JXB Advance Access originally published online on August 1, 2005
Journal of Experimental Botany 2005 56(419):2335-2344; doi:10.1093/jxb/eri226
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
RESEARCH PAPER |
A soybean seed protein with carboxylate-binding activity
Southern Crop Protection and Food Research Center, Agriculture and Agri-Food Canada, 1391 Sandford Street, London, Ontario, Canada N5V 4T3
* To whom correspondence should be addressed. Fax: +1 519 457 3997. E-mail: dhaubhadels{at}agr.gc.ca
Received 5 October 2004; Accepted 13 May 2005
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionReferences |
|---|
The seed coat serves as a multifunctional organ with a role in protection and for the supply of nutrients to the embryo sac during development. The composition of the legume seed coat differs from other seed tissues in many ways including its protein composition. An abundant 24 kDa protein (SC24) has been purified and identified from soybean (Glycine max [L.] Merr) seed hulls. The corresponding cDNA and genomic DNA clones for SC24 were isolated and characterized, and expression patterns were determined. The deduced protein sequence of 219 amino acids included an N-terminal signal peptide. Transcripts encoding SC24 were present in the seed coat from 30 days after pollination (DAP) until maturity, but the protein was not detected until the final stages of seed maturation. In mature seeds, most of the SC24 protein was localized to the parenchyma and aleurone layers of the seed coat. The expression of SC24 was also induced in vegetative tissues by pathogen infection and by wounding. The SC24 protein bound to an affinity column containing an isophthalic acid ligand, and was eluted with 7 mM citrate. Polyclonal antibodies raised against recombinant SC24 cross-reacted with the seed coat peroxidase enzyme, suggesting that these two proteins may share an antigenic determinant. Overall, the results indicate that SC24 belongs to a novel class of plant defence proteins with carboxylate-binding activity.
Key words: Citrate binding protein, peroxidase, seed coat, soybean, wound stress
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Angiosperm seeds develop from fertilized ovules. The seed coat varies in structure but is essential for the development of seeds (Reiser and Fischer, 1993
). At maturity, the seed coat is primarily a protective structure, although its specific role may be varied and complex (Villiers, 1972). Legumes possess highly differentiated seed coats that arise from the inner and outer integuments of the ovule (Corner, 1951; Esau, 1977; Miller et al., 1999). The seed coat plays a vital role in legume seed architecture and is a feature that is used for identification. In addition to its protective function, the seed coat serves as a multifunctional organ and is involved in supplying nutrients to the embryo sac during seed development (Murray, 1987). Seed coats transport, transform, and secrete various metabolites to the embryo (Patrick and Offler, 2001). In the last stages of maturation, there is an immense reduction in the metabolic activity and rapid desiccation of the seed as well as compression of seed coat layers. The mature seed is covered with dead and hard tissues providing the seed with structural protection and mechanical support. It also protects the seed against pathogens, insects, wind, frost, and other stresses (Boesewinkel and Bouman, 1995). In leguminous crops such as soybean, the seed coat also has a significant impact in the agricultural marketplace as a component or by-product of processed seeds.
In mature soybean (Glycine max [L.] Merr.) seed, 4–8% of total seed mass is comprised of seed coat. The composition of the seed coat is very different from that of the embryo, including its soluble protein components. More than 100 proteins have been identified from whole seeds (Herman et al., 2003), and various isoforms of the 11S and 7S storage proteins, ß-conglycinin and glycinin, constitute approximately 70% of the total protein (Yaklich, 2001). Most of these seed proteins occur in the embryo and are far less abundant or absent from the seed coat (Gijzen et al., 2001). Protein components identified from extracts of soybean seed coats include proline-rich proteins (Percy et al., 1999), Kunitz and Bowman–Birk trypsin inhibitors (Gijzen et al., 2001; Sessa and Wolf, 2001), peroxidase (Gijzen et al., 1993), chitinase (Gijzen et al., 2001), and hydrophobic protein (Gijzen et al., 1999).
The purpose of this study was to identify and characterize a new and unusual 24 kDa protein, SC24, from soybean seed coats. SC24 was purified from seed coats and the corresponding cDNA and genomic clones encoding the protein were isolated. It is shown that SC24 and its corresponding transcript are most abundant in the seed coat at late stages of seed development. The SC24 mRNA is also prevalent in wounded tissues and is induced by infection with the soybean pathogen Phytophthora sojae. Biochemical tests indicate that SC24 has carboxylate-binding activity and that the protein may share antigenic domains with soybean seed coat peroxidase.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Purification of native SC24 from soybean seed hulls
Soybean seed hulls were obtained from a processing facility (ADM Agri-Industries, Windsor, Ontario). Proteins were extracted from 20 g seed hulls in 150 ml of 60% (v/v) ethanol by gentle shaking for 16 h. This extract was filtered (Miracloth, Calbiochem, La Jolla, CA) and clarified by centrifugation for 20 min at 14 000 g. The supernatant was collected and mixed with 2 vols of acetone, prechilled to –20 °C. Precipitated proteins were collected by centrifugation for 10 min at 14 000 g, and redissolved in 10 ml of 25 mM TRIS–HCl, pH 8.0. A 2.0 ml sample of this concentrated protein extract was separated on a gel filtration column (Superdex-75 16/60, Amersham Biosciences, Baie d'Urfé, Quebec) installed on a liquid chromatography apparatus (FPLC, Amersham Biosciences, Baie d'Urfé, Quebec). Elution was performed with 50 mM TRIS–HCl pH 8.0 and 100 mM NaCl. Fractions were analysed by SDS–PAGE using 12.5% acrylamide gels and visualized by silver staining (Blum et al., 1987
). For amino-terminal microsequencing, fractions containing SC24 protein were concentrated by ultrafiltration (Centricon, Millipore, Bedford, MA) followed by electrophoresis and transferred to PVDF membrane (Moos et al., 1988).
Dissection of seed coat
Soybean seeds were soaked in distilled water for 3 h to remove the seed coats. The seed coat tissues were separated by dissection into two fractions. For photography, pieces of tissue were cut and immersed in 3% agarose gel. Cubes containing the tissues were cut from the gel and sectioned using a vibrating blade microtome (Leica VT 1000S), stained with 0.05% (w/v) toluidine blue and observed under an inverted microscope. Pictures were taken with a DXM 1200 Nikon digital camera.
Library screening and DNA sequencing
Construction and screening of soybean cv. Harosoy 63 cDNA and genomic 
libraries were performed according to Gijzen (1997). Positive cDNA clones in phagemid vector (pBK-CMV, Stratagene, La Jolla, CA) were sequenced on both strands by primer walking. Positive genomic DNA clones were plaque purified and subcloned into plasmid vector (pBluescript, Strategene, La Jolla, CA) for sequence analysis. Automated sequencing of DNA was performed (Model 377, Applied Biosystems, Foster City, CA) using dye-labelled terminators. A 15.8 kb EcoRI genomic clone was shotgun sequenced by random transposon insertion (GPS-1, New England Biolabs, Beverly, MA), and gaps were filled by primer walking.
Citrate binding assay
An affinity column was prepared with 5-aminoisophthalic acid coupled to N-hydroxysuccinimide-activated agarose beads (Affi-Gel 15, Bio-Rad, Mississauga, Canada). Coupling was performed in the presence of dimethylformamide at 30 °C for 3 h. The gel was washed three times with 0.1 M triethanolamine pH 8. The column was equilibrated with 0.1 M triethanolamine pH 8 and was washed with 20 mM Tricine pH 7.5, 0.1% Triton X-100 before use.
Soybean seed hulls were extracted in 20 mM Tricine pH 7.5, 0.1% Triton X-100 and centrifuged at 10 000 g for 30 min at 4 °C. The supernatant was filtered (Acrodisc, HT Tuffryn, 0.2 µm) and loaded to the affinity column. The column was washed with 20 mM Tricine pH 7.5, 0.1% Triton X-100. Proteins bound to the column were eluted with 20 mM Tricine pH 7.5, 0.1% Triton X-100 and a citrate gradient from 0–20 mM. Fractions were collected and analysed by 12.5% SDS–PAGE, followed by silver staining. Confirmation of SC24 in the fraction was done by western blotting using 
-SC24 antibody.
Southern blot analysis
Soybean genomic DNA was isolated from frozen tissue according to Murray and Thompson (1980) with some modifications and analysed by Southern blotting. Samples of DNA (30 µg) were digested with restriction enzymes, separated on a 0.7% agarose gel, and blotted to nylon membrane using standard protocols (Sambrook et al., 1989). Following hybridization at 65 °C for 16 h in hybridization buffer (0.25 M Na2HPO4 pH 7.2, 1% BSA, 1 mM EDTA, and 7% SDS) with a radiolabelled SC24 gene fragment, the membrane was washed four times for 15 min each at 68 °C in a high stringency wash solution containing 20 mM Na2HPO4 pH 7.2, 1 mM EDTA, and 1% SDS, followed by autoradiography.
Transcript profile of SC24 using EST database
The SC24 cDNA sequence was used to search soybean expressed sequence tags (ESTs) in the SoyBase (http://stadler.agron.iastate.edu/blast/blast.html) by BLASTN (Altschul et al., 1997). Transcripts matching SC24 were categorized according to source cDNA library and tissue type. The frequency of ESTs matching the SC24 cDNA sequence was calculated for corresponding source cDNA library.
Plant treatments
Etiolated soybean hypocotyls were challenged with P. sojae according to Qutob et al. (2000). Each hypocotyl was inoculated with a 10 µl droplet of zoospore suspension, containing approximately 103 free swimming zoospores. Care was taken not to wound the tissues. For the wounding experiment, etiolated hypocotyls were cut with a razor blade by scarring the surface, and tissues were collected after 3, 24, or 48 h. The tissues were frozen in liquid N2 and stored at –80 °C.
RT-PCR analysis of gene expression
Total RNA was isolated from soybean tissues following the procedure of Wang and Vodkin (1994). RNA samples were treated with DNase I (Promega, Madison, WI) for 30 min at 37 °C prior to RT-PCR. Samples were further purified by phenol:chloroform 3:1 (v:v) extraction and precipitated with ethanol. Samples of total RNA (2 µg each) were electrophoretically separated in formaldehyde gels and stained with ethidium bromide to ensure equal loading of samples prior to RT-PCR. The RT-PCR reactions were performed using a kit (ThermoscriptTM RT-PCR System, Life Technologies) following the manufacturer's instructions. Primer sequences for PCR were as follows: SC24-8 5'-CGCCGATCCCACATTTG-3', SC24-9 5'-TATTTGTAAACCGTGCTTCTCA-3'. The conditions for PCR were as follows: 94 °C for 12 s, 51 °C for 15 s, and 72 °C for 2 min (35 cycles).
For real time PCR, total RNA (5 µg) was reverse transcribed to cDNA with random hexamers (ThermoScript Reverse Transcriptase Kit, Invitrogen, USA). Real time PCR was performed on a thermocycler (LightCycler, Boehringer Mannheim/Roche Diagnostics, Laval, PQ) using the fluorescent dye, SYBR Green. Cycling conditions were performed using 2 µl of 1:10, 1:100, or 1:1000 (v/v) dilutions of first strand cDNA in a final volume of 20 µl, using buffers and conditions supplied by the manufacturer (FastStart SYBR Green I, Boehringer Mannheim/Roche Diagnostics, PQ). Gene-specific primers and PCR conditions were as described previously. Amplification rate for SC24 cDNA was evaluated from cycle threshold numbers obtained from serial cDNA dilutions. Optimal primer and template concentrations were determined by analysis of the dissociation curves and the amplified products were verified by agarose gel electrophoresis. Amplification profiles monitoring relative amounts of PCR product were visualized on graphs where the log of fluorescence was plotted against the number of cycles. Relative fold change in accumulation of SC24 under a given experimental treatment was standardized against cDNA derived from water-inoculated soybean tissue.
Production and purification of rSC24
Two primers, 5'-AATTCCATGGTTGCCGATCCCACAT-3' and 5'-CGGCCGCTCTAGAAGTACTCTCGAGT-3', were designed to produce a 776 bp product from cDNA that included most of the SC24 ORF, but lacked the signal peptide. The PCR product was digested with NcoI and XhoI and cloned into the corresponding sites of an E. coli expression vector (pET-30b, Novagen) to create an rSC24 ORF that included a 5 kDa amino-terminal His-tag. The construct was transformed into E. coli (XL1Blue, Stratagene, La Jolla, CA). Recombinant plasmids were sequence-verified prior to transformation into E. coli strain BL21(DE3) for rSC24 production.
For rSC24 protein production, bacteria from a fresh colony were grown overnight at 37 °C in Luria-Bertani (LB) medium containing 100 µg ml–1 kanamycin. An aliquot of the overnight-grown culture was used to inoculate 25 ml of fresh LB medium containing kanamycin. The culture was grown at 37 °C to an OD600 of 0.4–0.6. At this time, isopropylthio-ß-D-galactoside was added to a final concentration of 0.4 mM to induce the expression of rSC24. The culture was grown overnight at 20 °C. Bacterial cells were collected by centrifugation, resuspended in lysis buffer (BugBuster, Novagen) containing 100 µg lysozyme and 25 units of Benzonase nuclease ml–1. The cell lysate was clarified by centrifugation at 16 000 g for 20 min at 4 °C. The supernatant containing poly-His tagged rSC24 protein was filtered through 0.2 µm filter (Acrodisc, nylon) and was applied to a chelated Ni2+ affinity column (HiTrap chelating HP, Amersham Pharmacia) using FPLC system. The column was washed with 20 mM TRIS pH 7.4 and 500 mM NaCl. The rSC24 was eluted from the Ni2+ column using 20 mM TRIS pH 7.4, 500 mM NaCl, and a gradient of imidazole from 50–500 mM. Fractions were collected and analysed by 12.5% SDS–PAGE. Fractions containing rSC24 were pooled together and concentrated using a centrifugal filter device with a 10 kDa molecular weight cut off (Amicon, Millipore).
Antibody generation
A rabbit was bled to collect control serum prior to immunization. The preimmune serum was determined to be free of antibodies that cross-react with soybean proteins. Purified rSC24 with His tag (200 µg) was mixed 1:1 (v/v) with complete Freund's adjuvant and injected into the rabbit. Booster injections of rSC24 with incomplete Freund's adjuvant were performed 14 d and 35 d after the first injection. Antiserum to SC24 (-SC24) was collected on days 24 and 45, and stored at –80 °C.
Western blotting
Frozen plant tissue was ground in extraction buffer (25 mM TRIS–HCl pH 8.0, 1 mM EDTA pH 8.0, 20 mM NaCl, and protease inhibitor cocktail (Roche Applied Sciences) as described in Dhaubhadel et al. (1999) with some modifications. The protein concentration was determined by dye-binding assay (Bradford-Coomassie reagent, Bio-Rad Ltd., Mississauga, Canada).
For western blot analysis, proteins (15 µg from each sample) were separated on SDS–PAGE according to the method of Laemmli (1970) and transferred onto PVDF membranes by electroblotting using a semi-dry electrophoretic transfer device (Bio-Rad Ltd., Mississauga, Canada). The SC24 protein was detected by sequential incubation with -SC24 and horseradish peroxidase (HRP)-conjugated anti-rabbit IgG, each at a dilution of 1:7500, followed by the chemiluminescent detection (ECL system, Amersham Biosciences). For the wounding experiment, proteins (25 µg from each sample) were separated on SDS–PAGE and western blotting was performed as described above. The proteins were detected by using SuperSignal west femto maximum sensitivity substrate (Pierce Biotechnology Inc., IL) according to the manufacturer's instructions.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
SC24 is an abundant protein in mature soybean seed coats
To purify SC24 from soybean seed hulls, protein extracts were precipitated with acetone and separated by gel filtration chromatography. Shown in Fig. 1 are representative results from such a purification. The SC24 protein is abundant in seed hulls and may be purified to near-homogeneity with relative ease. The purification was assisted by some unusual characteristics of SC24 and another protein that co-eluted with SC24 from the gel-filtration column. First, SC24 eluted relatively late from the gel-filtration column for a protein of its size. The mass of SC24 was estimated to be 24 kDa based upon SDS–PAGE analysis, but the elution volume of the native protein from the gel-filtration column suggested an apparent mass of 11.5 kDa. It is not clear what delays the elution of SC24, but it may be interacting with the agarose- and dextran-based column matrix. Second, the main contaminating protein that co-eluted with SC24 was determined to be the soybean hydrophobic protein (HPS). This protein spontaneously crystallized and precipitated out after chromatography, leaving highly purified SC24 in solution (Fig. 1). Further, analysis of SC24 by peptide micro-sequencing revealed an amino-terminal sequence identical to that reported for ‘Protein V’ from seed hulls (Gijzen et al., 2001
), and similar to a partial sequence reported for a protein released by soybean seeds in hot water (Hirano et al., 1992). Databases of ESTs (http://stadler.agron.iastate.edu/blast/blast.html) were searched for reading frames matching the SC24 amino-terminal peptide sequence to identify transcripts encoding protein. Several corresponding ESTs were identified, but none of these encoded a complete open reading frame (ORF). The EST sequences were used to design primers and construct probes for screening a seed coat cDNA library (Gijzen, 1997). Six cDNA clones were isolated, including a transcript that apparently encoded a complete ORF of SC24. This SC24 cDNA was used as a hybridization probe to isolate a 15 841 bp EcoRI genomic fragment containing the SC24 gene.
|
SC24 binds to polycarboxylates
The cDNA encoding SC24 was 815 bp in length and included 34 bp of 5' untranslated region (UTR), an ORF of 219 amino acids, and 140 bp of 3' UTR. The 219 amino acid preprotein consists of a 26 amino acid signal peptide leader sequence that is cleaved during processing (Fig. 2). The search for protein domain architecture did not reveal any known domain within the protein sequence. However, a protein fold recognition server, 3D-PSSM (Kelly et al., 2000
), suggested that SC24 may share structural similarity with mouse fibronectin protein with 70% certainity and a PSSM e-value of 0.265. The full-length ORF encoded a preprotein of calculated molecular mass of 24.5 kDa and a mature protein of 21.8 kDa with a pI of 9.3.
|
The SC24 amino acid sequence (accession number ASS59524) was similar to a citrate binding protein (CBP) isolated from latex of the rubber tree (Rentsch et al., 1995
), and to predicted proteins from potato and Angelica dahurica (Fig. 2). Transcripts potentially encoding proteins similar to SC24 were also detected in numerous plant species, including monocots (barley, rice, sugar cane) and dicots (alfalfa, ginseng, poplar, potato) by analysis of EST databases (not shown). Most EST hits originated from cDNA libraries constructed from wounded or pathogen-infected tissues. However, no counterpart to SC24 is present in Arabidopsis. Thus, SC24-like proteins appear to be widely distributed among plants, but apparently are dispensable. The decision was taken to test whether SC24 could be purified using an affinity column containing an isophthalic acid ligand, since this method had been used to isolate the CBP from latex. The results demonstrate that SC24 binds to this affinity column (Fig. 3). Application of a citrate gradient resulted in the elution of SC24 in the 5–7 mM range. These properties are similar to those described for the CBP, indicating that SC24 and CBP share functional characteristics with regard to their affinity towards polycarboxylates.
|
SC24 is a low copy gene and contains a single intron
Sequence analysis of the genomic clone revealed that the SC24 gene is encoded within a 1762 bp stretch that contains a single intron of 784 bp and two exons of 417 and 387 bp lengths (Fig. 4A). The SC24 cDNA sequence exactly matched sequences encoded within exons of the genomic clone. The cDNA transcript was nearly full length, missing only 19 bp of the 5' UTR downstream of the predicted transcription start site. A TATA box was located at –30 from the predicted transcription start site, which is consistent with the distance of the TATA box from the transcription initiation site in many plant genes (Joshi, 1987
). No other genes were present within the 15.8 kb genomic clone, as determined by comparison to known EST and protein sequences, and by a gene prediction programme (Burge and Karlin, 1997). To investigate the copy number of SC24, a 32P-labelled cDNA probe was hybridized to soybean genomic DNA under conditions of high stringency. Hybridization patterns were consistent with that of a single copy gene, since all bands could be accounted for and predicted from analysis of the 15 841 bp EcoRI genomic fragment (Fig. 4D). Furthermore, no evidence of additional SC24-like transcripts could be found by searching the extensive soybean EST databases.
|
The SC24 transcript is abundant in seed coats and is induced by stress
As a first step to determine the expression pattern of the soybean SC24 gene, ESTs were analysed for sequences exhibiting similarities to SC24. Nucleotide sequences corresponding to SC24 were used in BLASTN searches of 309 385 soybean ESTs (http://stadler.agron.iastate.edu/blast/blast.html) originating from more than 80 different cDNA libraries (Shoemaker et al., 2002
). This resulted in the identification of 39 matching ESTs from 13 different source cDNA libraries. These data were pooled according to the tissue-type of the source cDNA library (Fig. 5A). The results show that ESTs corresponding to SC24 occurred most frequently in cDNA libraries constructed from wounded cotyledons. Several ESTs matching the SC24 gene sequence were also found in cDNA libraries from plant organs exposed to other stresses, such as elicitor or toxin treatment, or pathogen infection.
|
To study in detail the expression pattern of SC24 in different soybean plant tissues during development, RT-PCR was performed using SC24 gene-specific primers. Results indicate that SC24 transcript accumulation is prevalent in the seed coat from 30 DAP to maturity (Fig. 5B). The SC24 transcript was also detected in the flower buds, flower, leaf, embryo, and pod wall during later stages of development. Analysis by RNA blot hybridization yielded similar results and indicated that SC24 mRNA is most abundant in the seed coat during late stages of seed maturation (not shown). It was also determined that SC24 mRNA may be induced by pathogen infection (Fig. 5C). Transcripts from P. sojae-infected soybean hypocotyls at 3, 6, 12, 24, or 48 h after inoculation with a virulent race of the pathogen were compared by quantitative, real-time RT-PCR using primers specific for SC24. The expression of SC24 was induced within 6 h of infection and peaked at 24 h.
SC24 accumulates late in seed development and in response to wound stress
To produce recombinant SC24 (rSC24), the complete ORF from the SC24 cDNA, without the signal peptide, was cloned into an E. coli expression vector. The rSC24 possessed an N-terminal histidine (His)-tag for purification by Ni2+ affinity chromatography. Using these methods, large amounts of highly purified, soluble rSC24 could be recovered from E. coli cultures suitable for polyclonal antibody production (Fig. 6).
|
To determine the apparent molecular mass of rSC24, purified protein was analysed by gel filtration chromatography (data not shown). The apparent molecular mass of His-tagged rSC24 was 26 kDa, in close agreement with the deduced molecular mass of 25.9 kDa. However, after cleavage of the 4.1 kDa amino terminal His-tag of rSC24, the apparent molecular mass of the protein was 11.5 kDa, much less than its calculated mass of 21.8 kDa, but identical to the apparent mass of SC24 isolated from seed hulls (Fig. 1). Thus, only after cleavage of its His-tag does rSC24 display an anomalous molecular mass as determined by gel filtration chromatography.
To measure SC24 protein accumulation in different soybean tissues and during seed development, polyclonal antibodies raised against purified rSC24 were used to probe soluble protein extracts by western blotting analysis. Trace amounts of SC24 were detected in protein extracts from flower buds, young pod tissue, and seeds, but not in any other soybean organs. In the seed, SC24 accumulated in seed coat tissues at the late stages of development (Fig. 7A). It was first detected in seed coat at 50 DAP, at a time when the seeds were near maturity. Polyclonal antibodies raised against rSC24 also bound to a 43 kDa protein present in seed coat tissues. The 43 kDa protein was first detected at 30 DAP and continued to accumulate to seed maturity. This cross-reacting protein was identified as the seed coat peroxidase by comparison with purified enzyme purchased from commercial sources. To verify that the 43 kDa signal was not due to endogenous peroxidase activity, the western blotting analysis was performed using alkaline phosphatase conjugated anti-rabbit IgG in place of horseradish peroxidase-conjugated anti-rabbit IgG as the secondary antibody, and similar results were obtained (data not shown). Surprisingly, the seed coat peroxidase enzyme appears to share a common antigenic domain with SC24, despite the fact that the two proteins are unrelated and do not show any significant similarity in amino acid sequence.
|
To determine which cell layers of the seed coat contain SC24, two fractions of seed coat tissues that naturally separate from one another when the tissue is sectioned were collected and placed in water (Fig. 7B). Fraction I consisted of the palisade and hourglass cell layers while Fraction II contained the remaining tissues of the seed coat, including the parenchyma and aleurone layers. The results indicate that SC24 is present in parenchyma and aleurone layers, but not in palisade and hourglass cell layers, whereas peroxidase was detected in both fractions (Fig. 7C).
Since SC24 transcripts were abundant in tissues that were subjected to various stresses, the level of SC24 was determined in soybean tissues that were either challenged by pathogen or by wounding. A slight increase in the level of SC24 accumulation was observed after 48 h of P. sojae infection (data not shown). However, a significantly high level of SC24 accumulation was detected in response to wound stress (Fig. 8). After 3 h of wound treatment, the amount of SC24 doubled compared with the control samples. The amount of SC24 protein present in wounded tissues increased to 8.8 and 9.5 times that in unwounded tissues after 24 h and 48 h, respectively, as determined by image intensity analysis (Scion Image Beta program, Scion Corporation).
|
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Previously, the composition of proteins present in soybean seed hulls was analysed and five major soluble components were identified (Gijzen et al., 2001
). A 24 kDa protein (SC24) was also purified and partially sequenced from extracts of seed hulls. This 24 kDa protein possessed a highly basic pI and was abundant in soybean seed hulls. In this study, the SC24 protein has been characterized further, its citrate binding activity has been demonstrated, the corresponding cDNA and genomic clones have been isolated, and the amount of SC24 mRNA and protein present in various plant tissues during development and in response to stresses have been measured.
The amino acid sequence of SC24 shares similarity with the rubber tree CBP, an abundant protein occurring in the latex, a defensive secretion that is rich in anti-microbial constituents (Rentsch et al., 1995; Subroto et al., 2001). The CBP protein was first identified based upon photoaffinity labelling using analogues of the dicarboxylic acids citrate and malate, in an attempt to identify citrate transport proteins (Rentsch et al., 1995). The rubber tree CBP is considered as a vacuolar protein, possesses a C-terminal extension that is not present in SC24 and related proteins from potato and Angelica.
Plants have developed various responses to environmental stresses by inducing the synthesis of a wide array of defence-related proteins. The expression pattern of SC24 at mRNA level in response to pathogen infection suggests that it may have a role in defence. However, the pattern of SC24 transcript accumulation did not correlate directly with those of protein accumulation in pathogen-infected tissues. Rapid protein turnover and/or degradation may result in lower levels of SC24 accumulation in pathogen challenged tissues. By contrast, a significant increase in SC24 level was detected in response to wound stress. There was slight increase in SC24 accumulation after 3 h of wound treatment, compared with unwounded tissues. The level of SC24 increased dramatically at later time points. The results suggest that SC24 may have a role in the wound repair of damaged tissues and not in the production of wound signals. The appearance of SC24 in seed coat tissues near seed maturity and its localization in the inner layers of seed coat are consistent with a defensive or structural role for the protein.
The SC24 protein displayed unusual properties when subjected to gel filtration chromatography on polydextran media, and was retarded in its elution compared with other proteins of comparable size. Furthermore, SC24 showed affinity towards an isophthalic acid-coupled activated agarose column and could be eluted with increasing concentration of citrate. Citrate is often considered to be an exogenous siderophore, even though it has a simpler chemical composition and weaker iron-binding affinity compared to other siderophores (Yue et al., 2003). Siderophores are crucial for the virulence of various pathogens in animal models of diseases (Ratledge and Dover, 2000) and are a vital factor for the growth and survival of many bacteria and fungi in the soil and in aqueous environment (Guerinot, 1994). It is possible that binding of citrate by SC24 affects the availability of iron to the pathogens which negatively influences their growth and development.
Polyclonal antibodies to rSC24 cross-reacted with the soybean seed coat peroxidase, a surprising result that suggests shared structural and possibly functional characteristics of these two proteins. Higher plant peroxidases are haem-containing oxidoreductases that catalyse the reduction of hydrogen peroxide and oxidation of various hydrogen donors. They function in cell wall biosynthesis and defence, and are often induced by wounding or pathogen infection (Campa, 1991; Moerschbacher, 1992; Hiraga et al., 2001). There are no obvious or significant similarities in the aminio acid sequences of SC24 and the seed coat peroxidase that can account for the common antigenic domain(s) that these two proteins must share. However, the three-dimensional structure of soybean seed coat peroxidase has been solved by X-ray crystallography (Henriksen et al., 2001), and may provide a reference for predicting the structure of SC24, once the common antigenic domains have been identified.
Many characteristics of SC24, such as its abundance, stability, expression pattern, and citrate binding activity suggest that this protein may have a role in defence. Past studies have also shown that the seed coat is particularly rich in defence-related proteins and peptides (Gijzen et al., 2001). However, no anti-microbial activity of SC24 could be detected when tested against a variety of fungal, oomycete, and bacterial plant pathogens (not shown). Transgenic Arabidopsis lines expressing SC24 driven by a constitutive promoter (cauliflower mosaic virus 35S) were also created. Transgenic plants expressing the protein were recovered, but did not display any obvious phenotype that differentiates them from control plants (not shown).
To summarize, a major soluble protein present in soybean seed coats, SC24, has been isolated and characterized. The main characteristics of SC24 suggest that it is a defensive protein, thus supporting the biological role of seed coat in seed protection and defence. In addition, soybean seed coat is one of the crucial factors for embryo development and establishment of many agronomic traits such as seed size, lustre, colour, and composition. Therefore, identifying seed coat constituents and characterizing their corresponding genes is important, since soybean is the largest legume crop in the world.
|
Acknowledgements |
|---|
We thank Vaino Poysa (Agriculture and Agri-Food Canada, Harrow) for soybean seed; ADM Agri-Industries (Windsor, ON) for processed seed hulls; Canadian Food Inspection Agency (Ottawa, ON) for antibody production, the Biotechnology Service Centre (University of Toronto, ON) for peptide microsequencing; Dinah Qutob for wounded samples, and Aldona Gaidauskas-Scott, Pat Moy, Alex Molnar, and Mana Farhangkhoee for technical assistance.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ. 1997. Gapped BLAST and PSI-BLAST: a new generation of proteins database search programs. Nucleic Acids Research 25, 3389–3402.
Blum H, Beier H, Gross HJ. 1987. Improved staining of plant proteins, RNA and DNA in polyacrylamide gels. Electrophoresis 8, 93–99.[CrossRef][Web of Science]
Boesewinkel FD, Bouman F. 1995. The seed: structure and function. In: Kigel J, Galili G, eds. Seed development and germination. New York: Marcel Dekker, 1–24.
Burge C, Karlin S. 1997. Prediction of complete gene structures in human genomic DNA. Journal of Molecular Biology 268, 78–94.[CrossRef][Web of Science][Medline]
Campa A. 1991. Biological roles of plant peroxidases: known and potential function. In: Everse J, Everse KE, Grisham MB, eds. Peroxidases in chemistry and biology, Vol. II. Boca Raton, FL: CRC Press, 25–50.
Corner EJH. 1951. The leguminous seed. Phytomorphology 1, 117–150.
Dhaubhadel S, Chaudhary S, Dobinson KF, Krishna, P. 1999. Treatment with 24-epibrassinolide, a brassinosteroid, increases the basic thermotolerance of Brassica napus and tomato seedlings. Plant Molecular Biology 40, 333–342.[CrossRef][Web of Science][Medline]
Esau K. 1977. Anatomy of seed plants. New York: Wiley.
Gijzen M. 1997. A deletion mutant at the ep locus causes low seed coat peroxidase activity in soybean. The Plant Journal 12, 991–998.[CrossRef][Web of Science][Medline]
Gijzen M, Kuflu K, Qutob D, Chernys JT. 2001. A class I chitinase from soybean seed coat. Journal of Experimental Botany 52, 2283–2289.
Gijzen M, Miller SS, Kuflu K, Buzzell RI, Miki BLA. 1999. Hydrophobic protein synthesized in the pod endocarp adheres to the seed surface. Plant Physiology 120, 951–959.
Gijzen M, Van Huystee R, Buzzell RI. 1993. Soybean seed coat peroxidase: a comparison of high-activity and low-activity genotypes. Plant Physiology 103, 1061–1066.[Abstract]
Guerinot ML. 1994. Microbial iron transport. Annual Review of Microbiology 48, 743–772.[CrossRef][Web of Science][Medline]
Henriksen A, Mirza O, Indiani C, Teilum K, Smulevich G, Welinder KG, Gajhede M. 2001. Structure of soybean seed coat peroxidase: a plant peroxidase with unusual stability and haem–apoprotein interactions. Protein Science 10, 108–115.[CrossRef][Web of Science][Medline]
Herman EM, Helm RM, Jung R, Kinney AJ. 2003. Genetic modification removes an immunodominant allergen from soybean. Plant Physiology 132, 36–43.
Hiraga S, Sasaki K, Ito H, Ohashi Y, Matsui H. 2001. A large family of class III plant peroxidases. Plant Cell Physiology 42, 462–468.
Hirano H, Kagawa H, Okubo K. 1992. Characterization of proteins released from legume seeds in hot water. Phytochemistry 31, 731–735.[Web of Science][Medline]
Joshi CP. 1987. An inspection of the domain between putative TATA box and translation start site in 79 plant genes. Nucleic Acids Research 15, 6643–6653.
Kelly LA, MacCallum RM, Sternberg MJE. 2000. Enhanced genome annotation using structural profiles in the program 3 D-PSSM. Journal of Molecular Biology 299, 499–520.[Web of Science][Medline]
Laemmli UK. 1970. Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227, 680–685.[CrossRef][Medline]
Miller SS, Bowman LA, Gijzen M, Miki BLA. 1999. Early development of the seed coat of soybean (Glycine max). Annals of Botany 84, 297–304.
Moerschbacher BM. 1992. Plant peroxidases: involvement in response to pathogens. In: Penel C, Gaspar T, Greppin H, eds. Plant peroxidases 1980–1990: topics and detailed literature on molecular, biochemical, and physiological aspects. Geneva: University of Geneva Press, 91–99.
Moos M, Nguyen NY, Liu TY. 1988. Reproducible high yield sequencing of proteins. Electrophoretically separated and transferred to an inert support. Journal of Biological Chemistry 263, 6005–6008.
Murray DR. 1987. Nutritive role of seed coats in developing legume seeds. American Journal of Botany 74, 1122–1137.[CrossRef][Web of Science]
Murray MG, Thompson WF. 1980. Rapid isolation of high-molecular weight plant DNA. Nucleic Acids Research 8, 4321–4325.
Patrick JW, Offler CE. 2001. Compartmentation of transport and transfer events in developing seeds. Journal of Experimental Botany 52, 551–564.
Percy JD, Philip R, Vodkin LO. 1999. A defective seed coat pattern (Net) is correlated with the post-transcriptional abundance of soluble proline-rich cell wall proteins. Plant Molecular Biology 40, 603–613.[CrossRef][Web of Science][Medline]
Qutob D, Hraber PT, Sobral BWS, Gijzen M. 2000. Comparative analysis of expressed sequences in Phytophthora sojae. Plant Physiology 123, 243–253.
Ratledge C, Dover LG. 2000. Iron metabolism in pathogenic bacteria. Annual Review of Microbiology 54, 881–941.[CrossRef][Web of Science][Medline]
Rentsch D, Gorlach J, Vogt E, Amrhein N, Marinoia E. 1995. The tonoplast-associated citrate binding protein (CBP) of Hevea brasiliensis. Journal of Biological Chemistry 270, 30525–30531.
Reiser L, Fischer RL. 1993. The ovule and the embryo sac. The Plant Cell 5, 1291–1301.
Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning: a laboratory manual, Cold Spring Harbor, New York: Cold Spring Harbor Laboratory Press.
Sessa DJ, Wolf WJ. 2001. Bowman–Birk inhibitors in soybean seed coats. Industrial Crops Production 14, 73–83.
Shoemaker R, Keim P, Vodkin L, et al. 2002. A compilation of soybean ESTs: generation and analysis. Genome 45, 329–338.[Medline]
Subroto T, de Vries H, Schuringa JJ, Soedjanaatmadja UMS, Hofsteenge J, Jekel PA, Beintema JJ. 2001. Enzymic and structural studies on processed proteins from the vacuolar (lutoid-body) fraction of latex of Hevea brasiliensis. Plant Physiology and Biochemistry 39, 1047–1055.[CrossRef][Web of Science]
Villiers TA. 1972. Seed dormancy. In: Kozlowski TT, ed. Seed biology, Vol. II. New York: Academic Press, 219–281.
Wang CS, Vodkin LO. 1994. Extraction of RNA from tissues containing high levels of procyanidins that bind RNA. Plant Molecular Biology Reporter 12, 132–145.[CrossRef]
Yaklich RW. 2001. ß-Conglycinin and glycinin in high-protein soybean seeds. Journal of Agricultural Food Chemistry 49, 729–735.
Yue WW, Grizot S, Buchanan SK. 2003. Structural evidence for iron-free citrate and ferric citrate binding to the TonB-dependent outer membrane transporter FecA. Journal of Molecular Biology 332, 353–368.[CrossRef][Web of Science][Medline]
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  S. Dhaubhadel, M. Farhangkhoee, and R. Chapman Identification and characterization of isoflavonoid specific glycosyltransferase and malonyltransferase from soybean seeds J. Exp. Bot., March 2, 2008; (2008) ern046v2. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||