RESEARCH PAPER |
An 
-L-arabinofuranosidase/ß-D-xylosidase from immature seeds of radish (Raphanus sativus L.)
1Department of Biochemistry and Molecular Biology, Faculty of Science, Saitama University, 255 Shimo-okubo, Sakura-ku, Saitama 338-8570, Japan
2Biological Function Division, National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan
3Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan
*To whom correspondence should be addressed. E-mail: kotake{at}molbiol.saitama-u.ac.jp
Received 14 October 2005; Accepted 16 March 2006
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Abstract |
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The carbohydrate moieties of arabinogalactan proteins (AGPs) are essential for their physiological functions and undergo rapid turnover in vivo. Degradation of the carbohydrate moieties of AGPs seems to occur by concerted action of several glycosidases, among them
-L-arabinofuranosidase, ß-D-galactosidase, and ß-D-glucuronidase. Here, a bifunctional -L-arabinofuranosidase/ß-D-xylosidase from immature seeds of radish (Raphanus sativus L.), which hydrolyses -L-arabinofuranosyl residues of the carbohydrate moieties of AGPs, has been cloned by reverse transcriptase-PCR. The gene, designated RsAraf1, contained an open reading frame of 2343 bp (780 amino acids), including a putative signal sequence (33 amino acids) at the N-terminus. RsAraf1 is highly similar to barley -L-arabinofuranosidase/ß-D-xylosidases and belongs to family 3 of the glycosyl hydrolases based on sequence homology. Southern blot analysis revealed that several related genes exist in the radish genome. RsAraf1 is expressed throughout seed development and weakly expressed in young seedlings. It was found that -L-arabinofuranosidase activity in a cell-wall protein fraction prepared from transgenic Arabidopsis plants with enhanced expression of RsAraf1 was significantly higher than that in a wild-type protein fraction; the crude enzyme preparation released L-arabinose from radish AGPs as well as -(1
5)-arabinan and arabinoxylan. Accordingly, the amount of L-arabinosyl residues in the cell walls of transgenic plants was significantly decreased. These results indicate that RsAraf1 encodes a bifunctional -L-arabinofuranosidase/ß-D-xylosidase and suggest that RsAraf1 is involved in the hydrolysis of the carbohydrate moieties of AGPs in immature radish seeds.
Key words:
Arabidopsis, -L-arabinofuranosidase, arabinogalactan protein, cell wall, family 3 glycoside hydrolase, polysaccharide, radish, seed development, transgenic plant, and ß-xylosidase
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Arabinogalactan proteins (AGPs), a family of proteoglycans, are commonly found in higher plants and implicated in many physiological processes such as cell-to-cell signalling, cell adhesion, cell elongation, cell death, and stress responses (Fincher et al., 1983; Nothnagel, 1997; Majewska-Sawka and Nothnagel, 2000; Shi et al., 2003). Several studies on AGPs have revealed the importance of the carbohydrate moieties for their physiological functioning. Xylogen, for example, a non-classical AGP from Zinnia (Zinnia elegans) mesophyll cells, induces the differentiation to tracheary elements, but the inductive function of xylogen is lost when the carbohydrate moieties are removed from the xylogen by chemical treatment (Motose et al., 2004). It is also well-known that the ß-glycosyl-Yariv reagent [chemical name, 1,3,5-tri(p-glycosyloxyphenylazo)-2,4,6-trihydroxybenzene] specifically binds to the carbohydrate moieties of AGPs and perturbs their molecular functions (Komalavilas et al., 1991; Majewska-Sawka and Nothnagel, 2000). In cultured cells of Arabidopsis (Arabidopsis thaliana), the ß-glycosyl-Yariv reagent induces programmed cell death, possibly by disrupting the plasma membrane-cell wall connections (Gao and Showalter, 1999). Moreover, the function of xylogen is suppressed when Zinnia cells are treated with the ß-glycosyl-Yariv reagent.
AGPs are characterized by large amounts (generally more than 90% of total weight) of carbohydrate components rich in galactose (Gal; sugars in this study are D series unless designated otherwise), L-arabinose (L-Ara), and protein components rich in hydroxyproline, serine, threonine, alanine, and glycine. Although there are many molecular species of AGPs differentiated by their core proteins, the carbohydrate moieties of AGPs have a common overall structure of ß-(13)-galactosyl backbones to which side chains of ß-(16)-linked galactosyl residues are attached through O-6. The ß-(16)-linked galactosyl chains are further substituted with L-Ara and lesser amounts of other auxiliary sugars such as glucuronic acid (GlcA), 4-O-methyl-glucuronic acid, L-rhamnose (L-Rha), and L-fucose (L-Fuc). The L-Ara residues are attached to the ß-(16)-linked galactosyl chains through -(13)-linkages. In spite of this complex structure, the carbohydrate moieties of AGPs are actively metabolized in planta. For example, an AGP from tobacco, stylar transmitting tissue (TTS protein), undergoes degradation of its carbohydrate moieties in pollen tubes, stimulating the elongation of the pollen tubes (Cheung et al., 1995; Wu et al., 1995). The precise structure of the carbohydrate moieties of radish AGPs varies depending on plant source and developmental stage (Tsumuraya et al., 1988). In proso millet (Panicum miliaceum) cells, the rate of turnover of the carbohydrate moieties of AGPs is extremely rapid (Gibeaut and Carpita, 1991). In carrot (Daucus carota), maternally derived AGPs are degraded in immature seeds (van Hengel et al., 2002). The monomeric sugars released from the carbohydrate moieties by glycosidases are incorporated into the cells, then converted to nucleotide sugars via monosaccharide 1-phosphates by the action of monosaccharide kinases and nucleotide sugar pyrophosphorylases (Reiter and Vanzin, 2001; Kotake et al., 2004). Since the carbohydrate moieties of AGPs have a complex structure with many branches, their structural modification and degradation require the concerted action of several glycosidases, such as -L-arabinofuranosidase (EC 3.2.1.55
[EC]
), ß-galactosidase (EC 3.2.1.23
[EC]
), and ß-glucuronidase (EC 3.2.1.31
[EC]
). It seems that the role of -L-arabinofuranosidase is to release terminal L-arabinofuranosyl residues of AGPs, making the carbohydrate moieties susceptible to ß-galactosidase (Sekimata et al., 1989; Kotake et al., 2005).
Based on the amino acid sequence and structural similarities, glycoside hydrolases are classified into more than 90 families (Henrissat, 1991; Henrissat and Bairoch, 1993), and -L-arabinofuranosidases fall into five families (family 3, 43, 51, 54, and 62). Recent studies reveal that higher plants have bifunctional -L-arabinofuranosidase/ß-xylosidases as well as monofunctional -L-arabinofuranosidases responsible for the hydrolysis of -L-arabinofuranosyl residues in plant polysaccharides. An -L-arabinofuranosidase from carrot (Daucus carota), classified as a family 51 glycoside hydrolase, acts on sugar beet arabinan and the pectic fraction from carrot (Tanaka et al., 2001). A family 3 bifunctional -L-arabinofuranosidase/ß-xylosidase (ARA-I) from barley hydrolyses both -L-arabinosyl and ß-xylosyl residues of arabinoxylan in collaboration with endo-ß-(14)-xylanase (EC 3.2.1.8
[EC]
). Interestingly, a family 3 ß-xylosidase (XYL) from barley, which is related to ARA-I, possesses distinct substrate specificity exhibiting much higher catalytic efficiency for ß-xylosyl residues than for -L-arabinofuranosyl residues (Lee et al., 2003). An -L-arabinofuranosidase gene, PpARF2, cloned from the Japanese pear (Pyrus pyrifolia) is specifically expressed in the ripening stage of the fruit and implicated in the degradation of pectic -(15)-arabinan (Tateishi et al., 2005). However, the -L-arabinofuranosidases responsible for the hydrolysis of -L-arabinofuranosyl residues of AGPs remain to be identified.
For the present study, a gene encoding a bifunctional -L-arabinofuranosidase/ß-xylosidase was isolated from immature seeds of radish and introduced into Arabidopsis. The changes in hydrolytic activity of -L-arabinofuranosidase in the transgenic plants toward polysaccharides containing -L-arabinofuranosyl residues were analysed. The role of the bifunctional -L-arabinofuranosidase/ß-xylosidase in the structural modification and degradation of the carbohydrate moieties of AGPs are discussed.
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Plant material
Seeds of radish (Raphanus sativus L. var. hortensis cv. aokubi) were purchased from Tokita Seed and Plant Co., Ltd. (Saitama, Japan). The radish seeds were sown on moist plastic mesh and grown at 25 °C for 6 d in the dark, then used for DNA and RNA preparations. Immature seeds were collected from developing siliques of radish. Because of the difficulty of dividing the immature seeds based on days after pollination, weight (mg–1 seed) was used as a proxy and the seeds separated into five groups.
Polysaccharides
AGPs from radish leaves (AGP R-II) and roots (AGP IV) were prepared in this laboratory (Tsumuraya et al., 1984, 1988). Arabinoxylan from wheat flour and -(15)-arabinan from sugar beet were purchased from Megazyme (Wicklow, Ireland). p-Nitrophenyl (PNP)-ß-glucoside, -ß-galactoside, --L-arabinofuranoside, -ß-xyloside, and -ß-glucuronide and xylan from birchwood were from Sigma (St Louis, MO, USA).
Peptide sequencing
The -L-arabinofuranosidase/ß-xylosidase was purified from radish seeds using ammonium sulphate fractional precipitation, ion exchange, hydroxylapatite, gel-filtration, and hydrophobic chromatographies as described previously (Hata et al., 1992). The purified enzyme (5 µg) was digested with 3 µg of V8 protease from Staphylococcus aureus (Wako, Osaka, Japan) at 25 °C for 15 min. The resulting peptide fragments (38 kDa and 35 kDa) were separated by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS–PAGE, Laemmli, 1970), blotted onto a PVDF-Plus membrane (Osmonics, Moers, Germany) and subjected to N-terminal amino acid analysis with a protein sequencer HP G1000A (Hewlett Packard, Houston, TX, USA).
Reverse transcriptase-PCR
The cDNA encoding radish -L-arabinofuranosidase/ß-xylosidase was cloned by reverse transcriptase-PCR (RT-PCR) from immature seeds of radish (average seed weight, approximately 6 mg seed–1). Immature seeds of radish were frozen in liquid nitrogen, homogenized with a mortar and pestle, and the RNA was extracted with an Isogen kit (Nippon Gene, Tokyo, Japan) according to the manufacturer's instructions. Single strand cDNA was synthesized from 2 µg of total RNA from the immature seeds using a reverse transcriptase, ReverTra Ace-- (Toyobo, Osaka, Japan) and oligo(dT)-adaptor primer (5'-GCGACATCATCGAATTCCGATGTTTTTTTTTTTTT-3'). Based on the protein sequences of the purified -L-arabinofuranosidase, two sets of degenerate primers were designed: F-1 (5'-CARCAYTAYACNAARACNCC-3', corresponding to QHYTKTP of the 38 kDa fragment) and R-1 (5'-GYTTYTTNGGRTCNCCRTC-3', corresponding to DGDPKKQ of the 35 kDa fragment), and F-2 (5'-CCNGCNGARGCNGC-3' corresponding to PAEAA of the 38 kDa fragment) and R-2 (5'-GCRTTRTCDATNGCNGC-3', corresponding to AAIDNA of the 35 kDa fragment). The first PCR was performed with the degenerate primers F-1 and R-1, using the single strand cDNA as a template under the following conditions: 0.5 min denaturing at 96 °C, 0.5 min annealing at 50 °C and 1.0 min amplification at 72 °C, 40 cycles. The PCR-product was diluted and used as a template for the second PCR. The second PCR was performed with the degenerate primers F-2 and R-2 under the following conditions: 0.5 min denaturing at 96 °C, 0.5 min annealing at 50 °C and 1.0 min amplification at 72 °C, 45 cycles. The amplified cDNA fragment encoding the 125 bp region of the -L-arabinofuranosidase/ß-xylosidase gene was subcloned into a pGEM T-Easy vector (Promega, Madison, WI, USA) and the nucleotide sequence was determined with an ABI PRISM 310 genetic analyser (Applied Biosystems, Foster City, CA, USA). The partial cDNA encoding 685 bp of the 5' region of the cDNA was amplified by PCR with an internal specific primer, R-3 (5'-TGATTCGCGGGCGTGCA-3') and a degenerate primer F-3 (5'-GARGCWYTHCAYGGNGT-3', corresponding to EALHGV) designed for the conserved sequence of family 3 -L-arabinofuranosidase/ß-xylosidases. The 5' end of the cDNA was amplified with a rapid amplification of 5' cDNA ends (5'-RACE) system (Invitrogen, Carlsbad, CA, USA) using the internal specific primers R-4 (5'-TGGCTTGAAACAGAGATACG-3') and R-5 (5'-TTGGCGGGAGAAACGAGT-3'). The 3' region of the cDNA was amplified with an internal specific primer, F-4 (5'-GGTTTGGACTTGAACTGTG-3') and an adaptor primer (5'-GCGACATCATCGAATTCCGATG-3') using the single strand cDNA as a template. The coding region for the radish -L-arabinofuranosidase/ß-xylosidase was amplified with proofreading polymerase (KOD-Plus-, Toyobo) and the nucleotide sequence was determined.
To detect the expression of the radish -L-arabinofuranosidase/ß-xylosidase gene in young radish seedlings, RT-PCR was performed using a set of specific primers, RsAraf1-7 (5'-GGTTTGGACTTGAACTGTG-3') and RsAraf1-11R (5'-CTTTAGGACCTAGCCCTCC-3'), under the following conditions: 0.5 min denaturing at 95 °C, 0.5 min annealing at 55 °C and 2.0 min amplification at 72 °C 30 cycles. The amplified cDNA fragment (181 bp) was separated on a gel containing 2.0% agarose and detected by staining with ethidium bromide.
Southern and northern blot analyses
Genomic DNA was extracted from cotyledons of radish by the method described in Murray and Thompson (1980). The genomic DNA (20 µg) was digested with restriction enzymes, separated on a gel containing 0.7% agarose and blotted onto nylon membrane (Hybond N+; Amersham Biosciences, Piscataway, NJ, USA). The blotted membrane was baked at 80 °C for 2 h, then hybridized with a digoxigenin (DIG)-labelled cDNA probe prepared with a DIG high prime DNA labelling and detection kit (Roche Diagnostics, Basel, Switzerland). The cDNA probe was the 600 bp fragment excised from RsAraf1 cDNA with the restriction enzymes PstI and SacI. Probe labelling, hybridization, and signal detection were carried out according to the manufacturer's instructions. Total RNA was extracted from developing immature seeds and dark-grown hypocotyls, cotyledons, and roots with an Isogen kit as described above. Approximately 20 µg of total RNA was separated on 1.2% formaldehyde-agarose gel and blotted onto nylon membrane. The blotted membrane was baked, then hybridized with the DIG-labelled cDNA probe. To verify the amount of loaded RNA, ribosomal RNA was stained with 1% (w/v) methylene blue.
Plasmid construction and generation of transgenic Arabidopsis plants
RsAraf1 cDNA including the 5' untranslated region (UTR, 24 bp) and the 3' UTR (124 bp) was amplified with specific primers, Araf1-F (5'-CGCGGATCCCAAGAATCAATACTG-3') and Araf1-R (5'-CATGTTCTTTTCTTTAACATGC-3'), then subcloned into a pGEM5zf+ vector (Promega). After confirmation of the nucleotide sequence, the cDNA fragment was excised using BamHI and SacI restriction sites and inserted between a cauliflower mosaic virus (CaMV) 35S promoter and a nopaline synthase terminator of the pBI121 plasmid to produce the plasmid construct RsAraf1/pBI121. The construct was introduced by electroporation into the Agrobacterium tumefaciens EHA105 strain. The resulting bacteria were used to transform wild-type Arabidopsis (Columbia) by in planta vacuum infiltration as described in Bechtold et al. (1993). The transgenic Arabidopsis lines were selected on MS medium containing 1% (w/v) sucrose and 50 mg l–1 kanamycin (Murashige and Skoog, 1962) and confirmed as transgenic by genomic PCR.
Hydrolytic activity of transgenic plants
Transgenic Arabidopsis plants were grown on rockwool fibres (Nittobo, Tokyo, Japan) under continuous light at 23 °C for 45 d. The following operations were carried out at 0–4°C. The aerial parts of transgenic Arabidopsis plants were homogenized with a mortar and pestle in 25 mM sodium acetate buffer (pH 5.0). The homogenate was centrifuged at 10 000 g for 5 min, and the resulting precipitate was washed with ice-cold water twice, then suspended in 20 mM sodium acetate buffer (pH 5.0) containing 1 M sodium chloride for 1 h to extract cell wall-bound proteins. After centrifugation at 10 000 g for 5 min, the supernatant was used as the cell-wall protein fraction. The concentration of protein was determined by the method of Bradford with bovine serum albumin as the standard (Bradford, 1976).
Hydrolytic activities of the cell-wall proteins extracted from transgenic plants toward synthetic substrates were determined using a reaction mixture (200 µl) consisting of the cell-wall proteins, 25 mM acetate buffer (pH 5.0), and 1 mM PNP-glycoside substrate. After incubation at 37 °C for 2 h, the reaction was terminated by the addition of 200 mM sodium carbonate (800 µl) and monitored at 420 nm. One unit of enzyme activity liberates 1 µmol of p-nitrophenol min–1. Hydrolytic activities of the cell-wall proteins of transgenic plants toward polysaccharides were determined using a reaction mixture (100 µl) consisting of the cell-wall proteins, 25 mM acetate buffer (pH 5.0), and 5 mg ml–1 polysaccharide. After incubation at 37 °C for the appropriate reaction time, the liberated sugars were determined reductometrically (Nelson, 1944; Somogyi, 1952). One unit of enzyme activity was defined as the amount of enzyme releasing 1 µmol of reducing sugar min–1. The reaction products of arabinoxylan from wheat flour and AGP from radish roots were analysed by high-performance anion-exchange chromatography (HPAEC) using a Dionex DX-500 liquid chromatograph fitted with a CarboPac PA-1 column (Dionex) and a pulsed amperometric detector (PAD, Dionex) as described by Ishikawa et al. (2000).
Extraction and analyses of cell-wall polysaccharides
Cell-wall polysaccharides were extracted from transgenic Arabidopsis plants grown for 45 d under the conditions described above. Transgenic Arabidopsis plants (approximately 0.5 g) were homogenized using a mortar and pestle with 20 mM sodium phosphate buffer (pH 7.0) at 4 °C. After centrifugation at 10 000 g for 5 min, the precipitate was washed with water twice. The precipitate was treated twice with a total of 1 ml of 17.5% (w/v) sodium hydroxide and 0.04% (w/v) sodium borohydride in a boiling-water bath for 5 min each, and centrifuged to remove insoluble materials. The extracted cell-wall polysaccharides were neutralized with 0.5 ml of glacial acetic acid, dialysed against water at 4 °C for 2 d, and lyophilized. The dried polysaccharides were hydrolysed with 72% (v/v) sulphuric acid at 4 °C for 1 h followed by heating in diluted (8%, v/v) acid solution at 100 °C for 4 h, then neutralized with barium carbonate and desalted with Dowex 50W (H+) resins. Quantification of monosaccharides of cell-wall polysaccharides was carried out by HPAEC-PAD (Ishikawa et al., 2000).
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Results |
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Peptide sequences of
-L-arabinofuranosidase/ß-xylosidaseNative
-L-arabinofuranosidase/ß-xylosidase from radish seeds was purified about 1000-fold using ammonium sulphate fractionation and successive chromatographies as in a previous study (Hata et al., 1992). The purified enzyme appeared as a single band with a relative molecular mass of 65 kDa on SDS-PAGE (Fig. 1). Since the N-terminal amino acid of the purified -L-arabinofuranosidase/ß-xylosidase was blocked, the enzyme was partially digested with protease V8 and two peptide fragments (38 and 35 kDa) were obtained. The N-terminal amino acid sequences of the purified -L-arabinofuranosidase/ß-xylosidase were YQHYTKTPAEAA AISILAGLDL N for the 38 kDa fragment, and AAIDNAISNN FLTLMRLGFF DGDPKKQIYG for the 35 kDa fragment. The peptide sequences of both fragments exhibited similarity to family 3 -L-arabinofuranosidase/ß-xylosidases and ß-xylosidases.
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Isolation of the cDNA clone
Based on the sequence determined for the purified enzyme from radish seeds, RT-PCR followed by 3' and 5' RACE procedures were performed. The cloned cDNA was designated RsAraf1 (Raphanus sativus
-L-arabinofuranosidase 1). Although the cDNA sequence obtained contained regions corresponding to the partial amino acid sequences determined for the native enzyme, three residues in the N-terminal sequence of the 38 kDa fragment and two residues in the 35 kDa fragment differed from the amino acid sequence deduced from RsAraf1 (Fig. 2). This discrepancy may be a consequence of contamination of the purified enzyme by closely related isozyme(s) with molecular mass very close to the purified enzyme. It is also possible that the obtained cDNA, RsAraf1, encodes a related enzyme other than the enzyme purified from radish seeds. In the process of isolation of the RsAraf1 cDNA fragment, a distinct but closely related cDNA fragment was also amplified by RT-PCR. However, the deduced amino acid sequence of the second cDNA was apparently different from that determined for the native enzyme (data not shown). Therefore it was not possible to isolate a cDNA fragment that completely matched the peptide sequences obtained from the native enzyme.
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Amino acid sequence of RsAraf1
RsAraf1 appeared to encode a polypeptide of 780 amino acids (molecular mass 83 802 Da) (Fig. 2). Its putative signal sequence (33 amino acids) was predicted by the SignalP 3.0 program (Bendtsen et al., 2004). The calculated molecular mass of the mature protein deduced from RsAraf1 (from Gln34 to Ile780, 80.2 kDa) was much larger than the relative molecular mass of the purified enzyme (65 kDa) determined on SDS–PAGE. The smaller molecular mass of the native enzyme is probably attributable to post-translational processing occurring on the enzyme protein. This hypothesis is suggested by the observation that the related enzymes, barley ARA-I and XYL, undergo post-translational processing in their C-terminal regions (Lee et al., 2003). If one assumes post-translational processing at the site of RsAraf1 corresponding to those of ARA-I and XYL, where it is known to occur, RsAraf1 should be cleaved between Phe640 and Ser641, resulting in a mature form of the protein with a molecular mass of 65 011 Da, which agrees with our measurements of the native enzyme. The calculated pI value for RsAraf1 was 9.45, which is quite different from the isoelectric point (4.70) of the native enzyme obtained by isoelectric focusing (Hata et al., 1992). However, assuming post-translational processing between Phe640 and Ser641, the calculated pI value for the mature protein (Gln34-Phe640) of RsAraf1 shifts to 6.39.
RsAraf1 has remarkable similarity to family 3 -L-arabinofuranosidase/ß-xylosidases and ß-xylosidases such as an -L-arabinofuranosidase/ß-xylosidase (ARA-I, 65% identical) and a ß-xylosidase (XYL, 46% identical) from barley, as well as with fungal ß-xylosidases such as XlnD (29% identical) from Emericella nidulans (Pérez-González et al., 1998), and bacterial ß-xylosidases such as BxlB (32% identical) from Clostridium stercorarium (Adelsberger et al., 2004). Phylogenetic analysis of RsAraf1 with other family 3 enzymes revealed that RsAraf1 is closely related to an Arabidopsis gene, At5g64570 (89% identical) (Fig. 3).
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Organization and expression pattern of RsAraf1
The expression pattern of RsAraf1 in young seedlings and immature seeds of radish was analysed (Fig. 4A). By northern blot analysis, the expression of RsAraf1 in the hypocotyls, cotyledons, and roots of dark-grown young seedlings was barely detectable, whereas relatively high expression of RsAraf1 was observed in immature seeds. To ascertain the expression of RsAraf1 in the seedlings, RT-PCR of RsAraf1 in the dark-grown young seedlings was performed and it was found that RsAraf1 was indeed expressed in the hypocotyls, cotyledons, and roots of the seedlings, if only at a very low level (Fig. 4B). The expression of RsAraf1 in the immature radish seeds was further examined at various developmental stages. The transcript levels of RsAraf1 were high throughout seed development (Fig. 4C), coinciding with the relatively high activity of
-L-arabinofuranosidase in radish seeds (Hata et al., 1992).
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The number of RsAraf1-related genes in the radish genome was examined by Southern blot analyses. A labelled cDNA probe hybridized to several restriction fragments of the genomic DNA digested with DraI, EcoRI, or HindIII. A few faint bands were also detected in the genomic DNA digested with BamHI or XbaI (Fig. 5). These results suggest that several related genes exist in the radish genome.
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DNA markers digested with HindIII are shown on the left side.Generation of transgenic Arabidopsis plants expressing RsAraf1
To address the properties and functions of RsAraf1, RsAraf1 cDNA was introduced into Arabidopsis by Agrobacterium-mediated transformation, and seven transgenic lines (numbers 1, 2, 3, 4, 5, 6, and 10) were confirmed to contain the RsAraf1 gene. The transgenic plants, in which RsAraf1 was expressed under the control of the CaMV 35S promoter, did not show any significant phenotype compared with the wild-type plants. The aerial parts of transgenic plants including flowers, immature siliques, inflorescence stems, cauline leaves, and rosette leaves were examined for
-L-arabinofuranosidase and ß-xylosidase activity in the cell-wall protein fraction (Fig. 6). Wild-type Arabidopsis has approximately 20 munit mg–1 protein of -L-arabinofuranosidase activity and 15 munit mg–1 protein of ß-xylosidase activity in the cell-wall preparation. The -L-arabinofuranosidase activity measured in the cell-wall proteins of the transgenic lines was significantly higher, at levels ranging from 45 to 189 munit mg–1 protein. The difference in -L-arabinofuranosidase activity between the transgenic lines is likely caused by variation in the number of copies of the transgene and/or an effect of the genomic location of the transgenes. Compared with the -L-arabinofuranosidase activity, the levels of ß-xylosidase activity in the cell-wall proteins were not drastically enhanced by the introduction of the RsAraf1 gene (ß-xylosidase activity, 28 to 49 munit mg–1 protein). These results are consistent with the properties of the native enzyme purified from radish seeds, which prefers PNP--L-arabinofuranoside to PNP-ß-D-xyloside at a ratio of 3:1 when reacted with 1 mM PNP-glycoside substrates (Hata et al., 1992). It seems that RsAraf1 encodes the native enzyme or at least an enzyme with properties very similar to the native enzyme.
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Hydrolytic activity of transgenic plants toward polysaccharides
For one of the transgenic lines, no. 10, with high
-L-arabinofuranosidase activity, several other glycosidase activities of the cell-wall proteins were compared with those of wild type plant proteins. Cell-wall proteins extracted from the transgenic line did not show a significant increase in ß-glucosidase, ß-galactosidase, or ß-glucuronidase activity (Table 1), suggesting that the introduction of the RsAraf1 gene did not affect other glycosidase activities. The ability of the transgenic plants to hydrolyse polysaccharides containing -L-arabinofuranosyl and ß-xylosyl residues was also examined (Table 1). The cell-wall proteins extracted from the transgenic line (no. 10) substantially hydrolysed AGPs from radish leaves and roots, showing more than twice the activity of proteins from the wild-type plants. The activities of the transgenic line toward -(15)-arabinan and arabinoxylan were also significantly enhanced compared with those of wild-type plants. To address the question of how RsAraf1 acts on arabinoxylan and AGPs, the reaction products from these polysaccharides, when hydrolysed by cell-wall proteins extracted from transgenic Arabidopsis plants, was analysed by HPAEC-PAD. The cell-wall proteins released monomeric L-Ara (15 mol%) together with Xyl (85 mol%) from the arabinoxylan, indicating that RsAraf1 hydrolyses both L-arabinofuranosyl and xylosyl residues of the arabinoxylan. The cell-wall proteins also released monomeric sugars, containing Gal (76 mol%) and L-Ara (24 mol%) from AGP from radish roots. The hydrolysis of the AGP by the cell-wall proteins is probably a consequence of the concerted action of RsAraf1 and endogenous ß-galactosidases. The result suggests that the action of RsAraf1 makes the AGP more susceptible to ß-galactosidases (Kotake et al., 2005). Whereas arabinoxylan has -L-arabinofuranosyl residues attached to ß-(14)-xylan chains through -(13)-linkages, -L-arabinofuranosyl residues of AGPs are linked to galactosyl residues by -(13)-linkages. On the other hand, -(15)-arabinan is mainly composed of -(15)-linked L-arabinosyl residues with branches of -(13)-linked L-arabinosyl residues. It was concluded that the enzyme encoded by RsAraf1 has rather broad specificity, hydrolysing several types of -L-arabinofuranosyl residues located in various polysaccharides, because it tolerates the presence of neighbouring sugar residues other than -L-arabinofuranosyl residues. The substrate specificity of RsAraf1 is thus consistent with that of the native enzyme purified from radish seeds (Hata et al., 1992). The high activity of the cell-wall proteins toward -(15)-arabinan in both wild-type and transgenic plants may be a consequence of the structure of -(15)-arabinan, which has branches of -L-arabinofuranosyl residues serving as a good substrate for -L-arabinofuranosidases. It is also possible that the degradation of the polysaccharides with the cell-wall proteins proceeds by synergistic action of several glycosidases including endo-acting enzymes not detectable with PNP-glycoside substrates. The activity toward polysaccharide substrates may not, therefore, be a result of the expression of the RsAraf1 gene alone. Indeed, the enhancement of the hydrolytic activity toward polysaccharides in the transgenic plants (usually about 2–3 times of wild type), was relatively weak compared with the enhancement of the activity toward PNP--L-arabinofuranoside (about 6 times the wild-type activity) (Fig. 6). Hydrolytic activity of the cell-wall proteins toward xylan was also enhanced by the RsAraf1 gene, but the level of activity was low in both wild-type and transgenic plants (3.2 and 6.4 munit mg–1 protein, respectively).
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Effect of the RsAraf1 gene on cell-wall polysaccharides in Arabidopsis
Cell walls of Arabidopsis contain
-L-arabinofuranosyl residues in many types of polysaccharides such as -(15)-arabinan, glucuronoarabinoxylan, and AGPs (Zablackis et al., 1995; Schultz et al., 2002). To evaluate the in vivo effects of the RsAraf1 gene on the metabolism of cell-wall polysaccharides, alkali-soluble polysaccharides were extracted from the aerial parts of transgenic plants including flowers, immature siliques, inflorescence stems, cauline leaves, and rosette leaves, and the sugar composition was examined (Fig. 7). In transgenic Arabidopsis plants, the L-Ara content in the alkali-soluble fraction of cell walls was significantly reduced by the action of RsAraf1 (16.2% for wild type and 11.6% for the transgenic plants), on the other hand, Xyl content remarkably increased in the transgenic plants (20.7% for wild type and 29.6% for transgenic plants). These results suggest that RsAraf1 expressed in Arabidopsis acts not only on -L-arabinofuranosyl residues of the cell-wall polysaccharides, but also influences the metabolism of sugar residues other than L-Ara.
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Discussion |
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It has been shown in a previous study that radish seeds contain a considerable amount of
-L-arabinofuranosidase, which is capable of hydrolysing -L-arabinofuranosyl residues of AGPs. For the present study, a cDNA, RsAraf1, encoding -L-arabinofuranosidase/ß-xylosidase, has been isolated from immature radish seeds. Although the deduced amino acid sequence from RsAraf1 did not completely coincide with the partial sequences determined for the native enzyme, it is suggested that RsAraf1 encodes the native enzyme or a closely related isozyme, because of the presence of high -L-arabinofuranosidase and ß-xylosidase activities in cell-wall protein fractions extracted from transgenic Arabidopsis plants.
The deduced amino acid sequence of RsAraf1 is closely related to an Arabidopsis gene, At5g64570 (89% identical at the amino acid level). Recently, two ß-xylosidases and one -L-arabinofuranosidase were purified from Arabidopsis. One of the enzymes, At5g64570 (XYL4), was identified as a ß-xylosidase that has specificity toward ß-xylosyl residues (Minic et al., 2004). This raises the intriguing possibility that these closely related enzymes possess distinct substrate specificities toward -L-arabinofuranosyl and ß-xylosyl residues and that the preference of family 3 -L-arabinofuranosidase/ß-xylosidase and ß-xylosidase for -L-arabinofuranosyl and ß-xylosyl residues may be determined by a few particular amino acid residues rather than the structure of the entire protein.
Expression of RsAraf1 in Arabidopsis decreased the level of L-Ara in the cell walls of transgenic plants, while the level of Xyl increased remarkably. The increase in Xyl content of cell walls by the introduction of a gene encoding bifunctional -L-arabinofuranosidase/ß-xylosidase may be attributable to the substrate specificity of RsAraf1 preferring -L-arabinofuranosyl residues to ß-xylosyl residues. In addition, the following mechanism is conceivable: L-Ara that is released by the action of RsAraf1 is incorporated into the plant cell and converted to UDP-L-Ara via L-Ara 1-phosphate in the salvage pathway of nucleotide sugars. UDP-L-Ara may then be converted to UDP-Xyl by the action of UDP-L-arabinose 4-epimerase (EC 5.1.3.5
[EC]
) (Reiter and Vanzin, 2001; Kotake et al., 2004). Therefore, the release of L-Ara from cell-wall polysaccharides may increase not only the level of UDP-L-Ara but also the level of UDP-Xyl in the cells, and thereby stimulate the synthesis of cell-wall polysaccharides containing xylosyl residues.
The introduction of RsAraf1 into Arabidopsis increased hydrolytic activity toward several polysaccharide substrates in the cell walls. It is highly probable that RsAraf1 has multiple functions in the structural modification and degradation of cell-wall polysaccharides including AGPs, pectic -(15)-arabinan, and glucuronoarabinoxylan. Previous research revealed that -L-arabinofuranosidase purified from radish seeds substantially hydrolyses AGPs extracted from leaves and seeds of radish (Hata et al., 1992), suggesting that this enzyme is involved in the degradation of AGPs. Recently, an exo-acting ß-galactosidase from radish (RsBGAL1) has been found, which appears specifically to hydrolyse the ß-(13),(16)-galactan backbones of AGPs by removing ß-galactosyl residues one by one (Kotake et al., 2005). Because the non-reducing terminal and internal ß-(16)-galactosyl side chains of AGPs are usually highly substituted with L-arabinosyl and glucuronosyl residues, the concerted action of ß-galactosidase, -L-arabinofuranosidase, and ß-glucuronidase is required for the degradation of the carbohydrate moieties of AGPs. It is conceivable that RsAraf1 and related enzymes release -L-arabinofuranosyl residues attached to the terminal and internal ß-(16)-galactosyl side chains of AGPs to make the AGPs susceptible to ß-galactosidases such as RsBGAL1 in immature seeds of radish.
AGPs are common cell-wall components in higher plants, and have been characterized in seeds of Brassica plants including radish and rape (Brassica campestris) (Siddiqui and Wood, 1972; Tsumuraya et al., 1987). In the radish, the structure of carbohydrate moieties of AGPs differs from those of AGPs from leaves in that L-fucosyl residues are found in AGPs from the leaves but missing in those from seeds. In the carrot, maternally derived AGPs undergo structural modification and degradation in a developmentally regulated manner in immature seeds (van Hengel et al., 2002). Recently, a non-classical AGP has been found in Arabidopsis, AtAGP30, which appears to be involved in the ABA-response controlling the proper timing of germination of the seeds (van Hengel and Robert, 2003). Since RsAraf1 showed relatively high expression in immature seeds, it seems possible that RsAraf1 is involved in seed germination or embryogenesis, where it may facilitate the structural modification or degradation of AGPs in radish seeds. Generally, dicotyledonous plants contain considerable amounts of pectin and arabinoxylan in the cell walls. Therefore, the possibility that RsAraf1 plays roles in the degradation of pectic arabinan and arabinoxylan in radish seeds can not be excluded.
The present study suggests that family 3 bifunctional -L-arabinofuranosidase/ß-xylosidases are involved in the structural modification and degradation of the carbohydrate moieties of AGPs in planta in collaborative action with family 35 ß-galactosidases. This is only a partial result. The identification of ß-glucuronidase(s) responsible for the hydrolysis of glucoronosyl and 4-O-methyl-glucuronosyl residues of AGPs would seem to be the next task in the elucidation of the AGP metabolism in plants.
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Acknowledgements |
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This research was supported in part by a Grant for Ground Research for Space Utilization to TK from the Japan Space Forum and a Grant-in-Aid for Scientific Research to TK (No. 17770028) from Ministry of Education, Culture, Sports, Science and Technology, Japan.
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Abbreviations |
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AGP, arabinogalactan protein; CaMV, cauliflower mosaic virus; DIG, digoxigenin; PNP, p-nitrophenyl; RACE, rapid amplification of cDNA ends; RT-PCR, reverse transcriptase-PCR.
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