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Osmotin purified from the latex of Calotropis procera: Biochemical characterization, biological activity and role in plant defense
Plant Physiology and Biochemistry 49 (2011) 738e743
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Research article
Osmotin purified from the latex of Calotropis procera: Biochemical characterization, biological activity and role in plant defense Cleverson Diniz Teixeira de Freitas a, *, Fábio César Sousa Nogueira b, Ilka Maria Vasconcelos a, José Tadeu Abreu Oliveira a, Gilberto Barbosa Domont b, Márcio Viana Ramos a, * a b
Departamento de Bioquímica e Biologia Molecular da, Universidade Federal do Ceará, Campus do Pici, Cx., Postal 6033, Fortaleza, Ceará, CEP 60451-970, Brazil Departamento de Bioquímica, Instituto de Química da, Universidade Federal do Rio de Janeiro, Rede Proteômica do Rio de Janeiro, Rio de Janeiro, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 June 2010 Accepted 26 January 2011 Available online 4 February 2011
A protein, similar to osmotin- and thaumatin-like proteins, was purified from Calotropis procera (Ait.) R.Br latex. The isolation procedure required two cation exchange chromatography steps on 50 mM Naacetate buffer (pH 5.0) CM-Sepharose Fast Flow and 25 mM Na-phosphate buffer (pH 6.0) Resource-S, respectively. The protein purity was confirmed by an unique N-terminal sequence [ATFTIRNNCPYTIWAAAVPGGGRRLNSGGTWTINVAPGTA]. The osmotin (CpOsm) appeared as a single band (20,100 Da) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as two spots in two-dimensional electrophoresis (pI 8.9 and 9.1). Both polypeptides were further identified by mass spectrometry as two osmotin isoforms with molecular masses of 22,340 and 22,536 Da. The CpOsm exerted antifungal activity against Fusarium solani (IC50 ¼ 67.0 mg mL1), Neurospora sp. (IC50 ¼ 57.5 mg mL1) and Colletotrichum gloeosporioides (IC50 ¼ 32.1 mg mL1). However, this activity was lost when the protein was previously treated with a reducing agent (DTT, Dithiothreitol) suggesting the presence of disulfide bounds stabilizing the protein. The occurrence of osmotin in latex substantiates the defensive role of these fluids. Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords: Amino acid sequence Antifungal activity Fusarium solani Laticifer proteins Mass spectrometry Thaumatin-like protein
1. Introduction Plants are constantly exposed to infection by pathogens such as fungi, bacteria and viruses or are attacked by insects and other herbivorous along their life cycle. However, they react to biotic stresses by triggering a set of defense mechanisms as hypersensitive response and systemic responses, including the synthesis of defensive substances as the pathogenesis-related (PR) proteins [25]. The already identified PR-proteins have been extensively reviewed and currently comprise 17 families, whose classification is based on their similarities in amino acid sequence and enzymatic and mechanistic activity [26]. Various PR-proteins are constitutive proteins that have their synthesis augmented in response to infection [27]. Osmotin- and thaumatin-like proteins belong to family 5 of PRproteins. These proteins have been purified from banana, tomato, grape, barley, wheat, tobacco and sorghum, and exhibit remarkable activity against several fungi [11,19,24]. On the other hand, osmotins- or thaumatin-like proteins from latex sources have not been
Abbreviations: DTT, Dithiothreitol; CpOsm, Calotropis procera osmotin. * Corresponding authors. Tel.: þ55 85 3366 9403; fax: þ55 85 3366 9789. E-mail addresses: [email protected] (C.D. Teixeira de Freitas), vramos@ ufc.br (M.V. Ramos). 0981-9428/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2011.01.027
purified and characterized extensively. The presence of an osmotin in Hevea brasiliensis latex was first reported [23]. Recently, a wound-inducible thaumatin-like protein was purified and characterized in Carica papaya latex [15]. Latex is a term used to describe a fluid with a milky aspect produced constitutively by more than 12,500 plants. This fluid is the cytoplasm of specialized cells called laticifers [16]. They are specialized circulating channels present in roots, barks, leaves and flowers. Latex is released from laticifers immediately after tissue damage caused by mechanical injury. Latices are a rich source of chemicals derived from secondary metabolism such as pyrrolidine alkaloid and sesquiterpene lactone [10]. Besides these proteins involved in basal cellular activities, laticifers are important sites of PR-proteins synthesis. Among purified and characterized PRproteins from latex, chitinases, b-1,3-glucanases, proteinase inhibitors, cysteine proteinases, lectins and anti-oxidative proteins have been reported [2,3,18,28]. These findings on the biochemical composition of laticifer fluids support the hypothesis of their defensive role against predators and invasive microorganisms [9,20]. Calotropis procera (Ait.) R.Br. is a lactiferous Apocynaceae species found in tropical and subtropical regions. This plant produces a large amount of latex which can be easily collected from green leaves and barks. The latex has been reported to comprise
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chitinases, proteinases and anti-oxidative enzymes [8]. Further studies have shown that laticifer proteins are involved in plant defense against crop pests [20,21]. Combined analyses of electrophoresis and mass spectrometry lead to the identification of two osmotins in laticifer proteins of C. procera, named CpOsm. This work reports the purification, partial biochemical characterization and activity of this new protein on three different phytopathogen fungi. 2. Results 2.1. CpOsm purification Three peaks were obtained when laticifer proteins from C. procera were subjected to cation exchange chromatography on a CM-Sepharose Fast Flow column, previously equilibrated with 50 mM Na-acetate buffer, pH 5.0 (Supplementary Fig. S1A). The proteins retained by the matrix and eluted after adding 0.2 M NaCl (PII-CM) presented antifungal activity to Fusarium solani, Neurospora sp. and Colletotrichum gloeosporioides (data not shown). PI-CM (non-retained proteins) was not active while PIII-CM (proteins eluted with 0.3 M NaCl) inhibited only Neurospora sp. Based on these findings, PII-CM was further fractionated and examined for antifungal activity. This fraction was chromatographed again by ion exchange chromatography using a Resource-S matrix equilibrated in 25 mM Na-phosphate buffer, pH 6.0; and it was further washed on a linear ascendant gradient of NaCl. This procedure yielded an unbound fraction and five separated peaks within salt gradient (Supplementary Fig. S1B). The antifungal activity against F. solani was concentrated in the eluted peak with 0.1 M NaCl (P2-R), while a negligible activity appeared in P3-R. The electrophoresis pattern of P2-R showed an unique protein band of a relative mass of 20.1 kDa, which was not reactive to Schiff’s reagent; thus revealing its non glycoprotein nature (Supplementary Fig. S1C and S1D). The presence of carbohydrate on osmotin also was not detected by the phenol-sulfuric acid method. Supplementary Table S1 summarizes the steps to purify CpOsm. 2.2. Physicochemical characterization and identification of CpOsm Automated amino acid sequence determination of P2-R (CpOsm) yielded a unique sequence of 40 N-terminal amino acid residues confirming the homogeneity of the purified protein. This sequence was similar to thaumatin-like proteins or osmotins of different plants, especially with that of H. brasiliensis latex (Table 1). Mass spectrometry analysis of CpOsm exhibited two mono protonated peaks with closely related masses (22,340 and 22,536 Da) suggesting the presence of two isoforms, and a double protonated peak with a mass corresponding to half of those mono protonated (data not shown). The presence of two isoforms with pI corresponding to 8.9 and 9.1 was confirmed by two-dimensional electrophoresis (Fig. 1). The isoform 1 represented about 35% and the isoform 2 about 65%. Both isoforms presented very similar tryptic patterns (Fig. 2). The mass difference of 196 Da between the
Fig. 1. (A) Two-dimensional polyacrylamide gel electrophoresis (12.5%) of CpOsm. Proteins were visualized by staining with 0.1% Coomassie Brilliant Blue R-350. Samples of 30 mg of protein were applied to the strips. (B) Tridimensional view of two spots using ImageMaster 2D PlatinumÒ Software (Amersham Biosciences). The Isoeletric points of the spots 1 and 2 were estimated as 8.9 and 9.1, respectively.
two isoforms can be due to small differences in their primary amino acid sequence (as no carbohydrate moieties have been detected). The detailed differences will be possible after complete sequence of both proteins. It is therefore interesting that a unique N-terminal amino acid sequence comprising almost 40 residues was observed. Both protein spots present in 2-D gels were also examined by tandem mass spectrometry (MALDI-TOFeTOF) after trypsin digestion. Two distinct peptides of each isoform were sequenced (Table 2). These results show that amino acid sequences of both isoforms are very close. All the determined sequences were matched with known osmotin proteins. 2.3. Antifungal activity of CpOsm CpOsm inhibited drastically the germination of the fungi tested (Fig. 3). CpOsm also exhibited mycelial growth inhibitory activity against F. solani, Neurospora sp. and C. gloeosporioides with IC50, ranging from 32.1 up to 67.0 mg mL1 (Supplementary Table S1). Inhibition was clearly seen when assays were performed by agar diffusion (Fig. 3). However, after treating with DTT, a reducing agent, the protein lost its inhibitory activity on the mycelial growth. 3. Discussion Proteins implicated in plant defense have been widely described in literature. They are grouped together on the sole basis of their participation as protective agents against damage caused by predators such as insects or invaders like viruses, bacteria and fungi. These proteins are referred to as Pathogenesis-Related Proteins (PR-Proteins). This diversified group of proteins includes
Table 1 The N-terminal amino acid sequence of an osmotin from Calotropis procera latex compared with other osmotins- and thaumatin-like proteins. Identification
Organism
Sequence
ID (NCBI)
% similarity
E-value
Osmotin PR-protein 5 Osmotin Thaumatin-like protein Osmotin Osmotin
Calotropis procera Helianthus annuus Piper colubrinum Capsicum annuum Arabidopsis thaliana Hevea brasiliensis
1-ATFTIRNNCPYTIWAAAVP-GGGRRLNSGGTWTINVAPGTA-40 22-AVFTIRNNCPYTVWAGAVP-GGGRQLNSGQTWSLTVAAGTA-61 30-ANFLIRNNCPYTVWAAAVP-GGGRRLDRGATWSLNVPAGT-68 22-ATFEVRNNCPYTVWAASTPVGGGRRLDRGQTWTINAPPGTA-62 23-ATFEILNQCSYTVWAAASP-GGGRRLDAGQSWRLDVAAGT–61 1-ATFTIRNNXPYTVWAAASP-GGGRRLDMARIW————————31
e gij20385169 gij161375756 gij15419836 gij21537409 gij32363250
e 87 84 82 79 74
e 4e10 2e09 6e09 5e07 6e07
740
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Fig. 2. Mass spectra of peptides generated by the digestion of the spots 1 (Top) and 2 (Bottom) from Fig. 1a with trypsin. Sample was mixed with 3,5-dimethoxy-4-hydroxycinnamic acid (matrix) dissolved in 0.1% trifluoroacetic acid. A MALDI-TOFeTOF analysis in linear mode was performed on ABI 4700 Proteomics Analyzer.
chitinases, proteolytic enzymes, and proteinase inhibitors [26]. Thaumatins and osmotins belong to PR-5 group. These proteins are representatives of the PR-Proteins group because experimental evidence suggested changes in their mRNA expression level under plant stress [12]. Thaumatin was first observed on Thaumatococcus daniellii and osmotin was first isolated from salt adapted tobacco cell culture [6,14]. Osmotins are also reported to be active against phytopathogenic fungi [5]. Despite the number of osmotins already described, a few osmotins were extensively purified or characterized from latex fluids. The N-terminal amino acid sequence of an osmotin from the latex of H. brasiliensis, which was determined after purification in reversed-phase chromatography, was the first report on latex osmotin. However, this osmotin was not characterized in any aspect [23]. Recently, a 22 kDa protein constituent of fresh latex of C. papaya was reported [15]. This protein was identified as a thaumatin-like protein on the basis of its partial amino acid sequence. Latex occurs in an important number of plants in a wide range of botanical groups [10]. Chitinases, proteolytic enzymes and proteins involved on cellular anti-oxidative machinery were detected in the latex of C. procera. In additional studies the proteins of the latex were shown to be deleterious to insects [20,21].
In this work a protein exhibiting antifungal activity, named CpOsm, was purified to homogeneity from the latex of C. procera. The protein, obtained after two chromatography steps, appeared as two basic isoforms of nearly 20.1 kDa, as judged by electrophoresis. As reported in literature, osmotins possess molecular mass ranging from 20 to 30 kDa [7,15,23,24]. Acidic, neutral and basic isoforms of osmotins have been found in plants which allow to separate osmotins in three sub-groups included in PR-5 [12]. In leaves of barley (Hordeum vulgare) eight different cDNAs encoding osmotins were identified and characterized with theoretical molecular mass ranging from 15,626 to 21,855 Da and pI from 4.3 to 8.1 [22]. The sequence of the 40 N-terminal amino acid residues of CpOsm was very similar to osmotins- or thaumatin-like proteins from different plant species and suggested that CpOsm is a new member of PR-5 family. To further highlight this hypothesis, purified CpOsm was assayed for antifungal activity. CpOsm was capable of inhibiting spore germination of different phytopathogenic fungi and also restricted fungal growth, with very low IC50 values (32 mg mL1). The recombinant osmotin from Solanum nigrum purified from Escherichia coli was antifungal at doses as lower as 3 mg when assayed by agar diffusion [5]. A thaumatin-like protein purified from Castanopsis chinensis seeds with active doses of 100 mg also
Table 2 Amino acid sequence determination by MALDI TOFeTOF of the two spots obtained by two-dimensional gel electrophoresis of the osmotin from Calotropis procera latex. aSpots “1” and “2” are those labeled in Fig. 1a. a
Spot
Teoric/Experimental
Protein score
Parental ions
Sequences of identified peptides
ID (NCBI)
Protein description
8.1/8.9
137
Osmotin [Piper colubrinum]
137
APGGCNNPCTVFK DDPTSTFTCPGGTNYR APGGCNNPCTVFK DDPTSTFTCPGGTNYR
gij161375756
8.1/9.1
1421.60 1788.72 1421.60 1788.72
gij161375756
Osmotin [Piper colubrinum]
MW (kDa)
pI
1
25.6/21.9
2
25.6/21.5
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Fig. 3. Inhibition of spore germination and mycelia growth of CpOsm on F. solani, Neurospora sp., and C. gloeosporioides. CpOsm (10 mg) was prepared with 50 mM Na-acetate buffer, pH 5.0. Bars: 40 mm. Control: 50 mM Na-acetate buffer (pH 5.0). The inhibitory effect of CpOsm on mycelia growth was performed after 48 h of sample application. (1) Control 50 mM Na-acetate buffer (pH 5.0); (2) 50 mM Na-acetate buffer (pH 5.0) containing 10 mM DTT; (3) CpOsm plus 10 mM DTT and (4) CpOsm in 50 mM Na-acetate buffer (pH 5.0). It was added 50 mL (1 mg mL1) of protein in each well.
inhibited fungal growth [7]. These values differed from that determined for CpOsm (50 mg) but these osmotins were tested against different fungi and culture conditions avoiding direct comparison. CpOsm lost its antifungal activity by treatment with DTT. This finding is in agreement with other osmotins described. Osmotins are proteins rich in cysteine amino acid residues, ranging from 10 to 16 residues, which are involved in the formation of 5e8 disulfide bonds. The formation of these disulfide bonds is essential for their biological activity [22]. This study reports the purification and partial characterization of an osmotin from latex of C. procera. Based on its biochemical features and activity this protein would be recognized as a new member of PR-5. Structural characterization and mechanism of action of this protein is currently under investigation. 4. Materials and methods
buffer, pH 5.0. Samples of 100 mg in 10 mL (50 mM Na-acetate buffer, pH 5.0) were assayed. After washing the column with 50 mM Na-acetate buffer (pH 5.0) to elute the unbound proteins (PI-CM), the column was washed with 0.2 M NaCl and 0.3 M NaCl in 50 mM Na-acetate buffer (pH 5.0) sequentially to obtain two new peaks (PII-CM and PIII-CM), respectively. The three distinct protein peaks were recovered, dialyzed against distilled water and lyophilized. They were analyzed by 1D-SDS-PAGE and tested for antifungal activity. PII-CM with antifungal activity was submitted to ion exchange chromatography on a Resource-S column (Amersham Biosciences, Brazil) previously equilibrated with 25 mM Na-phosphate buffer (pH 6.0) assisted by a fast protein liquid chromatography (FPLC) system. After removal of unbound proteins, the column was eluted with a linear concentration gradient of 0e0.4 M NaCl in 25 mM Na-phosphate buffer (pH 6.0). Proteins were monitored at 280 nm.
4.1. Latex and laticifer proteins
4.3. Polyacrylamide gel electrophoresis (1D-SDS-PAGE)
The latex of C. procera was collected from the aerial parts of wild plants in tubes containing distilled water on a 1:2 (v/v) dilution rate. Protein fraction of the latex was obtained as previously described by Freitas et al. [8]. The native plants were located in the vicinity of Fortaleza-CE, Brazil. The plant voucher (sample specimen N.32663) was deposited at the Prisco Bezerra Herbarium of the Federal University of Ceara, Brazil, where the botanical material was identified.
LP and the peaks obtained after cation exchange chromatography were submitted to SDS-PAGE (12.5%, 12 11 0.2 cm), as previously described by Laemmli [13], with minor modifications. The gels were ran at 20 mA, 25 C and the proteins stained with 0.1% Coomassie Brilliant Blue (R-350) solution in water/acetic acid/ methanol (5/1/4, v/v/v). Protein bands were revealed after destaining of the gels with the same solution without the dye. Molecular weight markers were from GE-Healthcare Product N. 170446-01.
4.2. Purification of CpOsm 4.4. N-terminal sequence analysis Laticifer proteins (LP) were submitted to ion exchange chromatography on a CM-Sepharose fast flow column (Amersham Biosciences, Brazil) previously equilibrated with 50 mM Na-acetate
The N-terminal amino acid sequence of CpOsm, dissolved in Milli-Q grade water, was determined on a Shimadzu PPSQ-23A
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automated protein sequencer performing Edman degradation. PTH amino acids were detected at 269 nm after separation on a reversed-phase C18 column (4.6 2.5 mm2) under isocratic conditions, according to the manufacturer’s instructions. Searches for sequence similarity were performed with the BLASTp program [1]. The protein sequence data reported in this paper will appear in the UniProt Knowledgebase under the accession number P86363. 4.5. Two-dimensional polyacrylamide gel electrophoresis (2D-SDS-PAGE) Immobiline DryStrips (11 cm), pH 3e10 (GE-Healthcare), were rehydrated overnight in 7 M urea/2 M thiourea/1% CHAPS/2% IpGbuffer pH 3e10/bromophenol blue, containing 30 mg of CpOsm. Runs were performed in an EttanÔ IPGPhor IIÔ system from GEHealthcare. Electrical conditions were as described by the supplier. After the first-dimensional run, the IpG gel strips were incubated at 25 C in 3 mL of equilibration buffer (50 mM TriseHCl pH 8.8, 30% glycerol, 6 M urea, 2% SDS and traces of bromophenol blue) for 30 min. The second dimension electrophoresis was performed in a vertical system with uniform 12.5% separating gel (14 14 cm), at 8 C. Protein spots in 2-D gels were seen after treatment with 0.1% Coomassie Brilliant Blue R-350 solution as described before. The gels were scanned using an Imager Scanner (Amersham Biosciences) and facilities of LabScan software. All details of individual gels and comparative analysis were performed by using ImageMaster 2D Platinum Software 6.0 (Amersham Biosciences). 4.6. Protein identification and molecular weight determination by mass spectrometry Protein spots were excised from Coomassie-stained polyacrylamide gels, destained and digested with sequencing-grade modified trypsin (Promega). Matrix assisted laser desorption ionization-time of flight-tandem mass spectrometry (MALDI-TOFeTOFMS/MS) acquisition was performed by an ABI 4700 Proteomics Analyzer (Applied Biosystems) using 3,5-dimethoxy-4-hydroxycinnamic acid as matrix, and the data obtained were analyzed using the software 4000 Series ExplorerTM v. 3.0 (Applied Biosystems). The acquired mass spectral data were queried against the NCBI database using the MASCOT (Matrix Science Ltd., London, UK) search engine. To achieve molecular weight, CpOsm was dissolved in 1 mL 0.1% TFA and an aliquot of 0.5 mL (0.5 mg) was mixed with 0.5 mL of sinapinic acid matrix and spotted on a MALDI plate. A MALDI-TOFeTOF analysis in linear mode was performed on ABI 4700 Proteomics Analyzer. 4.7. Glycoprotein detection The presence of covalently bound carbohydrate on CpOsm was assayed in 1-D-SDS-PAGE and 2-D-SDS-PAGE by Schiff’s reagent (Sigma S5133). After the electrophoresis, the gels were incubated in 200 mL of 10% TCA, for 30 min, washed six times with distilled water and incubated in 200 mL of 1% periodic acid in 3% acetic acid for 60 min. The gels were washed again with distilled water and 200 mL of Schiff’s reagent was added. They were left in the dark for 50 min and the glycoproteins were observed by washing the gels with 5% acetic acid. Canavalia ensiformis seed lectin (ConA), a carbohydratefree protein, and bovine lactotransferrin (BLT), a classical glycoprotein, were used as negative and positive controls, respectively. Detection of carbohydrate was also performed by the phenol-sulfuric acid method using glucose as standard [17]. The reaction mixture contained 50 mL of CpOsm (2 mg mL1 in water), 200 mL of concentrated sulfuric acid (85%) and 30 mL of 5% phenol. After incubating at 90 C for 5 min in a static water bath, the color development was
measured by turbidimetry at 492 nm using an automated microplate reader (Biotrak II Plate Reader, Amersham Biosciences). 4.8. Antifungal assays with CpOsm The antifungal activity of CpOsm was assayed against isolates of F. solani, Neurospora sp. and C. gloeosporioides. Fungi were obtained from the local collection of the Microbiology Laboratory at the Department of Biology of the Federal University of Ceara and maintained on Sabouraud Dextrose Agar (0.5% enzymatic digest of casein, 0.5% enzymatic digest of animal tissue, 4% dextrose and 1.5% agareagar, final pH 5.6) at 27 C,12 h light/dark cycle and 70% relative humidity. All assays were performed in similar conditions and CpOsm solutions were prepared immediately before using and filtered through a 0.22 mm membrane (Millipore). Homogenous spore suspensions were obtained from 2 to 3 week-old cultures in sterile distilled water. Conidia were quantified on a Neubauer chamber with the aid of a BX60 Olympus Light Microscope. Spore solutions were adjusted to have an appropriate dilution of 2 105 cells mL1. 4.8.1. Inhibition of spore germination assay The effect of CpOsm on spore germination was evaluated by mixing 10 mL of a spore suspension (2 105 mL1 in water) with 10 mL (1 mg mL1) of CpOsm prepared in 50 mM Na-acetate buffer (pH 5.0). The plates were closed to avoid evaporation and maintained at 27 C, 12 h light/dark cycle and 70% relative humidity. After 24 h, spore germination was observed by using BX60 Olympus Light Microscope and spores were considered germinated if any hypha structure was present. Each experiment was performed twice, consisting on two replicates per treatment. 4.8.2. Inhibition of vegetative growth assay The inhibitory effect of CpOsm on the mycelia growth of fungi was performed on Petri dishes (100 15 mm) containing Sabouraud Dextrose Agar (15 mL). Mycelia were allowed to grow and aliquots of 50 mL (1 mg mL1) of CpOsm in 50 mM Na-acetate (pH 5.0) were deposited on wells placed at a distance of 0.5 cm away from the rim of the mycelia colony in the agar. Acetate buffer was used as negative control. The assay was developed in the same conditions described previously until the mycelial growth had enveloped the wells containing the control. Inhibitory effect was estimated by direct observation. A quantitative assay for vegetative growth inhibition of fungi was performed following a protocol developed previously by Broekaert et al. [4]. Initially, 10 mL of a spore suspension (2 105 mL1) was incubated in 96-well flat bottomed microplates with 90 mL Yeast Peptone Dextrose broth (5% w/v). After 16 h, 100 mL of CpOsm in 50 mM Na-acetate buffer at different concentrations was added to the plates. The fungal growth was monitored for 48 h by measuring the changes in turbidimetry at 620 nm using an automated microplate reader (Biotrak II Plate Reader, Amersham Biosciences). The CpOsm concentration that reduced fungal growth by 50% of control values after 48 h was taken as IC50 and inhibition was expressed as mg of protein mL1. Data were presented as the mean SD of triplicate points. Acknowledgments Biochemical, functional and applied studies of the latex from Calotropis procera have been supported by grants from FUNCAP, CNPq (Universal, RENORBIO and Brazil/India cooperation), and IFS (M.V.R.). Appendix. Supplementary material Supplementary material related to this article can be found online at doi:10.1016/j.plaphy.2011.01.027.
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Plant Physiology and Biochemistry journal homepage: www.elsevier.com/locate/plaphy
Research article
Osmotin purified from the latex of Calotropis procera: Biochemical characterization, biological activity and role in plant defense Cleverson Diniz Teixeira de Freitas a, *, Fábio César Sousa Nogueira b, Ilka Maria Vasconcelos a, José Tadeu Abreu Oliveira a, Gilberto Barbosa Domont b, Márcio Viana Ramos a, * a b
Departamento de Bioquímica e Biologia Molecular da, Universidade Federal do Ceará, Campus do Pici, Cx., Postal 6033, Fortaleza, Ceará, CEP 60451-970, Brazil Departamento de Bioquímica, Instituto de Química da, Universidade Federal do Rio de Janeiro, Rede Proteômica do Rio de Janeiro, Rio de Janeiro, Brazil
a r t i c l e i n f o
a b s t r a c t
Article history: Received 30 June 2010 Accepted 26 January 2011 Available online 4 February 2011
A protein, similar to osmotin- and thaumatin-like proteins, was purified from Calotropis procera (Ait.) R.Br latex. The isolation procedure required two cation exchange chromatography steps on 50 mM Naacetate buffer (pH 5.0) CM-Sepharose Fast Flow and 25 mM Na-phosphate buffer (pH 6.0) Resource-S, respectively. The protein purity was confirmed by an unique N-terminal sequence [ATFTIRNNCPYTIWAAAVPGGGRRLNSGGTWTINVAPGTA]. The osmotin (CpOsm) appeared as a single band (20,100 Da) in sodium dodecyl sulfate-polyacrylamide gel electrophoresis and as two spots in two-dimensional electrophoresis (pI 8.9 and 9.1). Both polypeptides were further identified by mass spectrometry as two osmotin isoforms with molecular masses of 22,340 and 22,536 Da. The CpOsm exerted antifungal activity against Fusarium solani (IC50 ¼ 67.0 mg mL1), Neurospora sp. (IC50 ¼ 57.5 mg mL1) and Colletotrichum gloeosporioides (IC50 ¼ 32.1 mg mL1). However, this activity was lost when the protein was previously treated with a reducing agent (DTT, Dithiothreitol) suggesting the presence of disulfide bounds stabilizing the protein. The occurrence of osmotin in latex substantiates the defensive role of these fluids. Ó 2011 Elsevier Masson SAS. All rights reserved.
Keywords: Amino acid sequence Antifungal activity Fusarium solani Laticifer proteins Mass spectrometry Thaumatin-like protein
1. Introduction Plants are constantly exposed to infection by pathogens such as fungi, bacteria and viruses or are attacked by insects and other herbivorous along their life cycle. However, they react to biotic stresses by triggering a set of defense mechanisms as hypersensitive response and systemic responses, including the synthesis of defensive substances as the pathogenesis-related (PR) proteins [25]. The already identified PR-proteins have been extensively reviewed and currently comprise 17 families, whose classification is based on their similarities in amino acid sequence and enzymatic and mechanistic activity [26]. Various PR-proteins are constitutive proteins that have their synthesis augmented in response to infection [27]. Osmotin- and thaumatin-like proteins belong to family 5 of PRproteins. These proteins have been purified from banana, tomato, grape, barley, wheat, tobacco and sorghum, and exhibit remarkable activity against several fungi [11,19,24]. On the other hand, osmotins- or thaumatin-like proteins from latex sources have not been
Abbreviations: DTT, Dithiothreitol; CpOsm, Calotropis procera osmotin. * Corresponding authors. Tel.: þ55 85 3366 9403; fax: þ55 85 3366 9789. E-mail addresses: [email protected] (C.D. Teixeira de Freitas), vramos@ ufc.br (M.V. Ramos). 0981-9428/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2011.01.027
purified and characterized extensively. The presence of an osmotin in Hevea brasiliensis latex was first reported [23]. Recently, a wound-inducible thaumatin-like protein was purified and characterized in Carica papaya latex [15]. Latex is a term used to describe a fluid with a milky aspect produced constitutively by more than 12,500 plants. This fluid is the cytoplasm of specialized cells called laticifers [16]. They are specialized circulating channels present in roots, barks, leaves and flowers. Latex is released from laticifers immediately after tissue damage caused by mechanical injury. Latices are a rich source of chemicals derived from secondary metabolism such as pyrrolidine alkaloid and sesquiterpene lactone [10]. Besides these proteins involved in basal cellular activities, laticifers are important sites of PR-proteins synthesis. Among purified and characterized PRproteins from latex, chitinases, b-1,3-glucanases, proteinase inhibitors, cysteine proteinases, lectins and anti-oxidative proteins have been reported [2,3,18,28]. These findings on the biochemical composition of laticifer fluids support the hypothesis of their defensive role against predators and invasive microorganisms [9,20]. Calotropis procera (Ait.) R.Br. is a lactiferous Apocynaceae species found in tropical and subtropical regions. This plant produces a large amount of latex which can be easily collected from green leaves and barks. The latex has been reported to comprise
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chitinases, proteinases and anti-oxidative enzymes [8]. Further studies have shown that laticifer proteins are involved in plant defense against crop pests [20,21]. Combined analyses of electrophoresis and mass spectrometry lead to the identification of two osmotins in laticifer proteins of C. procera, named CpOsm. This work reports the purification, partial biochemical characterization and activity of this new protein on three different phytopathogen fungi. 2. Results 2.1. CpOsm purification Three peaks were obtained when laticifer proteins from C. procera were subjected to cation exchange chromatography on a CM-Sepharose Fast Flow column, previously equilibrated with 50 mM Na-acetate buffer, pH 5.0 (Supplementary Fig. S1A). The proteins retained by the matrix and eluted after adding 0.2 M NaCl (PII-CM) presented antifungal activity to Fusarium solani, Neurospora sp. and Colletotrichum gloeosporioides (data not shown). PI-CM (non-retained proteins) was not active while PIII-CM (proteins eluted with 0.3 M NaCl) inhibited only Neurospora sp. Based on these findings, PII-CM was further fractionated and examined for antifungal activity. This fraction was chromatographed again by ion exchange chromatography using a Resource-S matrix equilibrated in 25 mM Na-phosphate buffer, pH 6.0; and it was further washed on a linear ascendant gradient of NaCl. This procedure yielded an unbound fraction and five separated peaks within salt gradient (Supplementary Fig. S1B). The antifungal activity against F. solani was concentrated in the eluted peak with 0.1 M NaCl (P2-R), while a negligible activity appeared in P3-R. The electrophoresis pattern of P2-R showed an unique protein band of a relative mass of 20.1 kDa, which was not reactive to Schiff’s reagent; thus revealing its non glycoprotein nature (Supplementary Fig. S1C and S1D). The presence of carbohydrate on osmotin also was not detected by the phenol-sulfuric acid method. Supplementary Table S1 summarizes the steps to purify CpOsm. 2.2. Physicochemical characterization and identification of CpOsm Automated amino acid sequence determination of P2-R (CpOsm) yielded a unique sequence of 40 N-terminal amino acid residues confirming the homogeneity of the purified protein. This sequence was similar to thaumatin-like proteins or osmotins of different plants, especially with that of H. brasiliensis latex (Table 1). Mass spectrometry analysis of CpOsm exhibited two mono protonated peaks with closely related masses (22,340 and 22,536 Da) suggesting the presence of two isoforms, and a double protonated peak with a mass corresponding to half of those mono protonated (data not shown). The presence of two isoforms with pI corresponding to 8.9 and 9.1 was confirmed by two-dimensional electrophoresis (Fig. 1). The isoform 1 represented about 35% and the isoform 2 about 65%. Both isoforms presented very similar tryptic patterns (Fig. 2). The mass difference of 196 Da between the
Fig. 1. (A) Two-dimensional polyacrylamide gel electrophoresis (12.5%) of CpOsm. Proteins were visualized by staining with 0.1% Coomassie Brilliant Blue R-350. Samples of 30 mg of protein were applied to the strips. (B) Tridimensional view of two spots using ImageMaster 2D PlatinumÒ Software (Amersham Biosciences). The Isoeletric points of the spots 1 and 2 were estimated as 8.9 and 9.1, respectively.
two isoforms can be due to small differences in their primary amino acid sequence (as no carbohydrate moieties have been detected). The detailed differences will be possible after complete sequence of both proteins. It is therefore interesting that a unique N-terminal amino acid sequence comprising almost 40 residues was observed. Both protein spots present in 2-D gels were also examined by tandem mass spectrometry (MALDI-TOFeTOF) after trypsin digestion. Two distinct peptides of each isoform were sequenced (Table 2). These results show that amino acid sequences of both isoforms are very close. All the determined sequences were matched with known osmotin proteins. 2.3. Antifungal activity of CpOsm CpOsm inhibited drastically the germination of the fungi tested (Fig. 3). CpOsm also exhibited mycelial growth inhibitory activity against F. solani, Neurospora sp. and C. gloeosporioides with IC50, ranging from 32.1 up to 67.0 mg mL1 (Supplementary Table S1). Inhibition was clearly seen when assays were performed by agar diffusion (Fig. 3). However, after treating with DTT, a reducing agent, the protein lost its inhibitory activity on the mycelial growth. 3. Discussion Proteins implicated in plant defense have been widely described in literature. They are grouped together on the sole basis of their participation as protective agents against damage caused by predators such as insects or invaders like viruses, bacteria and fungi. These proteins are referred to as Pathogenesis-Related Proteins (PR-Proteins). This diversified group of proteins includes
Table 1 The N-terminal amino acid sequence of an osmotin from Calotropis procera latex compared with other osmotins- and thaumatin-like proteins. Identification
Organism
Sequence
ID (NCBI)
% similarity
E-value
Osmotin PR-protein 5 Osmotin Thaumatin-like protein Osmotin Osmotin
Calotropis procera Helianthus annuus Piper colubrinum Capsicum annuum Arabidopsis thaliana Hevea brasiliensis
1-ATFTIRNNCPYTIWAAAVP-GGGRRLNSGGTWTINVAPGTA-40 22-AVFTIRNNCPYTVWAGAVP-GGGRQLNSGQTWSLTVAAGTA-61 30-ANFLIRNNCPYTVWAAAVP-GGGRRLDRGATWSLNVPAGT-68 22-ATFEVRNNCPYTVWAASTPVGGGRRLDRGQTWTINAPPGTA-62 23-ATFEILNQCSYTVWAAASP-GGGRRLDAGQSWRLDVAAGT–61 1-ATFTIRNNXPYTVWAAASP-GGGRRLDMARIW————————31
e gij20385169 gij161375756 gij15419836 gij21537409 gij32363250
e 87 84 82 79 74
e 4e10 2e09 6e09 5e07 6e07
740
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Fig. 2. Mass spectra of peptides generated by the digestion of the spots 1 (Top) and 2 (Bottom) from Fig. 1a with trypsin. Sample was mixed with 3,5-dimethoxy-4-hydroxycinnamic acid (matrix) dissolved in 0.1% trifluoroacetic acid. A MALDI-TOFeTOF analysis in linear mode was performed on ABI 4700 Proteomics Analyzer.
chitinases, proteolytic enzymes, and proteinase inhibitors [26]. Thaumatins and osmotins belong to PR-5 group. These proteins are representatives of the PR-Proteins group because experimental evidence suggested changes in their mRNA expression level under plant stress [12]. Thaumatin was first observed on Thaumatococcus daniellii and osmotin was first isolated from salt adapted tobacco cell culture [6,14]. Osmotins are also reported to be active against phytopathogenic fungi [5]. Despite the number of osmotins already described, a few osmotins were extensively purified or characterized from latex fluids. The N-terminal amino acid sequence of an osmotin from the latex of H. brasiliensis, which was determined after purification in reversed-phase chromatography, was the first report on latex osmotin. However, this osmotin was not characterized in any aspect [23]. Recently, a 22 kDa protein constituent of fresh latex of C. papaya was reported [15]. This protein was identified as a thaumatin-like protein on the basis of its partial amino acid sequence. Latex occurs in an important number of plants in a wide range of botanical groups [10]. Chitinases, proteolytic enzymes and proteins involved on cellular anti-oxidative machinery were detected in the latex of C. procera. In additional studies the proteins of the latex were shown to be deleterious to insects [20,21].
In this work a protein exhibiting antifungal activity, named CpOsm, was purified to homogeneity from the latex of C. procera. The protein, obtained after two chromatography steps, appeared as two basic isoforms of nearly 20.1 kDa, as judged by electrophoresis. As reported in literature, osmotins possess molecular mass ranging from 20 to 30 kDa [7,15,23,24]. Acidic, neutral and basic isoforms of osmotins have been found in plants which allow to separate osmotins in three sub-groups included in PR-5 [12]. In leaves of barley (Hordeum vulgare) eight different cDNAs encoding osmotins were identified and characterized with theoretical molecular mass ranging from 15,626 to 21,855 Da and pI from 4.3 to 8.1 [22]. The sequence of the 40 N-terminal amino acid residues of CpOsm was very similar to osmotins- or thaumatin-like proteins from different plant species and suggested that CpOsm is a new member of PR-5 family. To further highlight this hypothesis, purified CpOsm was assayed for antifungal activity. CpOsm was capable of inhibiting spore germination of different phytopathogenic fungi and also restricted fungal growth, with very low IC50 values (32 mg mL1). The recombinant osmotin from Solanum nigrum purified from Escherichia coli was antifungal at doses as lower as 3 mg when assayed by agar diffusion [5]. A thaumatin-like protein purified from Castanopsis chinensis seeds with active doses of 100 mg also
Table 2 Amino acid sequence determination by MALDI TOFeTOF of the two spots obtained by two-dimensional gel electrophoresis of the osmotin from Calotropis procera latex. aSpots “1” and “2” are those labeled in Fig. 1a. a
Spot
Teoric/Experimental
Protein score
Parental ions
Sequences of identified peptides
ID (NCBI)
Protein description
8.1/8.9
137
Osmotin [Piper colubrinum]
137
APGGCNNPCTVFK DDPTSTFTCPGGTNYR APGGCNNPCTVFK DDPTSTFTCPGGTNYR
gij161375756
8.1/9.1
1421.60 1788.72 1421.60 1788.72
gij161375756
Osmotin [Piper colubrinum]
MW (kDa)
pI
1
25.6/21.9
2
25.6/21.5
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Fig. 3. Inhibition of spore germination and mycelia growth of CpOsm on F. solani, Neurospora sp., and C. gloeosporioides. CpOsm (10 mg) was prepared with 50 mM Na-acetate buffer, pH 5.0. Bars: 40 mm. Control: 50 mM Na-acetate buffer (pH 5.0). The inhibitory effect of CpOsm on mycelia growth was performed after 48 h of sample application. (1) Control 50 mM Na-acetate buffer (pH 5.0); (2) 50 mM Na-acetate buffer (pH 5.0) containing 10 mM DTT; (3) CpOsm plus 10 mM DTT and (4) CpOsm in 50 mM Na-acetate buffer (pH 5.0). It was added 50 mL (1 mg mL1) of protein in each well.
inhibited fungal growth [7]. These values differed from that determined for CpOsm (50 mg) but these osmotins were tested against different fungi and culture conditions avoiding direct comparison. CpOsm lost its antifungal activity by treatment with DTT. This finding is in agreement with other osmotins described. Osmotins are proteins rich in cysteine amino acid residues, ranging from 10 to 16 residues, which are involved in the formation of 5e8 disulfide bonds. The formation of these disulfide bonds is essential for their biological activity [22]. This study reports the purification and partial characterization of an osmotin from latex of C. procera. Based on its biochemical features and activity this protein would be recognized as a new member of PR-5. Structural characterization and mechanism of action of this protein is currently under investigation. 4. Materials and methods
buffer, pH 5.0. Samples of 100 mg in 10 mL (50 mM Na-acetate buffer, pH 5.0) were assayed. After washing the column with 50 mM Na-acetate buffer (pH 5.0) to elute the unbound proteins (PI-CM), the column was washed with 0.2 M NaCl and 0.3 M NaCl in 50 mM Na-acetate buffer (pH 5.0) sequentially to obtain two new peaks (PII-CM and PIII-CM), respectively. The three distinct protein peaks were recovered, dialyzed against distilled water and lyophilized. They were analyzed by 1D-SDS-PAGE and tested for antifungal activity. PII-CM with antifungal activity was submitted to ion exchange chromatography on a Resource-S column (Amersham Biosciences, Brazil) previously equilibrated with 25 mM Na-phosphate buffer (pH 6.0) assisted by a fast protein liquid chromatography (FPLC) system. After removal of unbound proteins, the column was eluted with a linear concentration gradient of 0e0.4 M NaCl in 25 mM Na-phosphate buffer (pH 6.0). Proteins were monitored at 280 nm.
4.1. Latex and laticifer proteins
4.3. Polyacrylamide gel electrophoresis (1D-SDS-PAGE)
The latex of C. procera was collected from the aerial parts of wild plants in tubes containing distilled water on a 1:2 (v/v) dilution rate. Protein fraction of the latex was obtained as previously described by Freitas et al. [8]. The native plants were located in the vicinity of Fortaleza-CE, Brazil. The plant voucher (sample specimen N.32663) was deposited at the Prisco Bezerra Herbarium of the Federal University of Ceara, Brazil, where the botanical material was identified.
LP and the peaks obtained after cation exchange chromatography were submitted to SDS-PAGE (12.5%, 12 11 0.2 cm), as previously described by Laemmli [13], with minor modifications. The gels were ran at 20 mA, 25 C and the proteins stained with 0.1% Coomassie Brilliant Blue (R-350) solution in water/acetic acid/ methanol (5/1/4, v/v/v). Protein bands were revealed after destaining of the gels with the same solution without the dye. Molecular weight markers were from GE-Healthcare Product N. 170446-01.
4.2. Purification of CpOsm 4.4. N-terminal sequence analysis Laticifer proteins (LP) were submitted to ion exchange chromatography on a CM-Sepharose fast flow column (Amersham Biosciences, Brazil) previously equilibrated with 50 mM Na-acetate
The N-terminal amino acid sequence of CpOsm, dissolved in Milli-Q grade water, was determined on a Shimadzu PPSQ-23A
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automated protein sequencer performing Edman degradation. PTH amino acids were detected at 269 nm after separation on a reversed-phase C18 column (4.6 2.5 mm2) under isocratic conditions, according to the manufacturer’s instructions. Searches for sequence similarity were performed with the BLASTp program [1]. The protein sequence data reported in this paper will appear in the UniProt Knowledgebase under the accession number P86363. 4.5. Two-dimensional polyacrylamide gel electrophoresis (2D-SDS-PAGE) Immobiline DryStrips (11 cm), pH 3e10 (GE-Healthcare), were rehydrated overnight in 7 M urea/2 M thiourea/1% CHAPS/2% IpGbuffer pH 3e10/bromophenol blue, containing 30 mg of CpOsm. Runs were performed in an EttanÔ IPGPhor IIÔ system from GEHealthcare. Electrical conditions were as described by the supplier. After the first-dimensional run, the IpG gel strips were incubated at 25 C in 3 mL of equilibration buffer (50 mM TriseHCl pH 8.8, 30% glycerol, 6 M urea, 2% SDS and traces of bromophenol blue) for 30 min. The second dimension electrophoresis was performed in a vertical system with uniform 12.5% separating gel (14 14 cm), at 8 C. Protein spots in 2-D gels were seen after treatment with 0.1% Coomassie Brilliant Blue R-350 solution as described before. The gels were scanned using an Imager Scanner (Amersham Biosciences) and facilities of LabScan software. All details of individual gels and comparative analysis were performed by using ImageMaster 2D Platinum Software 6.0 (Amersham Biosciences). 4.6. Protein identification and molecular weight determination by mass spectrometry Protein spots were excised from Coomassie-stained polyacrylamide gels, destained and digested with sequencing-grade modified trypsin (Promega). Matrix assisted laser desorption ionization-time of flight-tandem mass spectrometry (MALDI-TOFeTOFMS/MS) acquisition was performed by an ABI 4700 Proteomics Analyzer (Applied Biosystems) using 3,5-dimethoxy-4-hydroxycinnamic acid as matrix, and the data obtained were analyzed using the software 4000 Series ExplorerTM v. 3.0 (Applied Biosystems). The acquired mass spectral data were queried against the NCBI database using the MASCOT (Matrix Science Ltd., London, UK) search engine. To achieve molecular weight, CpOsm was dissolved in 1 mL 0.1% TFA and an aliquot of 0.5 mL (0.5 mg) was mixed with 0.5 mL of sinapinic acid matrix and spotted on a MALDI plate. A MALDI-TOFeTOF analysis in linear mode was performed on ABI 4700 Proteomics Analyzer. 4.7. Glycoprotein detection The presence of covalently bound carbohydrate on CpOsm was assayed in 1-D-SDS-PAGE and 2-D-SDS-PAGE by Schiff’s reagent (Sigma S5133). After the electrophoresis, the gels were incubated in 200 mL of 10% TCA, for 30 min, washed six times with distilled water and incubated in 200 mL of 1% periodic acid in 3% acetic acid for 60 min. The gels were washed again with distilled water and 200 mL of Schiff’s reagent was added. They were left in the dark for 50 min and the glycoproteins were observed by washing the gels with 5% acetic acid. Canavalia ensiformis seed lectin (ConA), a carbohydratefree protein, and bovine lactotransferrin (BLT), a classical glycoprotein, were used as negative and positive controls, respectively. Detection of carbohydrate was also performed by the phenol-sulfuric acid method using glucose as standard [17]. The reaction mixture contained 50 mL of CpOsm (2 mg mL1 in water), 200 mL of concentrated sulfuric acid (85%) and 30 mL of 5% phenol. After incubating at 90 C for 5 min in a static water bath, the color development was
measured by turbidimetry at 492 nm using an automated microplate reader (Biotrak II Plate Reader, Amersham Biosciences). 4.8. Antifungal assays with CpOsm The antifungal activity of CpOsm was assayed against isolates of F. solani, Neurospora sp. and C. gloeosporioides. Fungi were obtained from the local collection of the Microbiology Laboratory at the Department of Biology of the Federal University of Ceara and maintained on Sabouraud Dextrose Agar (0.5% enzymatic digest of casein, 0.5% enzymatic digest of animal tissue, 4% dextrose and 1.5% agareagar, final pH 5.6) at 27 C,12 h light/dark cycle and 70% relative humidity. All assays were performed in similar conditions and CpOsm solutions were prepared immediately before using and filtered through a 0.22 mm membrane (Millipore). Homogenous spore suspensions were obtained from 2 to 3 week-old cultures in sterile distilled water. Conidia were quantified on a Neubauer chamber with the aid of a BX60 Olympus Light Microscope. Spore solutions were adjusted to have an appropriate dilution of 2 105 cells mL1. 4.8.1. Inhibition of spore germination assay The effect of CpOsm on spore germination was evaluated by mixing 10 mL of a spore suspension (2 105 mL1 in water) with 10 mL (1 mg mL1) of CpOsm prepared in 50 mM Na-acetate buffer (pH 5.0). The plates were closed to avoid evaporation and maintained at 27 C, 12 h light/dark cycle and 70% relative humidity. After 24 h, spore germination was observed by using BX60 Olympus Light Microscope and spores were considered germinated if any hypha structure was present. Each experiment was performed twice, consisting on two replicates per treatment. 4.8.2. Inhibition of vegetative growth assay The inhibitory effect of CpOsm on the mycelia growth of fungi was performed on Petri dishes (100 15 mm) containing Sabouraud Dextrose Agar (15 mL). Mycelia were allowed to grow and aliquots of 50 mL (1 mg mL1) of CpOsm in 50 mM Na-acetate (pH 5.0) were deposited on wells placed at a distance of 0.5 cm away from the rim of the mycelia colony in the agar. Acetate buffer was used as negative control. The assay was developed in the same conditions described previously until the mycelial growth had enveloped the wells containing the control. Inhibitory effect was estimated by direct observation. A quantitative assay for vegetative growth inhibition of fungi was performed following a protocol developed previously by Broekaert et al. [4]. Initially, 10 mL of a spore suspension (2 105 mL1) was incubated in 96-well flat bottomed microplates with 90 mL Yeast Peptone Dextrose broth (5% w/v). After 16 h, 100 mL of CpOsm in 50 mM Na-acetate buffer at different concentrations was added to the plates. The fungal growth was monitored for 48 h by measuring the changes in turbidimetry at 620 nm using an automated microplate reader (Biotrak II Plate Reader, Amersham Biosciences). The CpOsm concentration that reduced fungal growth by 50% of control values after 48 h was taken as IC50 and inhibition was expressed as mg of protein mL1. Data were presented as the mean SD of triplicate points. Acknowledgments Biochemical, functional and applied studies of the latex from Calotropis procera have been supported by grants from FUNCAP, CNPq (Universal, RENORBIO and Brazil/India cooperation), and IFS (M.V.R.). Appendix. Supplementary material Supplementary material related to this article can be found online at doi:10.1016/j.plaphy.2011.01.027.
C.D. Teixeira de Freitas et al. / Plant Physiology and Biochemistry 49 (2011) 738e743
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