Storage protein profiles in Spanish and runner market type peanuts and potential markers
© Liang et al; licensee BioMed Central Ltd. 2006
Received: 15 June 2006
Accepted: 12 October 2006
Published: 12 October 2006
Proteomic analysis has proven to be the most powerful method for describing plant species and lines, and for identification of proteins in complex mixtures. The strength of this method resides in high resolving power of two-dimensional electrophoresis (2-DE), coupled with highly sensitive mass spectrometry (MS), and sequence homology search. By using this method, we might find polymorphic markers to differentiate peanut subspecies.
Total proteins extracted from seeds of 12 different genotypes of cultivated peanut (Arachis hypogaea L.), comprised of runner market (A. hypogaea ssp. hypogaea) and Spanish-bunch market type (A. hypogaea ssp. fastigiata), were separated by electrophoresis on both one- and two-dimensional SDS-PAGE gels. The protein profiles were similar on one-dimensional gels for all tested peanut genotypes. However, peanut genotype A13 lacked one major band with a molecular weight of about 35 kDa. There was one minor band with a molecular weight of 27 kDa that was present in all runner peanut genotypes and the Spanish-derivatives (GT-YY7, GT-YY20, and GT-YY79). The Spanish-derivatives have a runner-type peanut in their pedigrees. The 35 kDa protein in A13 and the 27 kDa protein in runner-type peanut genotypes were confirmed on the 2-D SDS-PAGE gels. Among more than 150 main protein spots on the 2-D gels, four protein spots that were individually marked as spots 1–4 showed polymorphic patterns between runner-type and Spanish-bunch peanuts. Spot 1 (ca. 22.5 kDa, pI 3.9) and spot 2 (ca. 23.5 kDa, pI 5.7) were observed in all Spanish-bunch genotypes, but were not found in runner types. In contrast, spot 3 (ca. 23 kDa, pI 6.6) and spot 4 (ca. 22 kDa, pI 6.8) were present in all runner peanut genotypes but not in Spanish-bunch genotypes. These four protein spots were sequenced. Based on the internal and N-terminal amino acid sequences, these proteins are isoforms (iso-Ara h3) of each other, are iso-allergens and may be modified by post-translational cleavage.
These results suggest that there may be an association between these polymorphic storage protein isoforms and peanut subspecies fastigiata (Spanish type) and hypogaea (runner type). The polymorphic protein peptides distinguished by 2-D PAGE could be used as markers for identification of runner and Spanish peanuts.
There is considerable variation in Arachis hypogaea L. subspecies hypogaea and fastigiata Waldron, which are further classified into four market types including runner, Virginia, Spanish, and Valencia . Most cultivated peanuts belong to Spanish and runner types. They exhibit genetically-determined variation for a number of botanical and agronomical traits including branching and flowering habits, seed dormancy, and maturation time. However, there are few categorical criteria for distinguishing subspecies because of the limited detectable molecular polymorphism. Recently, several molecular approaches have been employed to assess genetic diversity and taxonomic relationships. Among them are isozymes , restriction fragment length polymorphisms (RFLP), random amplified polymorphisms (RAPD), amplified fragment length polymorphisms (AFLP), and simple sequence repeats (SSR) [3–6]. However, very little genetic polymorphism between the two subspecies was detected. Singh et al. [7, 8] and Bianchi-Hall et al.  found very limited or no variation among cultivated peanut based on seed protein profiles.
To date, proteomic analysis has proven to be the most powerful method for describing plant species and lines , and identification for proteins (especially protein markers) in complex mixtures. The strength of this method resides in high resolving power of two-dimensional PAGE (2D-PAGE), coupled with polypeptide sequencing by highly sensitive mass spectrometry (MS) such as electrospray ionization tandem mass spectrometry (ESI-MS/MS), and sequence homology search in databases .
The aim of the research described in this paper was to investigate the ability of proteomic analysis to assess diversity of seed storage proteins in peanut for subspecies or cultivar identification. Subspecies or cultivar-specific proteins, if they exist, should be helpful for genetic studies, breeding, taxonomy and evolutionary relationships in peanut.
Analysis of gel electrophoresis
Polypeptide sequence analysis
List of cultivated peanut used in this study
Georgia Green 
Internal peptide and N-terminal sequences of some protein spots of cultivated peanut
Internal peptide sequence
N-terminal peptide sequence
QGGEENECQFQR FHLAGNQEQEFLR IESEGGYIETWNPNNQEFECAGVALSR
Peptide sequence of spot 1 was unique, and present only in Spanish-type peanuts. Two peptides sequenced after in-gel trypsin digestion were the same, while one fragment gave 100% (FYLAGNQEQEFLR) identity and another fragment gave 88% (14 out of 16 amino acids) identity with iso-Ara h3. The N-terminal sequence (VGQDDPSQQQ) of spot 1 was 100% identical with iso-Ara h3, whereas Ara h3 and Ara h4 have two amino acids missing in this region (Fig. 4). N-terminal sequencing for spot 2 and spot 3 resulted in the sequences containing VTFRQGG, identical with the sequence for iso-Ara h3 . The N-terminal sequence of spot 4 was GIEETICSASVK, 100% identical with iso-Ara h3 and one amino acid (S/T) different from Ara h3 and Ara h4, supporting that spot 4 is the C-terminal part of this protein which always starts with GIEETIC .
The initial intention of this study was to profile the storage proteins using improved protein extraction method and to identify protein markers that could be used to separate subspecies of peanut, such as hypogaea and fastigiata, in order to select diverse breeding lines for mapping population construction. Based on the preliminary protein profiles , we selected Tifrunner and GT-YY20 for development of recombinant inbred lines (RILs) for genetic mapping. On 2-D PAGE gels, several proteins, labelled as spots 1–4 with similar molecular mass and different pIs, were sequenced. The peptide sequences obtained from these spots were all aligned to peanut allergens, such as iso-Ara h3 (AAT39430), indicating that this single gene encoded protein may be processed differently in different peanut subspecies. The partial cDNA sequence (accession number AY618460) was deposited in GenBank by Kang and Gallo-Meagher  in 2004. A full-length cDNA sequence identified in our EST sequencing project has been submitted to GenBank (DQ855115). The internal and N-terminal sequences of peptide spot 1 suggest that the apparent rearrangement of the amino acid sequence has occurred (Fig. 4).
In peanut the majority of seed storage protein (about 87%) is globulin consisting of two major fractions, arachin and conarachin . The arachin subunits consist of the acidic polypeptides and the basic polypeptides . The uniformity of the one-dimensional SDS-PAGE protein profiles within the runner type and Spanish type cultivars and breeding lines is in agreement with the studies [7–9], indicating that very low variation in protein profiles was detected in cultivated peanut using SDS-PAGE gel electrophoresis.
Generally, SDS-PAGE is not a sufficiently-powerful technique to distinguish a specific cultivar. Therefore, we adopted the widely used protocol developed by Damerval et al.  and introduced some modifications including a preliminary de-fatting step of peanut seeds for 2-D PAGE separation. We were able to generate 2-D electrophoresis gel separations with superior resolution and recovery from peanut seeds. Bianchi-Hall et al.  reported that the polypeptides of acidic arachin using SDS-PAGE distinguish Spanish from other market type cultivars. In this study, we did not identify the four bands in the range of acidic arachin by SDS-PAGE (Fig. 1), but we could detect the fourth spot of protein on 2-D PAGE for Spanish type genotypes (Fig. 2). We also detected a 26 kDa polypeptide by SDS-PAGE; this polypeptide could be used to differentiate Spanish and runner.
This study demonstrated that two-dimensional electrophoresis (2-D PAGE) achieved a better resolution of protein profiles of peanut seeds, revealing polymorphisms between runner and Spanish genotypes. The basic arachin group, having one heavy band on SDS-PAGE gels at about 22 kDa, was resolved into several spots or subunits on the 2-D PAGE with distinct isoelectric points and slight differences in molecular weights. These proteins are isoforms (iso-Ara h3) of each other and the iso-allergens may be modified by post-translational cleavage. These results suggest that there may be an association between these polymorphic storage protein isoforms and peanut subspecies fastigiata (Spanish type) and hypogaea (runner type). Future studies could be designed to test the allergenic reactions of these peanut genotypes with different protein profiles and association with the resistance to aflatoxin contamination .
Twelve peanut genotypes were used in this study. There were six runner-type peanut genotypes: Georgia Green, A100, A104, GK7, A13 and Tifrunner, and six Spanish-bunch type peanut genotypes: ICGV 95435 (International Crops Research Institute for the Semi-Arid Tropics, Patancheru, India), MXHY and ZQ48 (Chinese landraces), and GT-YY20, GT-YY7 and GT-YY79 (Spanish derivatives with runner type peanut in their pedigrees, obtained from Crops Research Institute, Guangdong Academy of Agricultural Sciences, China). To avoid the effects of different locations, all genotypes were grown in Tifton, GA in 2003. Seeds were harvested at full maturity per normal production practices. After harvest, seeds were air-dried at 40°C and stored at 4°C before use.
Total protein extraction
The total protein extraction was modified from TCA/Acetone protein extraction protocol  with the first step of de-fatting using hexane. Dry peanut kernels (20 g) of each genotype were frozen in liquid nitrogen and ground to powder in a mill and defatted with hexane (10 ml/g dry weight) at -20°C overnight. The defatted samples were collected by centrifugation (15,000 × g for 10 min at 4°C), air-dried, and ground to a fine powder in a pre-chilled mortar and pestle in liquid nitrogen. Protein extraction and precipitation were performed in 10% (w/v) trichloroacetic acid in cold acetone with 0.07% (v/v) β-mercaptoethanol at -20°C for 2 h, followed by centrifugation at 10,000 × g for 10 min at 4°C. The pellets were washed twice with cold acetone containing 0.07% β-mercaptoethanol, followed by washing twice with cold 80% acetone and then centrifuged at 10,000 × g for 10 min at 4°C. The pellets were air dried and stored at 4°C overnight. The total proteins were dissolved in lysis buffer (10 μl/mg) containing 9.5 M urea, 4% Igepal CA-360 (Sigma, St. Louis, MO), 2.5% ampholytes (0.5% pH 3.0–10, 0.5% pH 4–6, and 1.5% pH 6–8) (Sigma), 5% β-mercaptoethanol, and kept at 35°C for 30 min. After centrifugation (15,000 × g, 20 min, 25°C), the supernatant was collected for loading in first-dimension gel electrophoresis, or alternatively, for storing at -20°C until use. The supernatant protein concentration was determined using the Bradford  assay. The experiment was conducted twice, and each genotype was run at least three times.
SDS-PAGE and two-dimensional PAGE electrophoresis
Total protein samples from these twelve peanut genotypes were first profiled using SDS-PAGE (15% separating gel with 4% stacking gel) according to the method of Laemmli  with the Mini-PROTEIN ®II Dual Slab Cell System (BIO-RAD, Hercules, CA) . Total proteins (100 μg) from each sample were loaded onto SDS-PAGE gels. Low-range protein markers (Sigma) were used as molecular mass standard. The gels were electrophoresed (120 V, 1.5 h), stained with 0.125% Coomassie blue R-250 in 40% methanol and 10% acetic acid. For 2-D PAGE, total seed proteins (1 mg) were loaded into tube gels (8 M urea, 4% acrylamide, 2% Igepal CA-630, 0.5% ampholyte pH 3.0–10, 0.5% ampholyte pH 4–6, 1.5% ampholyte pH 6–8, 0.01% ammonium persulfate, and 0.1% TEMED), and overlaid with 20 μl sample overlay buffer (4 M urea, 0.25% ampholyte pH 3.0–10, 0.25% ampholyte pH 4–6, 0.75% ampholyte pH 6–8, 2.5% β-mercaptoethanol, 1% Igepal CA-360, and 0.05% Bromophenol blue). Isoelectric focusing (IEF) was conducted by using Mini-Protean® 2-D Electrophoresis Cell (BIO-RAD). The upper and lower chamber buffers were 100 mM NaOH and 10 mM H3PO4 respectively. IEF conditions were 200 V for 15 min, 300 V for 15 min, 400 V for 30 min, and 750 V for 6 h. The focused tube gels were equilibrated immediately for 30 min in 10 ml SDS equilibration buffer (60 mM Tris-HCl, pH6.8, 2% SDS, 10% glycerol, and 0.05% Bromophenol blue), or kept at -20°C until use. After equilibration, the tube gels were embedded in a 1% agarose solution at the top of the 2-D gel. The second dimension was run on 15% polyacrylamide-SDS gels in a Mini-Protean® 3 Cell (BIO-RAD), 120 V for 90 min. The gels were stained with Coomassie Brilliant Blue R250 and all gels were scanned and the spot intensities were analyzed using the software Image Master-2D (BIO-RAD). The interesting spots of seed protein among the genotypes were identified by gel-to-gel comparison. For molecular weight determination, low molecular weight standard (Sigma) was used.
Protein peptides were excised from the 2-D gels and PVDF membranes for peptide sequencing using electrospray ionization tandem mass spectrometry (ESI-MS/MS) to obtain internal peptide sequences and using the conventional Edman degradation method to obtain N-terminal sequences. Protein spots from the gels were excised with combined total protein amount up to 10 pg, and were subjected to in-gel digestion and analysis by ESI-MS/MS to obtain peptide sequence information at the Protein Chemistry Core Facility, Baylor College of Medicine (Houston, TX). When peptide sequences could not be obtained unambiguously by using ESI-MS/MS, Edman degradation was performed using an Applied Biosystems Procise cLC sequencer to obtain sequence information for protein identification.
Electrobloting and N-terminal sequence
To prevent N-terminal blockage during second-dimension gel electrophoresis, gels were poured at least 24 hr prior to running and 0.1 mM thiodiglycolate was added as a scavenger in the upper running buffer. 2-D gels were equilibrated for 30 min in 25 mM Tris, 192 mM glycine, 10% MeOH (pH 8.3), and then electroblotted to Immobilon-p PVDF-membrane (Millipore, Bedford, MA, USA) at 300 mA for 4 hr in a Mini Trans-Blot® Electrophoretic Transfer Cell (BIO-RAD). The membrane was subsequently equilibrated for 5 min in deionized water and proteins stained with 0.05% Coomassie Blue in 1% acetic acid and 50% methanol for a few min, destained in 50% methanol until background was pale blue. The membrane was rinsed for 5–10 min in deionized water and air-dried. Spots were excised and used for N-terminal amino acid microsequencing at Baylor Medical School (Houston, TX).
Database sequence homology analysis
Internal and N-terminal peptide sequence homology identification was performed using basic local alignment search tool (BLAST)  against known or translated open reading frames of expressed sequence tags (ESTs) in the databases at the National Center for Biotechnology Information (NCBI) and SWISS-Prot.
We thank Ernest Harris and Kippy Lewis for technical assistance in the field and the laboratory. This research was supported partially by funds provided by USDA Agricultural Research Service and Peanut Foundation, and by funds provided by Scientific Cooperation Research Program of U. S. Department of Agriculture-Foreign Agricultural Service between U.S. and China. Mention of trade names or commercial products in this publication is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture.
- Krapovickas SA, Rigoni VA: Taxonomia del genero Arachis (Leguminosae). Bonplandia. 1994, 8: 1-186.Google Scholar
- Stalker HT, Philips TD, Murphy JP, Jones TM: Variation of isozyme patterns among Arachis species. Theor Appl Genet. 1994, 87: 746-755. 10.1007/BF00222901.PubMedView ArticleGoogle Scholar
- Kochert G, Halwart T, Branch WD, Simpson CE: RFLP variability in peanut (Arachis hypogaea L.) cultivars and wild species. Theor Appl Genet. 1991, 81: 565-570. 10.1007/BF00226719.PubMedView ArticleGoogle Scholar
- Halward TM, Stalker TH, LaRue E, Kochert G: Genetic variation detectable with molecular markers among unadapted germplasm resources of cultivated peanut and related wild species. Genome. 1991, 34: 1013-1020.View ArticleGoogle Scholar
- He GC, Prakash S: Identification of polymorphic DNA markers in cultivated peanut (Arachis hypogaea L.). Euphytica. 1997, 97: 143-149. 10.1023/A:1002949813052.View ArticleGoogle Scholar
- He G, Meng R, Gao H, Guo B, Gao G, Newman M, Pittman R, Prakash CS: Simple sequence repeat markers for botanical varieties of cultivated peanut (Arachis hypogaea L.). Euphytica. 2005, 142: 131-136. 10.1007/s10681-005-1043-3.View ArticleGoogle Scholar
- Singh AK, Sivaramakrishnan S, Mengesha MH, Ramaiah CD: Polygenetic relation in section Arachis based on seed protein profile. Theor Appl Genet. 1991, 82: 593-597.PubMedGoogle Scholar
- Singh AK, Santosh G, Jambunathan R: Phylogenetic relationship in the genus Arachis based on seed protein profiles. Euphytica. 1994, 74: 219-225. 10.1007/BF00040404.View ArticleGoogle Scholar
- Bianchi-Hall CM, Keys RD, Stalker TH: Use of seed protein profiles to characterize peanut cultivars. Peanut Sci. 1994, 21: 152-159.View ArticleGoogle Scholar
- Thiellemmet H, Bahrnman N, Damerval C, Plomion C, Rossignol M, Santony V, Vienne D, De Zivy M: Proteomics for genetic and physiological studies in plant. Electrophoresis. 1999, 20: 2013-2026. 10.1002/(SICI)1522-2683(19990701)20:10<2013::AID-ELPS2013>3.0.CO;2-#.View ArticleGoogle Scholar
- Park OK: Proteomic studies in plants. J Biochem Mol Biol. 2004, 37: 133-138.PubMedView ArticleGoogle Scholar
- Shokraii EH, Esen A, Mozingo RW: Relation of a 36,000-dalton arachin subunit to blanch ability in peanut (Arachis hypogaea L.). J Agric Food Chem. 1985, 33: 1114-1116. 10.1021/jf00066a024.View ArticleGoogle Scholar
- Boldt A, Fortunato D, Conti A, Petersen A, Ballmer-Weber B, Lepp U, Reese G, Becker W-M: Analysis of the composition of an immunoglobulin E reactive high molecular weight protein complex of peanut extract containing Ara h 1 and Ara h 3/4. Proteomics. 2005, 5: 675-686. 10.1002/pmic.200401150.PubMedView ArticleGoogle Scholar
- Guo BZ, Liang XQ, Maleki SJ, Chung SY, Holbrook CC, Ozias-Akins P: Characterization of five seed-proteins missing in one peanut genotype and the allergic nature of these proteins. Proceeding of the thirty-sixth annual meeting of the American Peanut Research and Education Society (APRES). San Antonio, Texas. 2004, 28-July 13–16, 2004Google Scholar
- Kang I-H, Gallo-Meagher M: Cloning and characterization of a novel ara h 3, a major peanut (Arachis hypogaea L.) allergen gene. GenBank. 2004, National Center for Biotechnology Information, Accession Number AY618460, [http://www.ncbi.nlm.nih.gov].Google Scholar
- Basha SM, Pancholy SK: Polypeptide composition of arachin and non-arachin proteins from early bunch peanut (Arachis hypogaea L.) seed. Peanut Sci. 1981, 8: 82-88.View ArticleGoogle Scholar
- Krisha TG, Mitra R: Arachin polymorphism in groundnut (Arachis hypogaea L.). Phytochemistry. 1987, 26: 897-902. 10.1016/S0031-9422(00)82313-4.View ArticleGoogle Scholar
- Damerval C, de Vienne D, Zivy M, Thiellement H: The technical improvements in two-dimensional electrophoresis increase the level of genetic variation detected in wheat-seedling proteins. Electrophoresis. 1986, 7: 52-54. 10.1002/elps.1150070108.View ArticleGoogle Scholar
- Liang XQ, Guo BZ, Holbrook CC: Identification of peanut seed-storage proteins associated with resistance against Aspergillus flavus infection and aflatoxin production. Proceeding of the thirty-sixth annual meeting of the American Peanut Research and Education Society (APRES). San Antonio, Texas. 2004, 74-July 13–16, 2004Google Scholar
- Bradford M: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principal of protein-dye binding. Anal Biochem. 1976, 72: 248-254. 10.1016/0003-2697(76)90527-3.PubMedView ArticleGoogle Scholar
- Laemmli UK: Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970, 227: 239-251. 10.1038/227680a0.View ArticleGoogle Scholar
- Guo BZ, Brown RL, Lax AR, Cleveland TE, Russin JS, Widstrom NW: Protein profiles and antifungal activities of kernel extracts from corn genotypes resistant and susceptible to Aspergillus flavus. J Food Prot. 1998, 61: 98-102.PubMedGoogle Scholar
- Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local alignment search tool. J Mol Biol. 1990, 215: 403-410. 10.1006/jmbi.1990.9999.PubMedView ArticleGoogle Scholar
- Rabjohn P, Helm EM, Stanley JS, West CM, Sampson HA, Burks AW, Bannon GA: Molecular cloning and epitope analysis of the peanut allergen Ara h3. J Clin Invest. 1999, 103: 535-542.PubMedPubMed CentralView ArticleGoogle Scholar
- Branch WD: Registration of 'Georgia Green' peanut. Crop Sci. 1996, 36: 806.View ArticleGoogle Scholar
- Guo B, Chen H, Dang P, Chen X, Liang X, Holbrook C: Identification of a new potential peanut seed allergen iso-Ara h3. GenBank. 2006, National Center for Biotechnology Information, Accession Number DQ855115, [http://www.ncbi.nlm.nih.gov].Google Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.