysis The 412 pb cDNA fragment obtained by RT-PCR and cloned in pGEM-T vector was used as specific probe in
Northern analysis. The amplified product of 412 bp fragment was gel purified and labelled with a32P dCTP (3000 Ci mmol-1) by using the random priming Neblot kit (New England Biolabs, St Quentin Yvelines, France). Ten micrograms of total RNA was fractionated on a 1·2% agarose gel containing formaldehyde and transferred to Hybond-N+ blotting membranes (Amersham) using 10 ¥ SSC (1·5 M NaCl, 0·15 M sodium citrate, pH 7), and cross-linked onto the membranes by UV treatment (Biorad). After an overnight hybridization at 60 ∞C in Church buffer (Church & Gilbert 1984), blots were washed under stringent conditions and then were exposed to Kodak BioMax MS film (Eastman Kodak Company, New York, USA). The relative hybridization signal was analysed and quantified using a phosphoimager and then analysed by the Cyclone storage phosphor system and Optiquant software (Packard Instrument Company, Rungys, France).
Southern blotting Ten micrograms DNA was digested with 10 U of restriction enzyme (Bam HI, EcoRI, Sph I, Hind III) in 20 mL of reaction volume. Each digestion was separated on a 0·8% agarose gel and stained with ethidium bromide. For blotting, the DNA was denatured in 0·25 M HCl for 15 min, 0·4 M NaOH/0·6 M NaCl for 30 min, 0·5 M Tris-HCl/1·5 M NaCl for 30 min prior to capillary blot transfer onto nylon membranes (Hybond N+ Amersham). The membrane was neutralized with 2 ¥ SSC before being hybridized with the same 32 P-labelled cDNA probe that was used for Northern blotting.
RESULTS Model of senescence of leaf sheaths of Lolium perenne L. Ryegrass represents a convenient model to study the process of ageing. After defoliation by cutting, it is possible to separate the stubble into different categories of tissues, namely from the inner (the youngest) to the outer parts of the regrowing tiller (Fig. 1). These are the elongating leaf bases (ELB), the inner leaf sheath (I), the internal medium leaf sheath (MI), the external medium leaf sheath (ME) and the outer leaf sheath (O). The biochemical analysis of these tissues allows the study of the biochemical events involved in the ageing of the sheaths which coincides with reserve mobilization from leaf sheaths to ELB. We analysed, during leaf sheath ageing, certain aspects of H2O2 metabolism. The quantification of H2O2 in remaining leaf sheaths revealed that H2O2 level increased from the inner to the outer leaf sheath with a maximum level in ME and O leaf sheaths with about 2000 mmol g-1 FW (Fig. 2a). This surprising result was inconsistent with the high concentrations of ascorbate and increased ascorbate peroxidase activity (Piquery et al. 2000). These results suggested that production of H2O2 might not be due to a default of the detoxification systems, but to an overproduction of H2O2.
© 2001 Blackwell Science Ltd, Plant, Cell and Environment, 24, 1033–1043
Expression of oxalate oxidase in ryegrass stubble 1037 hand, a dramatic increase in OxO activity was observed during ageing of leaf sheaths. Indeed, in comparison with the youngest leaf sheath (I), OxO activity was about fivefold higher in the older leaf sheath (O). Moreover, OxO activity was very low in the upper part of the leaves (above the ligule), and also in roots of 8-week-old ryegrass plants (data not shown).
H2O2 (mmol L-1 g-2 FW)
(a) 2000 1500 1000 500
Oxalate (mmol L-1 g-2 FW)
(a) (b) 4 3 2 1
ELB
I
MI
ME
O
Figure 2. Determination of H2O2 and oxalate concentrations in the different categories of leaf sheaths of ryegrass. The different tissues were separated from the stubble and homogenized for the assay of (a) H2O2 and (b) oxalate levels. ELB: elongating leaf base; I: inner leaf sheath; MI: internal medium leaf sheath; ME: external medium leaf sheath; 0: outer leaf sheath. Vertical bars indicate the mean ±SD for n = 3.
(b)
Figure 2b shows that the increase of oxalate concentration was strictly parallel to the production of H2O2 (as ME and O). These data implied that OxO which releases H2O2 and CO2 from oxalate, would be involved in H2O2 production in the oldest leaf sheaths of ryegrass. Concurrently, the presence of Ca-oxalate crystals observed by microscopy in the O leaf sheath (Fig. 3a) led us to determine the oxalate content in the different categories of leaf sheaths (Fig. 2b).
(c)
OxO activity during the senescence process An in situ assay of OxO activity, after tissue incubation in a buffer containing oxalate and a chromogenic substrate 4chloro-1-naphtol (Dumas et al. 1993) was used. A strong dark-blue staining was revealed (Fig. 3b & C) in the oldest leaf sheaths (O, Me and MI). Either a lesser or no staining was observed in I, ELB and lamina. In comparison, OxO activity was screened in ryegrass seedlings. As in barley seedlings (Dumas et al. 1995), OxO activity was observed only in the young roots (data not shown). In order to obtain further evidence of the involvement of OxO during the ageing of leaf sheaths, we determined the OxO activity in vitro. OxO activity was assessed in extracts of ELB and of different categories of leaf sheaths as shown in Fig. 4a. No activity was detected in ELB. On the other
Figure 3. Observation of Ca-oxalate crystals in external medium leaf sheath (ME) and in situ OxO activity in the different categories of leaf sheaths of ryegrass. (a) Crystals of Ca-oxalate within cells of ME.(b) In situ assay of OxO activity. Blue staining corresponds to detection of OxO activity. (c) Enlarging of the stained vascular bundles of O leaf sheaths. ELB: elongating leaf bases; I: inner leaf sheath; MI: internal medium leaf sheath; ME: external medium leaf sheath; 0: outer leaf sheath.
© 2001 Blackwell Science Ltd, Plant, Cell and Environment, 24, 1033–1043
(mmol min-1g-2 FW)
1038 C. Davoine et al.
(a)
(b)
(c)
Figure 4. Quantification of OxO in the different categories of leaf sheaths. (a) Activity of OxO. Vertical bars indicate the mean ±SD for n = 3. (b) Immunodetection of OxO by Western blot after separation by SDS-PAGE. Five micrograms of soluble proteins are loaded per lane. (c) Expression of OxO transcripts after Northern blot and hybridization of 32P-labelled cDNA of OxO. Ten micrograms of total mRNA from the different leaf sheaths are loaded per lane. ELB: elongating leaf bases; I: inner leaf sheath; MI: internal medium leaf sheath; ME: external medium leaf sheath; 0: outer leaf sheath.
Expression of ryegrass oxalate oxidase (oxO1) during ageing of leaf sheaths In order to verify OxO expression, soluble proteins and mRNA of OxO was followed in the different senescing leaf sheaths (Fig. 4b & c). Western blot analysis of protein extracts of leaf sheaths, performed with antibodies raised against OxO of wheat (Dumas et al. 1993), revealed a protein of about 28 kDa which corresponds to the monomer of OxO wheat. Furthermore, OxO was not detected by antibodies in ELB but was shown to accumulate during ageing (Fig. 4b) with the same progression as OxO activity (Fig. 4a). It is therefore assumed that the increase of OxO activity arises from synthesis of OxO protein during ageing and not from activation of an inactive form. Similarly, we studied OxO mRNA synthesis during ageing of the leaf sheaths by Northern blot (Fig. 4c) and the results were in agreement with the induction of OxO synthesis. Consequently the overall result reveals that increase of OxO activity during ageing of leaf sheaths is concomitant to activation of the OxO gene transcription and that posttranscriptionnal regulation is unlikely.
Cloning and sequence analysis of oxO1 In order to isolate the cDNA for oxO1, degenerate primers were synthesized on conserved sequence motives of other OxO cDNA’s. Using these primers in a RT-PCR reaction on mRNA’s extracted from leaf sheaths, we obtained a 412 bp fragment whose deduced protein sequence showed a strong homology (89%) to the OxO gene of Hordeum vulgare (EMBL Y14203) in the Blast Program. From this sequence new primers (OxOGSP2 and OxOGSP1) were synthesized and employed in 5¢ and 3¢ RACE reactions to obtain two overlapping sequences, producing a consensus sequence of 934 bp. The full length of oxO1 cDNA and its deduced amino acid sequence of 224 amino acids (EMBL AJ291825) are shown in Fig. 5. The cDNA contains a canonical polyadenylation signal site at 44 bp downstream from the stop codon and a poly (A+) tail beginning 159 nucleotides after the stop codon. The deduced amino acid sequence, when analysed with signal P and the Psort software of the expasy proteomic tools website (http://www.expasy.ch/tools) and the prodom software (http://www.protein.Toulouse.INRA.fr/ prodom.html) showed a putative 23 amino acid hydrophobic signal peptide upstream of mature oxO1 N terminus. This N terminal signal sequence displayed some typical features with the REG and Golgi transit peptide present in all the germin-like proteins and revealed a predicted signal peptide with a cleavage site between 23 and 24 (Fig. 5). The presence of a signal peptide, the absence of the vacuolar targeting sequence (KDHEL) and the presence of two putative N-glycosylation sites (NTST, NGSA) supports the hypothesis of an apoplastic localization of oxO1. Indeed, most of the OxO activity is recovered in the cell-wall and in the apoplast fluid. In situ immunogold localization has shown an apoplastic localization of the enzyme (data not shown). It is interesting that a putative peroxisomal targeting sequence (SKF) and an N-myristoylation site (GX(3)S/TX(2)) exist in oxO1. The predicted molecular mass for this protein was 22 kDa. This result was near to the 28 kDa molecular mass which was deduced from the Western blot. The difference of molecular mass might be due to the presence of a glycan moiety in OxO.
Southern blotting In order to investigate the complexity of the organization of the OxO gene family in Lolium perenne, Southern analysis was performed. Genomic DNA was digested with four different restriction enzymes which did or did not cut within the OxO gene sequence. Hybridization was carried out under low stringency conditions with a short cDNA fragment of the OxO gene sequenced. This fragment represents a region of the oxO1 that is highly conserved between germin-like genes and was selected to maximize the chance of detecting all members of the germin/germin-like family. Each lane of restriction digest, as shown in Fig. 6a, indicated that the genome contains as many as one to two genes
© 2001 Blackwell Science Ltd, Plant, Cell and Environment, 24, 1033–1043
Expression of oxalate oxidase in ryegrass stubble 1039 TCG AGC GGC CGC CCG GGC AGG TCG GAG ATA CCT AAC ACT CTC AGC AGC TCT AGC CGA TCA AAT AGC CCT AGC TAA TTA AGC TTG TTT CAT AGT GAG CTC CAA ATG GCG TAC TTC AAA ACC CTA GCG GCT GGC CTC TTC M
A
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GCC TTG CTA TTC CTT GCT CCA TTT ATC ATG GCC ACC GAT CCG GAC CCA CTT CAG GAT TTT TGC GTG GCT A
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GAC CTT GAT GGC AAG GAG GTG TCC GTG AAC GGG CAC CCA TGC AAG CCC ATG TCA GAG GCC GGC GAT GAC D
L
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TTC CTC TTC TCC TCC AAG CTC GCT AAG GCC GGC AAC ACC TCT ACC CCG AAC GGC TCG GCC GTG ACA GAG F
L
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CTG GAT GTG GCA GAG TGG CCC GGT ACC AAC ACG CTA GGC ATG TCT ATG AAC CGC GTC GAC TTC GCG CCT L
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GGA GGC ACA AAC CCG CCG CAC ATC CAC CCA CGT GCC ACT GAG ATT GGC ATC GTC ATG AAA GGT GAG CTC G
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CTC GTT GGC ATC ATC GGT AGC CTC GAC TCT GGA AAC AAG CTC TAT TCC AAG GTG CTG CGT GCT GGA GAG L
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ACG TTC CTC ATC CCG CGT GGG CTC ATG CAC TTC CAA TTC AAC GTT GGC AAG ACA GAG GCT TCC ATG GTC T
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GTA TCC TTC AAT AGC CAG AAC CCT GGC ATC GTC TTT GTG CCA CTC ACC GTC TTT GGC TCC AAC CCA CCA V
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V
F
V
P
L
T
V
F
G
S
N
P
P
ATC CCC ACG GCC GTA CTC ACC AAG GCG CTC CGT GTG GAT GCC GGG GTC GTA GAA CTT CTC AAG TCT AAG I
P
T
A
V
L
T
K
A
L
R
V
D
A
G
V
V
E
L
L
K
S
K
TTT GCC GGT GGG TTT TAA CTT CAA TTC TAA GAC CGT GGA ATG ATC AAA TTA ATA ATT CAA ATA AGC ATG F
A
G
G
F
*
CTT GCC AGA GTT TAT AAT TGT GTC ACC ACA AGT CGT GTA GTT AAG CTT TTG TTC GAT TGA CGA ACC CCC AGG TTC GAC CAA TGT GGG AAG TTT ACT TGT ATT GTC AAA AAA
closely related to OxO. A phylogenic tree constructed with complete amino acid sequences of some germin, OxO and germin-like proteins from complete amino acid sequences shows that OxO genes can be subdivided into five clusters (Fig. 6b) corresponding to different plant families. The oxO1 gene product makes up a distinct cluster from the other OxO gene members. This fact suggests that during the ageing process of ryegrass leaf sheaths, some new types of OxO genes might play different roles.
Vascular tissue expression Tissue immunolocalization of OxO was performed by light microscopy in the different categories of leaf sheaths (Fig. 7). The strongest immunolabelling was observed in the oldest leaf sheaths (ME) and especially in cells located in the bundle sheath surrounding xylem and phloem tissues (inset, Fig. 7). Immunolabelling was also found, but at a low extent, in the upper and lower epidermis. No labelling was detected in the youngest leaves (ELB) and in the youngest (MI) leaf sheath (Fig. 7) as expected from the previous results (Figs 3b and 4a). Control experiments performed with TBST (TBS, 0·15% Tween 20) instead of anti-OxO serum confirmed the specificity of the reaction.
DISCUSSION The aim of the present study was to investigate whether or not the accumulation of H2O2, observed during the ageing of ryegrass leaf sheaths (Piquery et al. 2000), was due to the co-ordinated increases of OxO gene expression and oxalate synthesis. Until now, OxO gene expression has been studied only in two physiological processes, germination (Dumas et al. 1993) and induction of plant defences against patho-
Figure 5. Nucleotide sequence deduced amino-acid sequence of oxalate oxidase (oxO1) gene of ryegrass (EMBL AJ291825). The signal peptide is shown in italic and bold, the germin box is shown in bold and underlined and putative N-glycosylation (NTST and NGSA), N-myristylation (GX(3)S/TX(2)) sequences and putative peroxisome targeting signals (SKF) are shown in bold.
gens (Dumas et al. 1995). The role of the oxalate/OxO system has not been defined completely. Although OxO seems to be involved in the cross-linking of cell wall components via H2O2 synthesis (Dumas et al. 1993; Lane 1994), no correlation has been made with the metabolism of oxalate. Indeed, studies on plant oxalate are focused mainly on its anti-nutritive and toxic effects (Libert & Franceschi 1987) and its role in calcium regulation (Webb 1999). Interestingly, oxalate crystals have been found in epidermal tissues (Brubaker & Horner 1989) and in cells surrounding the vascular bundles (Zindler-Frank 1995), which are major sites of infection by virus and pathogens. Ca-oxalate crystals are always found in the oldest (and the more vulnerable) leaf sheath of Lolium perenne (Fig. 3a). Therefore, it is important to determine whether the degradation of oxalate of the leaf sheaths contributes to the adaptation to cutting (and cropping by herbivores) and to the capacity of (re)growth of the young tissues surrounded by the leaf sheaths of the stubble. In the present study, evidence is presented that the increasing accumulation of H2O2 (Fig. 2a) during ageing of leaf sheaths is significantly correlated to the constitutive and organ-specific expression of the OxO activity and OxO gene (Fig. 4a,c), as well as to an increased concentration of oxalate. The contribution of OxO and oxalate to H2O2 accumulation is amplified by the collapse of H2O2 detoxification by the ascorbate/glutathion cycle, which has been previously demonstrated during ageing of leaf sheaths of ryegrass (Piquery et al. 2000). Indeed, the accumulation of H2O2 during ageing of leaf sheaths rises gradually until the remobilization process of N and C nutrients to the developing sinks (ELB) has been completely achieved (Ourry et al. 1988; Morvan et al. 1997). The co-ordinated increases of H2O2, oxalate and OxO activity from the inner to the outer leaf sheaths could be considered as representative of
© 2001 Blackwell Science Ltd, Plant, Cell and Environment, 24, 1033–1043
1040 C. Davoine et al. epidermis and in the bundle sheath surrounding the vascular bundles of ryegrass leaf sheaths (insert, Fig. 7). Such a localization of OxO gene expression was also found in the coleoptile and the coleorhiza of cereal embryos (Dumas et al. 1995; Caliskan & Cuming 1998). According to these authors, the H2O2 generated by OxO in these differentiating tissues might be involved in cell wall cross-linking. This hypothesis cannot be supported in the case of ryegrass leaf sheaths where vascular bundles are already completely differentiated. The co-ordinated synthesis of Ca-oxalate and of OxO might therefore explain the over accumulation of H2O2 in these tissues. These biochemical events, never described before, establish a working link (Fig. 8) which might lead to resistance against pathogens. The establishment of constitutive defences is certainly crucial at critical phases of plant life such as reserve mobilization, for instance during germination (Terras et al. 1995) or senes-
(a)
A. Ientiformis
A. thaliana
P. sativum
O. sativa
T. aestivum_b
H. vulgare_a
H. vulgare
T. aestivum
L. perenne
(b)
Figure 6. Southern analysis of the OxO gene and Phylogenetic tree of OxO and germin-like proteins. (a) Genomic DNA (10 mg) was digested separately with restriction enzymes, as indicated in each lane, and analysed by hybridization with the radio-labelled OxO insert probe. (b) Phylogenetic tree was constructed with the following amino acid sequences: Lolium perenne (this study), Atriplex lentiformis (GenBank BAA78563), Arabidopsis thaliana (GenBank AC06829), Pisum sativum (EMBL AJ250832), Orysa sativa (GenBank AF032974), Triticum aestivum and Triticum aestivum_b (GenBank M63223 and M63224, respectively), Hordeum vulgare and Hordeum vulgare_a (EMBL Y14023 and GenBank U01963). Phylogenic tree was obtained using the CLUSTALW and PHYLIP programs (Infobiogen).
the sequential events leading to senescence, which is known to involve reactive oxygen species (Del Rio et al. 1998) and programmed cell death (Jabs 1999). Another important finding shown by immunocytochemistry and by the in situ assay of OxO activity, was that the OxO protein is located specifically in the cells of the lower
Figure 7. Light microscopy immunolocalization of OxO in the different leaf sheaths of ryegrass. Immunogold labelling of transverse section of ryegrass stubble. (Inset) Immunogold labelling of transverse section of external medium leaf sheath (ME). Lp: lower epidermis; Vb: vascular bundle; ELB: elongating leaf bases; I: inner leaf sheath; MI: internal medium leaf sheath; ME: external medium leaf sheath; 0: outer leaf sheath. Bars indicate 5 mm.
© 2001 Blackwell Science Ltd, Plant, Cell and Environment, 24, 1033–1043
Expression of oxalate oxidase in ryegrass stubble 1041
Constitutive resistance
Induced resistance
Production of H2O2
Production of H2O2
Constitutive synthesis of oxalate by plant
Constitutive expression of oxalate oxidase by plant
Induction of oxalate oxidase expression in plant
Susceptibility
No induction of oxalate oxidase expression in plant
Secretion of oxalic acid
Pathogen
cence (Buchanan-Wollaston 1997). This interplay between Ca-oxalate and OxO in the stubble of rye grass differ from the well-known mechanisms (Cessna et al. 2000. Dunwell et al. 2000) of induced resistance or susceptibility (Fig. 8), both involving oxalic acid secretion by pathogens. The function of the OxO/oxalate system in the senescing leaf sheaths would be, according to its activity, the production of high levels of H2O2, setting up a sterile shield around elongating leaf bases. Therefore, this would favour the mobilization of reserves, via sieve elements also protected by the OxO/oxalate system against viral infection. The specific expression of the OxO/oxalate system in the lower epidermis and near the perivascular zone of the senescing sheaths would contribute to the survival of the plant especially after cutting. In addition to the putative role of the OxO/oxalate system in protection of nutrient remobilization and growth of leaf bases, oxalate could itself be a toxic and repellent compound against the grazing of leaf sheaths by predators. Interestingly, insect resistance seems to be associated with an elevated oxalate level in rice leaf sheaths (Yoshihara et al. 1979). Therefore, the OxO/oxalate system might be very useful at critical steps of the plant life by settling constitutive defences against pathogens and foraging animals. Despite the apparent paradox between germination and senescence, a parallel might be drawn between the two developmental processes occurring at both ends of a plant’s life ensuring nutrient mobilization and therefore an efficient protection for tissues against pathogens and predators. Our future objectives will be to determine the transduction signals involved in OxO gene expression during senescence; to establish, after virus inoculation, whether the centripetal expression of the OxO/oxalate in leaf sheaths of ryegrass actually prevents the youngest tissues from pathogen and viral attacks; and to elucidate the carbon pathway leading to oxalate during ageing and nutrient remobilization of ryegrass leaf sheaths.
Secretion of oxalic acid
Pathogen
Figure 8. Interplay between plant and oxalatesecreting micro-organisms, settling of a putative constitutive resistance, pathogeninduced resistance or susceptibility to pathogens.
ACKNOWLEDGMENTS The authors are grateful for the skilled technical assistance provided by C. Le Dantec throughout this study.
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