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Journal of Plant Physiology 167 (2010) 848–854

Contents lists available at ScienceDirect

Journal of Plant Physiology

0176-16

doi:10.1

Abbre

sulphonn Corr

E-m

journal homepage: www.elsevier.de/jplph

SHORT COMMUNICATION

Detection of novel trypsin inhibitors in the cotyledons ofPhaseolus vulgaris seeds

Marta Alves a, Ines Chaves a, Dina Carrilho a, Manuela Veloso b, Candido Pinto Ricardo a,n

a Instituto de Tecnologia Quımica e Biologica, Universidade Nova de Lisboa, Av. da Republica – EAN, 2780-157 Oeiras, Portugalb Instituto Nacional de Recursos Biologicos, Quinta do Marques, 2784-505 Oeiras, Portugal

a r t i c l e i n f o

Article history:

Received 13 November 2009

Received in revised form

5 January 2010

Accepted 6 January 2010

Keywords:

a-Amylase inhibitors

Lectins

Phaseolus vulgaris

Trypsin inhibitors

Two-dimensional electrophoresis reverse

zymography

17/$ - see front matter & 2010 Elsevier Gmb

016/j.jplph.2010.01.007

viations: CHAPS, 3-[3-cholamidoproplyl(dim

ate; TIs, trypsin inhibitors; 2-DE, two-dimen

esponding author. Tel.: +351 214 449 654; fa

ail address: ricardo@itqb.unl.pt (C.P. Ricardo)

a b s t r a c t

Protease inhibitors play important roles in plants in association with stress. Trypsin inhibitors (TIs) in

particular are known to act as protective agents against insect and pathogen attacks. The growing

relevance of these inhibitors requires expedited techniques for their detection. By using the two-

dimensional electrophoresis (2-DE) reverse zymography technique, we identified, from the crude

extract of bean seeds, nine novel polypeptides that showed trypsin inhibitor activity. One of these

polypeptide inhibitors yielded no homology in the database, which can be an indication that we are

found a new protein with unique TI properties. The remaining showed homology with proteins

annotated in the UniProt database and form, together with a Kunitz type inhibitor, a new TI cluster for

Phaseolus spp. Three of these polypeptides showed additional high homology with lectins, likely

indicating that they have lectin properties, while the other five showed high homology with a-amylase

inhibitors, indicating that they probably have a dual inhibitory effect against trypsin and the a-amylase

enzyme. These bifunctional inhibitors can be highly useful for crop management, since the two

inhibitory activities are important for plants when coping with pathogen and pest attacks.

& 2010 Elsevier GmbH. All rights reserved.

Introduction

There are thousands of legume species, but among them, thecommon bean (Phaseolus vulgaris L.) seed is the primary source ofprotein for human and domesticated animal consumption. Thisseed is a good source of vitamins, minerals and complexcarbohydrates. In addition to these nutritional components, italso contains some non-nutritional factors, such as trypsininhibitors (TIs), which, in the raw seed, have the ability to inhibitthe proteolytic activity of the digestive enzymes (Liener, 1962).Since heat denaturation normally inactivates these proteins, theymay even play a positive nutritional role due to their highsulphur-containing amino acid content (Ryan, 1990). In plants,these protease inhibitors are known to act in defensive mechan-isms against biotic stress. It has been previously reported that atobacco transgenic plant expressing a cowpea TI gene showedplant-derived insect control (Hilder et al., 1987). Soybean TI hasalso been shown to reduce the growth of Helicoverpa armigera, aglobally problematic crop pest (Johnston et al., 1993; Wang andQin, 1996), and it was observed that the overexpression of corn TI

H. All rights reserved.

ethylammonio)]-1-propane-

sional electrophoresis

x: +351 214 433 644.

.

in Escherichia coli caused growth inhibition of plant pathogenicfungi (Chen et al., 1999). Despite P. vulgaris being an importantcrop, only a few TIs have been described for this plant and themajority of them belong to the Bowman–Birk family (Galassoet al., 2009). It is therefore important to investigate new TI in P.

vulgaris.With respect to abiotic stresses, TIs are also known to react in

response to drought stress, salt stress and wounding (Gosti et al.,1995; Kim et al., 2001). Thus, TIs are important candidates for themodern biotechnology used in crop management. Therefore,improved techniques for new TIs isolation and identification areneeded. By using an improved reverse zymography technique, wewere able to identify nine novel P. vulgaris TIs from a crudeprotein mixture of cotyledon seeds.

Materials and methods

Plant material

Common bean (Phaseolus vulgaris L. cv. 233H) seeds wereobtained from the bean collection of Departamento de RecursosGeneticos e Melhoramento (Instituto Nacional de RecursosBiologicos, Oeiras, Portugal). The seed cotyledons were separatedfrom the embryonic axis and carefully peeled.

ARTICLE IN PRESS

Fig. 1. 2-DE reverse zymography gels from P. vulgaris seeds showing detection of polypeptides with TI activity. The protein samples resulting from the water extraction of

the seed cotyledons were separated by 2-DE using a pH range of 3–10 NL: (a) colloidal Coomassie blue stained gel, (b) details of the gel copolymerized with azoalbumin and

colloidal Coomassie blue stained after incubation in a buffer solution with 0.005% (w/v) trypsin. The marked spots (TI1–TI9) refer to those spots having TI activity.

M. Alves et al. / Journal of Plant Physiology 167 (2010) 848–854 849

ARTICLE IN PRESS

Table 1MS/MS obtained sequences of the polypeptides with TI activity for database homology search. Polypeptide IDs (as marked in the 2-DE gel of Fig. 1a), protein identifications,

accession numbers, MMs and PIs are indicated. Sequences showing high homology to the respective protein annotated in the databases (EMBL servera) are in bold.

SptID

Protein ID (Ac. no.)a [PI; MM]b

Ionc Sequenced

TI1 Alpha-amylase inhibitor (Fragment) (P. coccineus) (Q9FPW9) [PI 4.7;

MM 7.3 kDa]

2363.738 LSGGGGGDSPGLSLK-LSGGGGGETPNDLK-LSGGGGGVGCDAAHK-LSGGNGVGCDAAHK-

LSGGGGGTEPNDLK

2404.361 LSLDVNNNDLK-LSLDVNNGGDLK-LSLDVNNGDGLK-LSLDVNNDGGLK-LSLDVGGNNDLK

2459.52 VVPRFPR-VVPRMPR-TPPRFPR-VVPVGFPR-VVPGVFPR

2493.943 FVQCTSALPGHK-FVQCSTALPGHK-FVQCGTSALPGHK-FVQCGSTALPGHK-

FVQCSGTALPGHK

2526.121 LSLDVNLDDLK-LSLDVCGPAELK-LSLDVCGPPCLK-LSLDVCAPADLK-LSLDVCPAADLK

2542.852 EGPSLSLDCGCSMCNAEVR-EGPSLSLDCGSCMCNAEVR-EGPSCPLAMGCSMCNAEVR-

EGPSCPLTCASCMCNAEVR-VSPSGCCAGSCSLSCNAEVR2545.153 CSSVSLCVTGGVR-SCSVSLCVTGGVR-CGSSVSLCVTGGVR-GCSSVSLCVTGGVR-

TGKFSLCVTGGVR

2619.233 VFGCYAHDYNR-VFCYAHDYNR-VFGYCAHDYNR-VFCGYAHDYNR-VFYCAHDYNR

2794.595 HVVPAAK-VHVPAAK-VVHPAAK-TPVPVPK-VVVPVPK

2847.525 EGEGGCMDMSQLGCPEVR-EGEGGCGMDMSQLGCPEVR-EGESACACTFSQLGCPEVR-

EGEGGCMDSMQLGCPEVR-EGESAACCTFSQLGCPEVR

2908.644 KGDDVMAQCSADMNTNVR-KGDDVMAGACSADMNTNVR-KGDDVFAAGCSADMNTNVR-

KGDDVMAAGCASDMNTNVR-KGDDVMAAGCGTDMNTNVR

3122.122 FAPSSGCAGGTLQK-AFPSSGCAGGTLQK-MAPSSGCAGGTLQK-FAPACGCAGGTLQK-

AFPACGCAGGTLQK

3294.358 AMYSAWSSCK-AFYSAWSSCK-MAYSAWSSCK-FAYSAWSSCK-AFYSAQPPER

3303.763 FPFGGPAAYDPVGAEPSK-FPFGGPAAYDVPGAEPSK-FPFGGPAADYVPGAEPSK-

FPFGGPAADYPVGAEPSK-FPFGGPAAYDPVQEPSK

3339.868 FYASAPLGLAR-YFASAPLGLAR-YAFSAPLGLAR-FFSSAPLGLAR-FSFSAPLGLAR

3356.661 ASGSTAGNSHVLLNAEVR-SAGSTAGNSHVLLNAEVR-GTGSTAGNSHVLLNAEVR-

TGGSTAGNSHVLLNAEVR-ASGSTAGNHSVLLNAEVR3407.556 AFYSAWSSCK-AFYSAPAPGER-AFYSASDSSCK-AFYSADSSSCK-AMYSAWSSCK

3463.385 TGEAAASGCPVAVACCCSK-TGEAAASGCGPVAVACCCSK-TGEAAASGPCVAVACCCSK-

TGEAAASGGCPVAVACCCSK-TGEGDQGGTVVAVACCCSK

3492.461 CCCCECTDPTK-CCCCCETDPTK-CCCECCTDPTK-CCCCECESPTK-CCCCCEESPTK

TI2 Alpha-amylase inhibitor-4 precursor (Alpha-AI-4) (Lectin) (Fragment)

(P. vulgaris) (P93504) [PI 5.0; MM 27.1 kDa]

2109.48 KLAASGGK-LKAASGGK-QLAASGGK-APAGSSGGK-AGLAASGGK

2261.515 LSDGPDGGAELK-LSDGPDGGEALK-LSDGPDGGANAGK-LSDGPDGGPCLK-LSDGPDGQELK

2424.908 VVPCAEPR-VVLSAEPR-VVPCATHK-VVTVAEPR-TPPCATHK

2441.59 NSLDVDNNDLK-GGSLDVDNNDLK-NSLDVDNGGDLK-NSLDVDGGNDLK-

NSLDVDNGDGLK

2500.764 VTLNVTTGRGLK-LSLNVTTGRGLK-SLLNVTTGRGLK-VTLENATGRGLK-VTLNLSTGRGLK

2513.542 TVLEEGDASPLK-LSLEEGDASPLK-TVLEEGADSPLK-VTLEEGDASPLK-TVLEEGGESPLK

2610.959 DGMDSAPGACHNK-DLCCSAPGACHNK-DCESSAPGACHNK-DGMDSAGPACHNK-

DGDMSAPGACHNK

2643.317 VATVEPAR-VATVEPVK-VATLDPAR-VACPEAPR-LGTVEPAR

2656.758 ASNAKCPYCLVR-ASGGAKCPYCLVR-GTNAKCPYCLVR-TGNAKCPYCLVR-ASNAAGCPYCLVR

2741.537 SYAPAHASK-YSAPAHASK-HPAPAHASK-CFAPAHASK-SYAPAAHSK

2821.184 FVSVSLSNLATGK-VFSVSLSNLATGK-FVSVSLSGGLATGK-VFSVSLSGGLATGK-

MVSVSLSNLATGK2862.812 AFYSAPLQLR-AFYSAPLGALR-FAYSAPLQLR-AFYSAGHQLR-AFYSAHGQLR

3309.56 LVGSYPALTVFVSNCSK-VLGSYPVADVFVSNCSK-LVGSYPVADVFVSNCSK-

DPGSYPVGEVFVSNCSK-DPGSYPVEGVFVSNCSK

3324.964 AFYSANHALR-FAYSANHALR-AFYSANAHLR-AMYSANHALR-AFYSAPAVALR

3334.41 KPPKPVVK-KKVPVPPK-KVVPKPPK-KVKPVPPK-KVVKPPPK

3500.296 QGLGVAVCGPEEGKPESK-AGGLGVAVCGPEEGKPESK-GAGLGVAVCGPEEGKPESK-

KGLGVAVCGPEEGKPESK-QGLGVAVCGPSGNGQPESK3560.31 DVVGGSGSGTLAHSGGLLGK-DVVGGSGGSTLASHGGLLGK-DVVGGSGGSLTASHGGLLGK-

DVVGGSGGSTLAHSGGLLGK-DVVGGSGGSATLSHGGLLGK

3602.784 ASSADSLCDGPDGK-ASSADSLCDGDPGK-GTSADSLCDGPDGK-SASADSLCDGPDGK-

TGSADSLCDGPDGK

4745.727 VAAVCGANK-GLAVCGANK-AVAVCGANK-LGAVCGANK-VAAVCGAGGK

TI3 Alpha-amylase inhibitor (P. maculatus) (Q40915) [PI 5.1; MM 29.3 kDa]

1893.708 ATNMVSEK-ATNFVSEK-ATGGMVSEK-ATNMVTDK-ATGGFVSEK

1965.7 ATNFVESK-TANFVESK-GDNFVESK-DGNFVESK-ATNFVSEK

2279.134 LASLDHGK-LASLDLPK-LATVDHGK-LASLEVPK-LASDLHGK

2320.022 EGDTLFPK-GEDTLFPK-ADDTLFPK-DADTLFPK-EGDTLMPK

2353.398 FVEAEDGSER-MVEAEDGSER-FAGGNEDGSER-FVEAEDGCLR-FANNEDGSER

2397.493 LSLDVNNATGLK-LSLDVNNAASLK-LSLDVNNAGTLK-LSLDVNNASALK-LSLDVGGNATGLK

2436.949 VATVEVPK-VATVEPVK-VATASHLK-LGTVEVPK-GLTVEVPK

2443.44 LSNNVNLDDLK-LSNGGVNLDDLK-LSGGNVNLDDLK-LSGNGVNLDDLK-LSLDVNLDDLK

2498.683 VATVLVLK-LGTVLVLK-AVTVLVLK-GLTVLVLK-VATVDPLK

2504.726 LSLDVNNTAGLK-LSLDVNNATGLK-LSLDVNNDGGLK-LSLDVNNGDGLK-LSLDVNGGTAGLK

2564.272 LSLDVNNATGLK-LSLLTNNATGLK-LSLTLNNATGLK-LSLDVNNDGGLK-LSLDVNGGATGLK

M. Alves et al. / Journal of Plant Physiology 167 (2010) 848–854850

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Table 1 (continued )

SptID

Protein ID (Ac. no.)a [PI; MM]b

Ionc Sequenced

2576.522 VPAGGSYVDGGTHGK-VPAGSGYVDGGTHGK-VPAGGSYTLGGTHGK-

VPAGGSYLTGGTHGK-VPAGGSYDVGGTHGK

2627.645 LSLDVNTAGNLK-LSLDVNTAGGGLK-LSLDVCEKPLK-LSLDVNTANGLK-LSLDVNMVPLK

2691.877 AFYSAPLKLR-AFYSAPLGALR-FAYSAPLKLR-FAYSAPLGALR-AFYSAPLQLR2691.877 GLVTSLPR-LGVTSLPR-VAVTSLPR-AVVTSLPR-GLVTEAPR

2740.812 GYLGGVSSNPSGTK-YGLGGVSSNPSGTK-GYLGGVSSNPCNR-YGLGGVSSNPCNR-

GYLGGVSSGGPSGTK

2872.045 AFYSAPTPRR-AFYSAPVVRR-AFYSAPPTRR-AFYSATPPRR-FAYSATPPRR

3340.144 VHESASAFSSK-HVESASAFSSK-MAYSACWSSK-MAYSACSDSSK-FAYSACWSSK

3448.083 CDDVNSK-TNTTESK-CDVDNSK-CDDVGGSK-TNAYPSK

TI4 Alpha-amylase inhibitor 2 precursor (Alpha-AI-2) (Alpha-AI-2) (Lectin)

(P. vulgaris) (Q41114) [PI 5.2; MM 26.6 kDa]

2200.761 VAVTEAPR-GLVTEAPR-AVVTEAPR-LGVTEAPR-VAVETAPR

2251.241 VVPMKHK-TPPMKHK-VVPMQHK-VVPCATHK-VVPFKHK2386.292 LSLDVNNNDLK-LSLDVGGNNDLK-LSLDVNNGGDLK-LSLDVNGGNDLK-LSLDVVENDLK

2454.386 SLARHGAK-APCRHGAK-DRRHGAK-ALSRHGAK-SALRHGAK

2469.848 VMSVSLSNSPTGK-VFSVSLSNSPTGK-MVSVSLSNSPTGK-FVSVSLSNSPTGK-

VFSADLSNSPTGK

2478.091 YKKCLGK-KYKCLGK-YVFVALGK-APALGGLLGK-YFVVALGK

3028.331 AFYSAPLGLVK-AFYSALPGLVK-AFYSAHGGLVK-AFYSAGHGLVK-AFYSAHLNVK

3179.079 FAGTYSSDSCK-FATGYSSDSCK-FASAYSSDSCK-FAASYSSDSCK-MAGTYSSDSCK

TI5 Alpha-amylase inhibitor 1 precursor (Alpha-AI-1) (Alpha-AI-1) (Lectin)

(P. vulgaris) (P02873) [PI 5.0; MM 27.2 kDa]

1846.018 ATNCSGASK-ATNCSQSK-ATNGCSGASK-ATGGCSGASK-ATNCGSGASK

1990.62 EASPATLSNR-LSSPATLSNR-TVSPATLSNR-SLSPATLSNR-VTSPATLSNR

2296.036 LATVGGPGGK-LATVGGGPGK-LATVGGNPK-ALTVGGPGGK-ALTVGGGPGK

2300.183 NGDMGEEK-GGGDMGEEK-NGDMGETR-GGGDMGETR-NGDFGEEK

2354.093 KLLSKRFK-LKLSKRFK-LLKSKRFK-KLLKSRFK-LKLKSRFK

2392.49 VATVEAPR-VATVEVPK-VATVEPVK-VAAEEAPR-VAVTEAPR

2409.941 AASPTHAGK-AASVVHAGK-AASTPHAGK-LATAAHAGK-ALTAAHAGK

2512.934 VAVTEAPR-VATVEPVK-VATVEAPR-VATVEVPK-VATVEPAR

2526.283 VFSVSLSNSPGTK-VFSVSLSNSPTGK-VFSVSLSNPSGTK-VFSVSLSNPSTGK-

VMSVSLSNSPTGK

2610.37 TPLQGYAK-TGHQGYAK-TPAAVGYAK-TPLAGGYAK-TPLKGYAK

2620.961 EASSVGSGGK-EASSRSGGK-TVSSRSGGK-VTSSVGSGGK-SLSSVGSGGK

2627.199 MCHCCDNR-MCCHCDNR-MHCCCDNR-MCHCDCNR-MHCCDCNR

3123.898 LPLPGGK-LLPPGGK-APGLPVK-LPLPNK-LLPPNK

TI6 Mannose lectin FRIL (P. vulgaris) (Q9M7M4) [PI 5.4; MM 31.1 kDa]

1739.594 LSYANSSK-LSYAGGSSK-LSYAMLR-LSYANACK-LSYANCAK

1974.374 SMGDVNTDSR-SFGDVNTDSR-SMDGVNTDSR-SMGDVGGTDSR-SFGDVGGTDSR

1989.774 VGLSASYAGPR-VGLSASAYGPR-VGLSAHPAGPR-VGLSASSMGPR-VGLSASSFGPR

2271.003 YLSAFNR-YSLAFNR-YVTAFNR-YLSAMNR-LYSAFNR

2331.633 VVTVSLPR-TPTVSLPR-VVTAYCSK-VVTVEAPR-TPTVEAPR

2404.713 VAVTAEPR-VATVAEPR-VATVAPER-VATVTVPR-VAVTTVPR

2659.859 VAVWVPR-VAVTLSPR-VAVTVTPR-VAVTAEPR-VAVTSLPR

2662.819 YSAPLQLR-YSAPLKLR-YSAPLAGLR-YSAPLGALR-YSAPLTPAK

2760.805 YGLDAAK-FVSDAAK-YTSPAAK-VFSDAAK-YGGGGKAK

2909.99 AFYSAPLGALR-FAYSAPLGALR-AMYSAPLGALR-AFYSALPGALR-MAYSAPLGALR

TI7 Mannose lectin FRIL (P. vulgaris) (Q9M7M4) [PI 5.4; MM 31.1 kDa]

1724.949 LSYNGTSK-LSYGGGTSK-LSYNSASK-LSYGGSASK-TVYNGTSK

1846.02 LSSYNASK-LSPHNASK-LSHPNASK-LSFCNASK-LSCFNASK

1915.871 VGLSASTEGEK-VGLSASTADEK-VGLSASTDAEK-VGLSASTGEEK-VGLSASDDGEK

1976.465 VGLSASTGEEK-VGLSASSAEEK-VGLSASGTEEK-VGLSASTGETR-VGLSASASEEK

1978.557 RTVASTEGDR-VGTVASTEGDR-RTGLSTEGDR-VGLSASTEGDR-RVTASTEGDR

2236.208 LGVTGSTFGPR-LGVASSTFGPR-VAVTGSTFGPR-AVVTGSTFGPR-GLVTGSTFGPR

2257.037 VATVEAPR-VATVEPAR-VAEAEAPR-VACPEAPR-VAPCEAPR

2313.526 VVPCTAHK-VVPCATHK-TPPCTAHK-VVAETAHK-VPVCTAHK

2317.668 VATVEVPK-VATVSGHR-AVTVEVPK-LGTVEVPK-GLTVEVPK

2374.333 VVTVLSPR-VVTVAEPR-TPTVLSPR-VVTVEAPR-VVAELSPR

2378.476 VATVEAPR-GLTVEAPR-LGVTEAPR-GLVTEAPR-LGTVEAPR

2401.208 RNVSLPR-VGNVSLPR-GVNVSLPR-RGGVSLPR-VGGGVSLPR

2439.323 VATGTYCK-AVTGTYCK-GLTGTYCK-LGTGTYCK-TVAGTYCK

2561.815 VAPCSLPR-VAPCEAPR-LGCPSLPR-VACPSLPR-LGPCSLPR

2643.056 AFSYAPVAALR-FASYAPVAALR-AFPHAPVAALR-AFHPAPVAALR-AFSYAPVVPSK

TI8 Mannose lectin FRIL (P. vulgaris) (Q9M7M4) [PI 5.4; MM 31.1 kDa]

1893.209 VGLSASTGEEK-VGLSASTEGEK-VGLSASTADEK-LGVSASTGEEK-VGLSASTSVEK

1954.71 VGLSASTMAPK-LGVSAELNMK-LGVSASTMAPK-RLSASTMAPK-VGLSAELNMK

TI9 Unknown

2213.451 YNMPTNQGGLQAK-YGGMPTNQGGLQAK-NYMPTNQGGLQAK-NYFPTNQGGLQAK-

YNMTPNQGGLQAK

2342.189 CASPDPR-ACSPDPR-DVGACHK-DVGAHCK-DVGHCAK

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Table 1 (continued )

SptID

Protein ID (Ac. no.)a [PI; MM]b

Ionc Sequenced

2366.887 YLVFSGGPTLGPK-YLVSFGGPTLGPK-YLVMSGGPTLGPK-LYVAYGGPTLGPK-

YLVAYGGPTLGPK

2427.908 LYVDELAMPLR-LYVDNQAMPLR-LYVDGGQAMPLR-LYVDNAGAMPLR-LYVDNKAMPLR

2442.196 VAVTEAPR-VATVEPAR-VATVEVPK-VATVEPVK-AVVTEAPR

2484.545 VVTVEAPR-VVTVEPAR-TPTVEAPR-TPTVEPAR-VVTVSHGR

2520.71 TLESPGVASAK-TLESPGLGSAK-TLESPGGLSAK-TLESPGAVSAK-DVESPGLGSAK

2562.99 VAPCAEPR-VAPCEAPR-LGPCAEPR-LGPCEAPR-GLPCAEPR

2673.322 YTAVWSK-GDYVWSK-ATYVWSK-TYAVWSK-GYDVWSK

2686.786 VATVEPVK-GLTVEPVK-AVTVEPVK-LGTVEPVK-VATVEVPK

a Proteins were identified by EMBL server http://dove.embl-heidelberg.de/Blast2/msblast.html.b pI and MW were calculated in Expasy database (http://www.exapsy.org).c Ion precursor (mass/charge unit).d Sequences obtained by PEAKS software.

M. Alves et al. / Journal of Plant Physiology 167 (2010) 848–854852

Protein extraction

The cotyledons were ground, washed for four periods of30 min with �20 1C acetone (3 mL/g) and defatted twice forperiods of 30 min with hexane (2 mL/g). After centrifugation at27,200g for 15 min at 4 1C, the final dried pellet was mixed withinsoluble polyvinylpyrrolidone [1% (w/w)] and extracted withdouble-distilled water [1/6 (w/v)]. After overnight extraction,with stirring at 4 1C, the sample was centrifuged at 27,200g for15 min at 4 1C and the supernatant was collected (Chougule et al.,2003).

Two-dimensional electrophoresis (2-DE) reverse zymography method

for TIs visualization

The protein content of the samples was determined accordingto the Bradford method, modified by Ramagli (Bradford, 1976),using bovine serum albumin as a standard. The proteins of thecrude extract were first separated by Isoelectric focusing in theIPGphor system with 3–10 NL IPG strips (GE Healthcare) loadedwith 250 mg of protein. The solubilization buffer had 8 M urea, 2%(w/v) 3-[3-cholamidoproplyl(dimethylammonio)]-1-propanesul-phonate (CHAPS) and 0.5% (v/v) IPG buffer 3–10 NL (GEHealthcare) and the IEF conditions were 30 V for 12 h, followedby 200 V for 1 h, 500 V for 1.5 h, 1000 V for 1.5 h and 8000 V for6.5 h, at 20 1C. The IPG strips were equilibrated for 15 min in abuffer solution containing 0.05 M Tris–HCl pH 8.8, 6 M urea, 30%(v/v) glycerol and 2% (w/v) SDS. The second dimension wasperformed on polyacrylamide gels of 150�120�1.0 mm3, 15% Tand 3.3% C (Laemmli, 1970). Two sets of gels were used, onewithout and the other one with azoalbumin [0.1% (w/v)]copolymerized with the polyacrylamide. The electrophoresiswas carried out at 4 1C with 30 mA until the sample entered thegel, and then the current was raised and maintained constant at60 mA until the end of the run.

After electrophoresis, the azoalbumin gel was immersed in awashing solution with 2.5% (v/v) Triton X-100 for 15 min toremove SDS and then incubated overnight at room temperature,in a buffer solution [0.1 M Tris–HCl at pH 7.6, 0.2 M NaCl, 0.01 MCaCl2, 0.02% (v/v) Brij-35] to which trypsin [0.005% (w/v)] wasadded. The two sets of gels were stained with colloidal CoomassieG-250 (Neuhoff et al., 1985). The undigested azoalbumin spotsthat resulted from the presence of TIs are observed in theazoalbumin gels, while all the polypeptides present in the crudeextract are visualized in the other gel. Both gels were scannedusing the ImageQuant v3.3 densitometer (Molecular Dynamics),and for the detection and matching of the spots the image analysis

software (ImageMaster 2D Platinum Software 5.0, GE Healthcare)was used.

MS/MS analysis

The spots detected as TIs were excised from colloidalCoomassie G-250 stained gels and analyzed by MALDI-TOF-TOFat the University of Durham School of Biological and BiomedicalSciences, Durham (UK). Mass peptide separation and sequencingwas carried out on an Applied Biosystems QSTAR PULSARiTquadrupole time of flight mass spectrometer coupled to an LCPackings UltiMateT nano HPLC workstation. TOF MS spectra werecollected between the mass range 100–2000 amu and precursorion selection and product ion spectra were generated usingApplied Biosystems BioAnalystT software’s fully automatedswitching and acquisition procedures. Only multiple chargedprecursor ion species were selected for fragmentation and peptidesequencing. The de novo sequences of the peptides were obtainedusing PEAKS software (Ma et al., 2003). The similarity searcheswere performed with the first five partial amino acid sequencesper fragment of the polypeptide, by using the EMBL server (http://dove.embl-heidelberg.de) with the standard settings (Shevchenkoet al., 2001).

Neighbor-joining tree

For the neighbor-joining tree analysis, the 55 entries for TI inPhaseolus spp. (P01056, P01058, P01062, P01060, P01061, P83311,P81484, P81483, Q93YY2, Q708V8, Q708V7, Q05G17, Q8L6E8,Q8L6E6, Q84RP7, Q8L6E7, A8KRM2, A4Q9H3, Q708V9, Q05G16,A0JKD0, Q05G15, Q05G14, A0JKC1, Q9FUP3, A4Q9H4, A4Q9H2,A4VAS0, B3W6M4, B0JFB2, B3GW40, B1VK41, B2RGA3, B0JFB3,A2VBN4, A0JKC2, A0AQW6, A0JKC9, A4D0C9, Q08G55, A4D0C8,B2RGA4, B2RG92, B2RG95, B2RGA5, B2RG94, B2RG93, A0JKC8,A0JKC7, B2RGA0, B2RGA2, B2RG99, B2RG91, B2RGA1 andB2RGA6) described in the UniProt database (http://www.uniprot.org – November 2009), and the six entries found for ourTIs (Q9FPW9, P93504, Q40915, Q41114, P02873 and Q9M7M4)were multiple sequence aligned by ClustalW, which wasperformed at http://align.genome.jp/ using the default settingfor the ACCURATE selection.

Results and discussion

From the crude extract of cotyledons of common bean seeds,nine novel TIs, with molecular masses of 20–30 kDa, were

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Fig. 2. Neighbor-joining tree of the Phaseolus spp. polypeptides that have TI activity. The cluster formed by the accession numbers of the TI1–TI8 (identified by the 2-DE

reverse zymography method) is shaded in grey. The multiple sequence alignment with ClustalW was used.

M. Alves et al. / Journal of Plant Physiology 167 (2010) 848–854 853

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M. Alves et al. / Journal of Plant Physiology 167 (2010) 848–854854

evidenced by the 2-DE reverse zymography method (Fig. 1). Themajority of the TIs that have been described for Phaseolus spp.have molecular weights lower than 10 kDa and belong to theBowman–Birk family (Galasso et al., 2009). After identification byMS/MS techniques (Table 1), we observed that five (TI1–TI5) ofthe novel TIs showed high homology with a-amylase inhibitorsfrom Phaseolus spp., and that TI2, TI4 and TI5 were additionallydescribed as lectins. For TI6–TI8, only high homology with lectins(mannose lectin FRIL proteins) was detected, while for theremaining inhibitor (TI9), no homology was found in thedatabases, probably because this is a new protein with uniqueTI properties.

Since TI1–TI5 showed homology with a-amylase inhibitors,they appear to be bifunctional inhibitors. Although not previouslyincluded for Phaseolus spp. in the UniProt database, they havebeen reported for other plant species (Ryan, 1990). Indeed, fourKunitz-like a-amylase inhibitors were recently described for thelegume tree Delonix regia (Alves et al., 2009). Similarly, Phaseolus

spp. TIs with lectin properties have not been annotated in thedatabase, but in the legume tree Peltophorum dubium seeds, a TIwith lectin-like properties was found (Troncoso et al., 2003).

When a neighbor-joining tree analysis was performed (Fig. 2),we observed that TI1–TI8 formed a new Phaseolus spp. cluster,together with the Kunitz type inhibitor (Q9FUP3) already presentin the UniProt database. It should be noted that, in the neighbor-joining tree, this is a quite different cluster from that of theBowman–Birk type inhibitors, which are TIs with distinctcharacteristics (Ryan, 1990). The scarcity of information in thedatabases for Phaseolus spp. Kunitz and bifunctional inhibitors isevident, as only one entry for a Kunitz type inhibitor (Q9FUP3)was found, and for the bifunctional inhibitors, only entries fortrypsin/chymotrypsin or elastase have been annotated. Thus,much information about Phaseolus spp. TI needs to be gathered.The 2-DE reverse technique, previously described for theseparation of a purified TIMP-2-like protein (Lødemel et al.,2004), proved to be a useful tool for the separation and

identification, from a crude protein extract, of nine novel TIsfrom P. vulgaris seeds. The additional information on bifunctionalactivity for the identified TIs can be useful for crop management,since a-amylase inhibition is also described to act againstpathogens and pests that attack plants (Ryan, 1990).

Acknowledgement

This work was supported by a fellowship from ITQB (006/BIC/2004). The help of Dr. Iain Rogers, Bioinformatics Solutions Inc.,Canada, in the analysis of the MS/MS data with PEAKS software isgratefully acknowledged.

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