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DOI 10.1007/s00425-016-2468-8

ORIGINAL ARTICLE

Proteomic analysis and purification of an unusual germin-like

protein with proteolytic activity in the latex of Thevetia peruviana
Cleverson D. T. de Freitas1 • Wallace T. da Cruz1 • Maria Z. R. Silva1 •
Ilka M. Vasconcelos1 • Frederico B. M. B. Moreno2 • Renato A. Moreira2 •
Ana C. O. Monteiro-Moreira2 • Luciana M. R. Alencar3 • Jeanlex S. Sousa3 •

Bruno A. M. Rocha1 • Márcio V. Ramos1

Received: 7 November 2015 / Accepted: 10 January 2016

Ó Springer-Verlag Berlin Heidelberg 2016

Abstract that peruvianin-I possesses 120 kDa, pI 4.0, and six subunits
Main Conclusion The latex from Thevetia peruviana is (20 kDa). A unique N-terminal amino acid sequence was
rich in plant defense proteins, including a 120 kDa obtained to oligomer and monomers of peruvianin-I
cysteine peptidase with structural characteristics simi- (1ADPGPLQDFCLADLNSPLFINGYPCRNPALAISDDF36).
lar to germin-like proteins. High-resolution images from atomic force microscopy
showed the homohexameric structure of peruvianin-I
More than 20,000 plant species produce latex, including
may be organized as a trimer of dimers that form a
Apocynaceae, Sapotaceae, Papaveraceae and Euphor-
central channel similar to germin-like proteins. Peru-
biaceae. To better understand the physiological role played
vianin-I exhibited no oxalate oxidase and superoxide
by latex fluids, a proteomic analysis of Thevetia peruviana
dismutase activity or antifungal effects. Peruvianin-I
(Pers.) Schum latex was performed using two-dimensional
represents the first germin-like protein (GLP) with cys-
gel electrophoresis and mass spectrometry. A total of 33
teine peptidase activity, an activity unknown in the GLP
proteins (86 %) were identified, including storage proteins, a
family so far.
peptidase inhibitor, cysteine peptidases, peroxidases and
osmotins. An unusual cysteine peptidase, termed peruvianin-
Keywords Atomic force microscopy  Apocynaceae 
I, was purified from the latex by a single chromatographic
Peptidase  Proteome
step involving gel filtration. The enzyme (glycoprotein) was
inhibited by E-64 and iodoacetamide and exhibited high
specific activity towards azocasein (Km 17.6 lM), with an
Introduction
optimal pH and temperature of 5.0–6.0 and 25–37 °C,
respectively. Gel filtration chromatography, two-dimen-
The evolution of plant defense-related traits includes a
sional gel electrophoresis, and mass spectrometry revealed
diverse array of morphological structures, such as the
development of specialized tissues as laticifers and secre-
Electronic supplementary material The online version of this tory channels (Pickard 2008). Laticifers have been found in
article (doi:10.1007/s00425-016-2468-8) contains supplementary
material, which is available to authorized users. a wide variety of plants, including herbs, shrubs and trees,
which grow in temperate, tropical and subtropical areas.
& Cleverson D. T. de Freitas More than 20,000 species belonging to 40 families produce
cleversondiniz@hotmail.com latex, including Apocynaceae, Cactaceae, Sapotaceae,
1
Departamento de Bioquı́mica e Biologia Molecular da
Papaveraceae, Euphorbiaceae and Leguminosae (Pickard
Universidade Federal do Ceará, Campus do Pici, 2008; Agrawal and Konno 2009). Laticifers can be found
Cx. Postal 6033, Fortaleza, CE CEP 60451-970, Brazil in all plant tissues, including the roots. However, these
2
Centro de Ciências da Saúde da Universidade de Fortaleza, structures are more abundant in green tissues, including the
Fortaleza, CE, Brazil leaves and stems (Agrawal and Konno 2009).
3
Departamento de Fı́sica da Universidade Federal do Ceará, Although a wide diversity of molecules have been
Campus do Pici, Fortaleza, CE, Brazil identified in laticifer fluids, the most well-studied

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molecules are proteins involved in plant defense, such as
peptidases, peptidase inhibitors, chitinases and anti-oxida-
tive enzymes (Konno 2011). Among them, peptidases have
been recognized as the major protein constituent of laticifer
fluids. Several latex cysteine peptidases have been purified
and these studies revealed some similarities among them
[i.e., small molecular mass (20–30 kDa) and kinetic
parameters such as optimum pH and temperature] (Dom-
salla and Melzig 2008). The role played by latex peptidases
has been studied, and the most accepted hypothesis
includes their participation in plant defense against insects
and fungi (Konno 2011; Souza et al. 2011).
In this study, a cysteine peptidase was purified from the
latex of Thevetia peruviana. It demonstrated uncommon
structural properties when it was compared to other latex
cysteine peptidases. The peptidase’s primary structure was
similar to that of germin-like proteins (GLPs). GLPs
exhibit structural similarity with germin, a protein that was
named based on its isolation from germinating wheat
grains (Bernier and Berna 2001). Germins are encoded by a
homogenous group of homologues genes present almost
exclusively in cereals and exhibit oxalate oxidase activity,
whereas GLPs are heterogeneous proteins without oxalate
oxidase activity found in different plants (Davidson et al.
2009). On the other hand, GLPs have been associated with
superoxide dismutase (Guicciardo et al. 2007), phospho-
diesterase (Rodriguez-Lopez et al. 2001), serine protease
inhibition (Mansilla et al. 2012) and polyphenol oxidase
activity (Cheng et al. 2014). A germin-like protein with
proteolytic activity has not yet been described in the lit-
erature so far. Currently, the presence of germin and ger-
min-like proteins has been reported in several tissues
including leaves, stems, root, flowers and seeds (Bernier
and Berna 2001; Davidson et al. 2009). Nevertheless, these
proteins have not been detected in latex fluids. Here, we
describe the first germin-like protein with proteolytic
activity isolated from the latex of Thevetia peruviana.
Materials and methods
Materials
IPG buffer, IPG strips, DTT (dithiothreitol), iodoacetamide
(IAA), urea, thiourea, CHAPS, molecular mass markers,
and Superdex-75 (10/300 GL) and Superose (12 HR 10/30)
columns were purchased from GE Healthcare (Piscataway,
NJ, USA). Schiff’s reagent, azocasein, BANA, BApNA,
pepstatin-A, PMSF (phenylmethylsulfonyl fluoride),
EDTA (ethylenediamine tetra acetic acid), E-64 [trans-
epoxysuccinyl-L-leucylamido(4-guanidino)butane], N,N-
dimethylaniline, 3-methyl-2-benzothiazolinone hydrazone
and horseradish peroxidase were purchased from Sigma
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(St. Louis, MO, USA). Sequencing-grade-modified trypsin
was purchased from Promega (Madison, WI, USA). All
other chemicals were of analytical grade and were pur-
chased from different companies.
Latex protein isolation
The latex of Thevetia peruviana (Pers.) Schum was col-
lected from the aerial parts of healthy plants that were
grown in the gardens of Fortaleza-Ce, Brazil (03° 430 0600 S
38° 320 3400 O). The latex was mixed with distilled water
for a 1:2 (v/v) final dilution, and the total protein content
was isolated (Freitas et al. 2007), quantified (Bradford
1976) and termed Thevethia peruviana latex proteins
(TpLP).
Proteomic analysis
One-dimensional polyacrylamide gel electrophoresis (1D
SDS-PAGE)
Polyacrylamide gel electrophoresis in the presence of SDS
was performed as described previously (Laemmli 1970).
The samples were dissolved in 62.5 mM Tris–HCl buffer
(pH 6.8) containing 2 % SDS in the absence or presence of
5 % 2-mercaptoethanol (non-heated and heated at 100 °C
for 10 min). The runs were performed for 3 h at 20 mA per
gel and at 25 °C. The proteins were stained with 0.1 %
Coomassie Brilliant Blue R-350.
Two-dimensional polyacrylamide gel electrophoresis (2D
SDS-PAGE)
Two-dimensional gel electrophoresis was performed as
described previously (Freitas et al. 2007). TpLP (300 lg)
was dissolved in 7 M urea/2 M thiourea/1 % CHAPS/1 %
DTT/0.1 % IpG buffer pH 3–10 or 4–7/bromophenol blue
and were applied to Immobiline DryStrips pH 3–10 or pH
4–7 (11 cm). The first-dimension separation was performed
using an Ettan IPGPhor II system from GE Healthcare, as
described by the supplier. After the second dimension
separation, the protein spots were stained using a 0.1 %
Coomassie Brilliant Blue R-350 solution and analyzed
using the Image Master 2D Platinum Software 6.0
(Amersham Biosciences). The experiments were repeated
at least 3 times using independent batches of plant as
biological replicates.
Protein identification by mass spectrometry (LC–MS/MS)
The protein spots were removed from the 2D SDS-PAGE
gel, destained and digested with sequencing-grade-modi-
fied trypsin (Hellman et al. 1995). The tryptic peptides
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were analyzed using a Synapt HDMS mass spectrometer
(Waters, Manchester, UK) coupled to a 2D NanoUPLC-
ESI system; the analysis was conducted using a 1 h
reversed phase gradient from 7 to 40 % acetonitrile con-
taining 0.1 % formic acid and a 500 nl min-1 flow rate.
Proteins were identified using the NCBI database and
MASCOT (Matrix Science Ltd., London, UK; http://www.
matrixscience.com) search engine.
Purification of a cysteine peptidase (peruvianin-I)
The latex proteins (TpLP) were subjected to gel filtration
chromatography on a Superdex-75 (10/300 GL) column
that had previously been equilibrated with 25 mM sodium
acetate buffer (pH 5.0) coupled to a high-performance
liquid chromatographic system (AKTA Purifier, GE
Healthcare). Five hundred microliter samples (4 mg ml-1)
were assayed, and the proteins were eluted from the col-
umn with 25 mM sodium acetate buffer (pH 5.0) at a
0.3 ml min-1 flow rate. The protein peaks were monitored
at 280 nm, recovered, dialyzed against distilled water,
lyophilized and analyzed by 1D SDS-PAGE. The purity of
the protein was also determined by 2D SDS-PAGE using
an amount of 100 lg of the sample and by mass spec-
trometry, as described above. The purified protein was
termed peruvianin-I.
N-terminal sequence analysis
Peruvianin-I (non-heated and heated at 100 °C for 10 min)
was separated by 1D SDS–PAGE and transferred to a
polyvinylidene difluoride (PVDF) membrane. Separately,
the 120 and 20 kDa bands were washed with water and
methanol and the N-terminal amino acid sequences were
determined using an automated protein sequencer (Shi-
madzu PPSQ-23A) performing Edman’s degradation. The
BLASTp program (http://blast.ncbi.nlm.nih.gov/Blast.cgi)
was used to identify the purified protein by searching for
similarities with other plant proteins. The protein sequence
data reported in this paper will appear in the UniProt
Knowledgebase under the accession number C0HJY2.
De novo peptide sequencing
After 2D SDS-PAGE gel, destained and digested with
sequencing-grade-modified trypsin, internal peptide
sequences of peruvianin-I were obtained by LC–MS/MS as
described above, using the Data-Dependent Acquisition
(DDA) function selected to perform MS/MS experiments
with doubly or triply charged precursor ions fragmented by
Collision-Induced Dissociation (CID). Data were pro-
cessed and analyzed with a Proteinlynx v2.4 (Waters)
using the peptide fragmentation pattern as search
parameter. Peptide sequences were obtained by manual de
novo sequencing.
Glycoprotein detection
The presence of carbohydrates on peruvianin-I was detec-
ted in gel (1D SDS-PAGE) using Schiff’s reagent (Freitas
et al. 2011) or using the phenol–sulfuric acid method, with
glucose as the standard (Masuko et al. 2005).
Molecular mass determination by gel filtration
The purity and the native molecular mass of peruvianin-I
were also determined by gel filtration using a Superose 12
HR column (1 9 30 cm; GE Healthcare) coupled to an
FPLC system (Pharmacia). The column was equilibrated
with 25 mM sodium acetate buffer (pH 5.0), and the pro-
tein was eluted at a 0.3 ml min-1 flow rate. The elution
profile was monitored at 230 nm. b-amylase (200 kDa), b-
galactosidase (116 kDa), bovine serum albumin (66 kDa),
egg albumin (45 kDa), and carbonic anhydrase (29 kDa)
were used as standards.
Atomic force microscopy (AFM)
The overall structure of peruvianin-I was acquired with an
AFM Nanoscope Multimode IIIa (Bruker-CA) in tapping
mode, with a scan rate of 0.5 Hz and a cantilever with a
nominal spring constant of 42 N/m. All measurements
were performed in air and at 25 °C. The protein sample
(2 lg ml-1) was deposited in freshly cleaved mica and
allowed to dry in a vacuum chamber for approximately
10 min. The z-limit of the piezoelectric positioner was
reduced for 170 nm to increase the resolution of the
images.
Enzymatic assays
Proteolytic activity
The proteolytic activities were evaluated using the sub-
strates azocasein, BANA and BApNA (Freitas et al. 2007).
For each individual reaction, TpLP or peruvianin-I (100 ll,
at 1 mg ml-1) was incubated with 200 ll of various buf-
fers (pH 3–10) and 200 ll of the substrate solution (1 %
azocasein, 1 mM BANA or 1.25 mM BApNA). All assays
were repeated at least three times.
Effect of pH, temperature and DTT
The effect of pH on proteolytic activity was measured with
azocasein, BANA and BApNA at 37 °C using the fol-
lowing buffers at 50 mM: glycine–HCl (pH 3.0); sodium
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acetate (pH 4.0 and 5.0); sodium phosphate (pH 6.0 and
7.0); Tris–HCl (pH 8.0, 9.0 and 10.0). The enzyme’s heat
stability was assayed after incubation at various tempera-
tures (25, 37, 45, 60, 75 and 90 °C) for 15 or 30 min. The
analysis of proteolytic activity was then performed using
azocasein as a substrate at 37 °C, pH 5.0 or 6.0. The effect
of different concentrations of DTT (0, 1, 2, 3, 5, 10, 15, 25
and 50 mM; 40 min at 37 °C) on proteolytic activity was
also determined using azocasein and BANA as substrates at
pH 5.0 or 6.0.
Effect of peptidase inhibitors
Different types of peptidase inhibitors were tested to
determine the mechanistic nature of the peptidases. TpLP
or peruvianin-I was pre-incubated separately with 5 mM
PMSF, 0.18 mM E-64, 1 mM iodoacetamide (IAA),
10 mM EDTA, 10 mM EGTA or 10 lM pepstatin-A
(PEP) at 25 °C for 30 min. Azocasein or BANA were used
to determine the remaining proteolytic activity at pH 5.0 or
6.0 and 37 °C.
Kinetic parameters
Azocasein was used to determine the Km of peruvianin-I.
The estimation of this parameter was performed using
20 lg of enzyme and a final concentration of 1–200 lM
substrate in 50 mM sodium phosphate buffer (pH 6.0)
containing 1 mM DTT at 37 °C. The Km, Vmax and Kcat
values were calculated from a Lineweaver–Burk plot.
Zymogram
The proteolytic activity was also determined using zymo-
gram containing 0.1 % gelatin (Freitas et al. 2007). After
1D SDS-PAGE, the gels were washed with 2.5 % Triton
X-100 (renaturing solution) and incubated in 50 mM
sodium phosphate buffer (pH 6.0) containing 1 mM DTT
for 2 h at 37 °C. After staining with 0.1 % Coomassie
Brilliant Blue R-350, the enzymatic activity was detected
as transparent bands in the blue gels.
Oxalate oxidase and superoxide dismutase activities
Because the N-terminal amino acid sequence of peru-
vianin-I was very similar to that of germin and germin-like
proteins (GLPs), enzymatic assays for oxalate oxidase
(in vitro, in situ and using nitrocellulose membranes) and
superoxide dismutase (in vitro and zymogram) were per-
formed (Beauchamp and Fridovich 1971; Giannopolitis
and Rieis 1977; Dumas et al. 1993; Zhang et al. 2013). The
evaluations of the enzymatic activities of the TpLP fraction
(2 mg ml-1) and peruvianin-I (1 mg ml-1) were
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performed at pH 7.0 and 37 °C. Rice leaves (Zhang et al.
2013) and latex proteins from Cryptostergia grandiflora
and Plumeria rubra (Freitas et al. 2010) were used as
positive controls for oxalate oxidase and superoxide dis-
mutase activities, respectively.
Antifungal activity
The effects of TpLP and peruvianin-I on Fusarium solani,
Rhizoctonia solani and Colletotrichum gloeosporioides
spore germination was also studied (Souza et al. 2011). The
samples (1 mg ml-1, 10 ll) were incubated in presence of
0.5 mM DTT (cysteine peptidase activator) with 10 ll of a
spore suspension (2 9 105 spores ml-1) for 24 h on
reticulated plastic plates at 27 °C and 70 % relative
humidity. The germination of the spores was evaluated
using a light microscope (Olympus BX60). The experi-
ments were repeated at least 3 times using independent
batches of TpLP and peruvianin-I.
Result and discussion
Latex composition and protein profile
After dialysis against water and centrifugation, the latex of
T. peruviana was separated into water-soluble and insol-
uble fractions. The latter represented 97 % of the dry mass
and corresponded to rubber. This result was very similar to
those found for latex fluids of other species such as
Calotropis procera, Cryptostergia grandiflora and Plume-
ria rubra, in which the rubber fraction corresponded to 82,
96 and 82 % of the dry mass, respectively (Freitas et al.
2007, 2010). Some works have indicated that rubber can be
involved in plant defense because, after mechanical dam-
age, laticifer fluids are released and clot upon exposure to
air, thus entrapping small insects (Konno 2011). In addition
to mechanical protection, rubber may repel insects and
exhibit a potentially toxic effect on mammals (Albu-
querque et al. 2009; Ramos et al. 2011).
The soluble protein content in T. peruviana latex was
estimated to be 380 lg ml-1. In some latices, such as that
obtained from Euphorbia tirucalli, proteins were not
detected (Freitas et al. 2010). In other laticifer fluids,
proteins can be present in small amounts, as is the case for
Criptostergia grandiflora (260 lg ml-1) and Plumeria
rubra (330 lg ml-1), or at high concentrations, as in
Calotropis procera (8.85 mg ml-1) (Freitas et al. 2007,
2010).
T. peruviana latex primarily contained proteins with
apparent molecular masses ranging from 28 to 100 kDa
(Fig. 1a). After heating (100 °C for 10 min) in the presence
of a reducing agent (5 % 2-mercaptoethanol), a new
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20 kDa protein band was observed, whereas the 100 kDa
protein band disappeared. This result suggests that the
100 kDa protein can be at least a pentamer. Two-dimen-
sional gel electrophoresis generated 37 protein spots, with
apparent molecular masses ranging from 20 to 100 kDa
and pI values of \5.0 (Fig. 1b).
Interesting, the protein profiles of laticifer fluids are very
dissimilar. For instance, 2D SDS-PAGE analysis of C.
procera latex detected 212 proteins (10–113 kDa, pI 3–10),
P. rubra 118 proteins (12–117 kDa, pI \6.5), C. grandi-
flora 196 proteins (10–75 kDa, pI 3–10) and Taraxacum
brevicorniculatum 582 proteins (10–150 kDa, pI 4–7)
(Freitas et al. 2007, 2010; Wahler et al. 2012). Latex has
been described in almost 21,500 plant species, and it rep-
resents the cytoplasm of specialized cells termed laticifers
(Pickard 2008). Although proteins involved in plant pri-
mary metabolism may be common and abundant, latex
proteins have primarily been studied in relation to plant
defenses against fungi and insects (Agrawal and Konno
2009; Konno 2011; Souza et al. 2011). Because latex
chemical composition is very diverse, with several sec-
ondary metabolites and proteins, laticifers have not been
defined by their contents but rather by their origin, devel-
opment and overall anatomy (Hagel et al. 2008).
Protein identification by mass spectrometry
The proteins in the T. peruviana latex were mainly acidic
proteins (pI \ 5.0) (Fig. 1b). Overall, 37 protein spots
were detected, excised, digested by trypsin and analyzed by
mass spectrometry. A total of 33 proteins were identified,
including storage proteins (5), a peptidase inhibitor (1),
cysteine peptidases (20), phosphotransferase (1), osmotins
(2) and peroxidases (4) (Table 1). Eighty-six percent of the
analyzed proteins were identified, which was a fascinating
Fig. 1 a 1D SDS-PAGE (12.5 %) of the latex proteins from Thevetia
peruviana (TpLp). Legend: 30 lg of TpLP non-heated and without
5 % 2-mercaptoethanol (1) and heated at 100 °C for 10 min with 5 %
2-mercaptoethanol (2). (b) 2D SDS-PAGE (300 lg) of TpLP, pH
result because very limited proteomic information (only
nine protein sequences) is available for T. peruviana. The
proteins found in the NCBI or UniProt databases were all
involved in plant primary metabolism, including ribulose-
1,5-bisphosphate carboxylase/oxygenase and NADH
dehydrogenase.
Although other proteomic studies of latex have identi-
fied a large number of proteins, as was the case for
Taraxacum brevicorniculatum (278 proteins) and Lactuca
sativa (587 proteins), almost all the identified proteins were
involved in plant primary metabolism processes, including
rubber biosynthesis, glycolysis, nitrogen assimilation, fatty
acid synthesis and ubiquitin-mediated proteolysis. There-
fore, these proteins provided no direct evidence of the
involvement of laticifer proteins in plant defense against
insects or microorganism (Cho et al. 2009; Wahler et al.
2012). In this work, almost all of the identified proteins (27
proteins) are related to plant defense (PR-proteins) against
fungi or insects. PR-proteins currently comprise 17 fami-
lies and include the following families: PR-5, osmotin or
thaumatin-like proteins; PR-6, peptidase inhibitors; PR-7,
peptidases; and PR-9, peroxidases. These proteins have
been found in several laticifer fluids (Konno 2011).
Therefore, our results corroborate with the hypothesis that
laticifer fluids are involved in plant defense. In contrast,
five storage proteins were identified in the T. peruviana
latex (Table 1). This result was interesting because latex
fluids have been described to function as a defensive
mechanism but not as a means of protein storage. On the
other hand, studies have demonstrated that H. brasiliensis
latex is relatively rich in sucrose (1–50 mM), and amylo-
plasts were found in some Euphorbia species (Kekwick
2001). Thus, these results can open a discussion about a
novel function of the laticifers as protein and carbohydrate
warehouses.
3–10 and pH 4–7. The proteins were stained with 0.1 % Coomassie
Brilliant Blue R-350, excised from the gels, digested by trypsin and
analyzed by mass spectrometry (numbers 1–37)
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Table 1 List of proteins identified in Thevethia peruviana latex by ESI-QUAD-TOF
Spot Noa Teoric/experimental Protein Parental ion Sequences of all identified peptides ID (NCBI) Protein description
score
Molecular pI
mass (KDa)

1 70.6/16.9 6.4/4.3 72 1127.6278 IPSGFISYILNR gi|1168390 7S seed storage protein (Arachis hypogaea)
1378.7662 GTGNLELVAVR
2 20.3/21.8 4.6/4.3 53 1162.6344 GIGTIISSPYR gi|3318877 Soybean trypsin inhibitor (Glycine max)
4 50.4/23.8 5.2/4.4 50 1595.7212 NSWGGSWGEKGYIR gi|514804351 Cysteine peptidase (Setaria italica)
8 29.7/25.7 5.4/3.8 57 919.4172 IANAFDNR gi|42561793 Phosphotransferase (Arabidopsis thaliana)
11 33.0/24.6 4.9/4.8 99 1595.7120 NSWGGSWGEQGYIR gi|357437721 Cysteine proteinase (Medicago truncatula)
12 40.3/24.7 5.6/4.7 65 2833.3132 CGVALDHGVVVVGYGTENGVDYWLVR gi|587864551 Germination-specific cysteine protease (Morus
notabilis).
12 26.0/24.7 6.1/4.7 62 1110.4532 TNCNFDGAGR gi|19315 Osmotin-like protein (Solanum lycopersicum)
13 33.0/25.8 4.9/4.6 93 1595.7186 NSWGGSWGEQGYIR gi|357437721 Cysteine proteinase (Medicago truncatula)
14 22.9/26.9 8.4/4.7 200 1788.8436 NNCPYTVWAAAVPGGGR gi|88193285 Osmotin-like (Theobroma cacao)
2901.1885 CPDAYSYPKDDQTSTFTCPGGTNYR
15 25.9/26.9 4.9/4.4 88 1485.6318 NGGIDSEEDYPYK NSWGSSWGENGYIR gi|42563538 Cysteine protease-like protein (Pelargonium x
1611.7096 hortorum)
16–17 33.0/26.9 4.9/4.3 80 1595.6818 NSWGGSWGEQGYIR gi|357437721 Cysteine proteinase (Medicago truncatula)
18–20 52.7/28.8 5.2/5.3 54 1525.8248 AVASQPISVAIEAGGR gi|398330370 Papain-like cysteine protease (Fagopyrum
esculentum)
21 33.0/30.0 4.9/4.7 93 2429.2165 DYWIVRNSWGGSWGEQGYIR gi|357437721 Cysteine proteinase (Medicago truncatula)
22–23 33.0/29.8 4.9 94 1594.7284 NSWGGSWGEQGYIR gi|357437721 Cysteine proteinase. (Medicago truncatula)
24 33.0/29.5 4.9/4.2 143 2429.2102 DYWIVRNSWGGSWGEQGYIR gi|357437721 Cysteine proteinase (Medicago truncatula)
25 36.7/42.8 4.8/4.2 83 1595.7168 NSWGGSWGEQGYIR gi|357437721 Cysteine proteinase (Medicago truncatula)
26 39.1/62.8 6.6/6.9 109 1627.8744 1071.5194 QVFQNDIGQAAGLLR gi|307949718 Peroxidase (Rubia cordifolia)
YYVDLMNR
27–29 39.5/63.4 5.9/6.8 243 757.4272/1048.5416/ IIEDLR/RDGLNFATR/YYVDLMNRQ gi|14031049 Peroxidase (Nicotiana tabacum)
1087.4724/1813.8535 QGLFTSDQDLYTDRR
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Proteolytic activity

Cysteine proteinase (Medicago truncatula).

Cysteine proteinase (Medicago truncatula).

Papain-like cysteine protease (Fagopyrum
In the latex of T. peruviana, 20 cysteine peptidases with

Storage protein (Arachis hypogaea).

Storage protein (Arachis hypogaea).
different molecular masses and pI values were identified
Storage protein (Arachis hypogaea)

Storage protein (Arachis hypogaea)

(Table 1). The presence of several peptidases was con-
firmed by zymogram analysis containing 0.1 % gelatin
(Supplementary Fig. S1b). More than 100 different latices
are known to contain at least one peptidase, and peptidases
Protein description

have been recognized as the major endogenous enzymes in

esculentum).
these fluids (Domsalla and Melzig 2008). Of these proteins,
papain, a cysteine peptidase from Carica papaya latex, is
the most known and studied. In addition to papain, the latex
of C. papaya contains three other cysteine peptidases
(Mezhlumyan et al. 2003). Similarly, the latex of C. pro-
gi|357437715

gi|357437715
gi|398330370

cera (Freitas et al. 2007), C. grandiflora (Ramos et al.

gi|37789212

gi|37789212
gi|3703107

gi|3703107
ID (NCBI)

2014) and Ficus carica (Zare et al. 2013) possess multiple

cysteine peptidases. In F. carica 90 % of all latex proteins
are cysteine peptidases. The multiple peptidases in latex
are not products of self-hydrolysis of one or more pepti-
dases because they exhibit clear difference in their struc-
tures (molecular masses and isoelectric points), stability
(pH and temperature) and substrate specificities (Domsalla
Sequences of all identified peptides

and Melzig 2008).

The presence of peptidases in T. peruviana latex (TpLP)
was also confirmed by colorimetric assays. The maximum
RPFYSNAPQEIFIQQGR

AVASQPISVAIEAGGR
NSWGGSWGEQGYIR

NSWGGSWGEQGYIR

proteolytic activity was achieved at pH 5.0 and 6.0 using

azocasein and BANA as substrates. No activity was
TANDLNLLILR

TANDLNLLILR
TANELNLLILR

TANELNLLILR

detected when BApNA was assayed (Supplementary

Fig. S2). In presence of low concentrations of DTT
(1–3 mM), the proteolytic activity of TpLP was increased
approximately fourfold, using BANA as substrate
The indicated spot numbers were from 2D SDS-PAGE (Fig. 1b)
Parental ion

2050.0345
1268.7512
1254.7346
1268.7494
1595.7154
1254.7362
1595.7120
1525.8242
Protein
score

102

83
90
76
78
80
53
5.5/5.1

5.6/5.1
5.5/4.5
5.9/4.4
5.6/4.8
5.9/5.0
5.2/5.1
Teoric/experimental
pI

Fig. 2 a Gel filtration chromatography of Thevetia peruviana latex

mass (KDa)

(TpLp) on a Superdex-75 column. The proteins were eluted with

Molecular

25 mM sodium acetate buffer (pH 5.0). Flow rate: 0.3 ml min-1.

61.8/66.3

58.5/66.3
61.8/73.9
53.4/75.0
58.5/84.3
53.4/84.3
52.7/83.3
Table 1 continued

Fractions: 0.5 ml. b 1D SDS-PAGE (12.5 %) of the peak P1

recovered from gel filtration chromatography. The proteins were
stained with 0.1 % Coomassie Brilliant Blue R-350. A total of 10 lg
of protein (without heating and reducing agent) was added in well.
Spot Noa

c Zymogram for peptidase detection in the P1 fraction. d Glycoprotein

33–34

detection in the gel using Schiff’s reagent. Ribonuclease B was used

30

31
32

35
36
37

as a standard glycoprotein (1)

a

123

Fig. 3 Effect of pH (a), temperature (b) and specific peptidase
inhibitors (c) on the proteolytic activity of peruvianin-I. Each point
represents n = 3. The error bars indicate standard error of the mean.
In b and c, the proteolytic activity was assayed using azocasein at pH
(Supplementary Fig. S2). Activation by reducing agents
such as DTT, L-cysteine, reduced glutathione and 2-mer-
captoethanol indicates that the affected enzymes belong to
the cysteine peptidase class (Freitas et al. 2007). Higher
DTT concentrations (5–50 mM) decreased the enzyme’s
proteolytic activity, which can be attributed to the reduc-
tion of intramolecular disulfide bonds and subsequent
enzyme denaturation (Supplementary Fig. S2). Overall,
DTT had slight effect on proteolytic activity when azoca-
sein was the substrate. The optimum proteolytic activity
was obtained at 45 °C and was observed up to 60 °C
(Supplementary Fig. S2). Only E-64 and iodoacetamide
(two cysteine peptidase inhibitors) were able to inhibit the
proteolytic activity of TpLP (Supplementary Fig. S2). All
these results confirm the presence of cysteine peptidases in
T. peruviana latex.
Purification of a cysteine peptidase (peruvianin-I)
The protein identification and zymogram analysis indicated
the presence of several peptidases in TpLP. Thus, to purify
123
Planta
6.0. d Effect of substrate concentration on the proteolytic activity of
peruvianin-I using azocasein as substrate at pH 6.0 and 37 °C. e The
Km value was calculated from the Lineweaver–Burk plot
a peptidase, different chromatographic steps, including the
use of DEAE- and CM-Sepharose columns (pH 5.0 and
6.0), were performed. However, these chromatographic
steps were not effective because only one peak was
obtained (data not shown). In contrast, four peaks were
obtained after gel filtration chromatography on a Superdex-
75 column (Fig. 2a). Only the P1 peak was homogeneous
in the 1D SDS-PAGE analysis (Fig. 2b). Zymogram con-
taining 0.1 % gelatin showed the protein in the P1 peak
(100 kDa) is a peptidase (Fig. 2c). The peptidase was a
glycoprotein (22 % carbohydrate, w/w) (Fig. 2d) and
exhibited a specific activity of 2.75 AU lg-1. The yield of
the purified protein was 12.7 % of the TpLP fraction. This
peptidase was termed peruvianin-I.
Proteolytic activity of peruvianin-I
Peruvianin-I was able to hydrolyze azocasein and BANA
but not BApNA (Fig. 3a). Azocasein is a nonspecific
substrate, therefore, all peptidases are able to hydrolyze
this molecule, whereas BANA and BApNA have been used
Planta
Fig. 4 a Mass spectrum of native peruvianin-I determined using ESI-
QUAD-TOF. [Inset 1D SDS-PAGE (12.5 %) of peruvianin-I heated
or non-heated at 100 °C for 10 min without (1) or with (2) 5 %
2-mercaptoethanol. b 2D SDS-PAGE (12.5 %) of peruvianin-I. A
as specific substrates for cysteine and serine peptidases,
respectively (Freitas et al. 2007, 2010). The optimal pH
range for the hydrolysis of azocasein and BANA by
peruvianin-I was 5.0–6.0, with maximal activity at
25–37 °C (Fig. 3a, b). Only E-64 and iodoacetamide
(IAA), which are both specific cysteine peptidase inhibi-
tors, were able to inhibit the proteolytic activity of peru-
vianin-I (Fig. 3c). All these results reinforce the
observation that peruvianin-I is a cysteine peptidase.
sample of 100 lg of protein was applied to the strip (pH 3–10). Inset
tridimensional view of the spots 2–6. The molecular masses, isoeletric
points (pI) and intensity of the spots 1–6 were estimated using
ImageMaster 2D Platinum Software 6.0 (Amersham Biosciences)
The proteolytic activity of peruvianin-I reached satura-
tion at 100 lM azocasein (pH 6.0 and 37 °C) and followed
Michaelis–Menten kinetics (Fig. 3d). The observed Km
value was 17.6 lM, as obtained from the Lineweaver–Burk
plot (Fig. 3e). This value was similar to the Km of other
latex cysteine peptidases such as procerain (Km = 22 lM),
procerain B (31 lM), ervatamin A (11 lM) and ervatamin
B (25 lM) (Supplementary Table 1). The Vmax and Kcat
values were 4.6 9 10-9 M s-1 and 0.23 s-1, respectively.
123

Fig. 5 Structural analysis of peruvianin-I by atomic force microscopy (AFM) in two different scales
In addition to the Km values, other characteristics of
peruvianin-I and latex cysteine peptidases, including the
optimal pH and temperature conditions, are compared in
Supplementary Table 1.
Structural analysis and other enzymatic activities
of peruvianin-I
Peruvianin-I was observed as a single band in the 1D SDS-
PAGE gel, with an estimated molecular mass of 100 kDa
(Fig. 2b, Inset Fig. 4a). Most of the latex cysteine pepti-
dases are relatively small, with molecular masses ranging
from 20 to 30 kDa (Supplementary Table 1). In contrast,
latex serine peptidases are large proteins, ranging from 33
to 117 kDa (Domsalla and Melzig 2008). Only after heat-
ing at 100 °C, independently of the reducing agent (2-
mercaptoethanol), peruvianin-I generated a single protein
band of approximately 20 kDa in the 1D SDS-PAGE gel
(Inset Fig. 4a). This fact was confirmed by mass spec-
trometry, which detected only peaks of approximately
20,522 Da (Fig. 4a).
Two-dimensional gel electrophoresis of peruvianin-I
indicated the presence of five spots with very similar
molecular masses and pI values (Fig. 4b). The five spots
are expected to be subunits of the 100 kDa protein released
in the presence of the strong denaturing medium (7 M urea
and 2 M thiourea) and not contaminating proteins. The
minor differences (pI and molecular masses) between the
monomers (Fig. 4a, b) are expected to be due micro
heterogeneity in the amino acid sequence of the subunits or
events of alternative splicing of the same gene. Even,
possible differences on subunits of peruvianin-I can be the
result of different glycosylation patterns because the mass
spectrum of the subunits of peruvinain-I showed the
123
Planta
presence of peaks (Fig. 4a) with differences corresponding
to the mass of a monosaccharide very common in plant
glycoproteins (Mannose) [162 Da = 180 (mannose) -18
(water)]. Attempts to identify positive or negative reaction
on each 2D spot with Schiff́s reagent failed. Only a single
and diffuse zone was observed (data not shown). It is not
clear whether all subunits are glycosylated or not, neither if
the same glycan is present.
Atomic force microscopy (AFM) and gel filtration
chromatography were used to clarify the oligomeric
structure of peruvianin-I. Atomic force microscopy was
used because it is a powerful technique with nanometer-
scale resolution that allows the analysis of molecules in
solution (native conditions). AFM is also routinely used as
a nanotool for various measurements such as the recogni-
tion and localization of specific molecules, visualization of
membrane proteins in their lipid bilayer and determination
of protein structure (Müller et al. 2002). The high-resolu-
tion images from atomic force microscopy (Fig. 5a, b)
showed the possible hexameric structure of peruvianin-I,
organized as a trimer of dimers forming a central channel.
The native molecular mass of peruvianin-I assessed by gel
filtration chromatography was estimated to be approxi-
mately 120 kDa (Fig. 6). Therefore, the most likely
hypothesis is that peruvianin-I is a homohexamer, as sug-
gested by atomic force microscopy and gel filtration
chromatography.
Structural analysis by X-ray crystallography demon-
strated that barley and wheat germins are homohexamers
organized as a trimer of dimers with a central channel
(Woo et al. 2000), very similar to peruvianin-I (Fig. 5a, b).
The interactions between the dimers are mainly
hydrophobic and involve aliphatic amino acids on surfaces
(Woo et al. 2000), which are likely responsible for the high
Planta

stability of germin to protease degradation and dissociation

by various denaturing agents such as detergents. Therefore,
germin does not dissociate when analyzed by SDS–poly-
acrylamide gel electrophoresis (SDS-PAGE) unless boiled
in the presence of a detergent or strong denaturing medium
(McCubbin et al. 1987). Similar to germins, peruvianin-I
can be a homohexamer that dissociated only after heating
at 100 °C or in presence of a strong denaturing medium

value

5e-08

9e-08

2e-07

2e-07

3e-07
E-

–
(Inset Fig. 4a, b).
The possibility that contaminating proteins might have
been co-purified with peruvianin-I was also excluded after

% Identity
N-terminal amino acid sequencing of the native (120 kDa)

64

58

66

58
58
–
and denatured (20 kDa) bands (Inset Fig. 4a), because a
unique sequence was obtained. The peruvianin-I N-termi-

XP_007044465.1
nal sequence showed high similarity to that of germin/

EMT00501.1

EMS56245.1

CAB55559.1
ABS44860.1
Accession
number
germin-like proteins (Table 2).

C0HJY2
Three internal sequences of peruvianin-I were obtained
by mass spectrometry after proteolysis of the protein with
trypsin (Table 3). These sequences comprised three pep-

Table 2 The N-terminal amino acid sequence of Peruvianin-I, compared with other plant proteins
tides that also have high identity with germin/germin-like

25SDPSPLQDFCVADLNSPVRVNGFVCKNPMNASADDF60

24SDPSPLQDFCVADMNSPVRVNGFVCKNPMEVNADDF59

23SDPGPLQDFCVADMQSPVRVNGFVCKNPMEVNADDF58

24SDPSPLQDFCVADMNSPVRVNGFVCKNPMEVNADDF59
DPDPLQDFCVADAKSPFFLNGAPCLNPSLALSSHF58
1ADPGPLQDFCLADLNSPLFINGYPCRNPALAISDDF36
proteins. The peptide EYPGLNTLGVSLNR showed high
identity with germin-like proteins from Cucumis melo
(92 %) and Prunus mume (85 %). The sequence NPA-
LATDDDFLYSGFK was very similar to germin-like
proteins from Erythranthe guttatus (75 %) and Selaginella Sequence
moellendorffii (79 %).
Germins are glycoproteins that were first identified
during the germination of wheat embryos (Bernier and
Berna 2001). After, germin-like proteins were identified in
different tissues, including seed, flowers, leaves and roots,
but not in latex fluids (Davidson et al. 2009). Germins
display oxalate oxidase activity (EC 1.2.3.4), catalyzing
the formation of one mole of H2O2 and two moles of CO2 24
Thevetia peruviana

Theobroma cacao

Triticum aestivum
Organism

Aegilops tauschii

Triticum urartu
Oryza sativa
Identification

(Peruvianin-I)
Germin-like

Germin-like

Germin-like

Germin-like

Germin-like
Peptidase

protein

protein

protein

protein

protein

Fig. 6 Native molecular mass determination of peruvianin-I by gel

filtration chromatography on Superose 12 HR column. The protein
profiles were monitored at 230 nm using a 0.3 ml min-1 flow rate.
Fractions 0.7 ml

123
 Planta

Table 3 Internal sequences of Peruvianin-I determined by ESI-QUAD-TOF

Spota Peptide Theoretical mass (Da) Experimental mass (Da) Delta (Da) Accession numberb Identity (%)

2 NPATVTADDFLYSGFK 1744.83 1744.82 0.01 ref|XP_012831332.1| 75

2 EYPGLNTLGVSLNR 1531.79 1531.78 0.01 ref|XP_008239680.1| 85
3 NPALATDDDFLYSGFK 1772.82 1772.87 -0.05 ref|XP_002975893.1| 79
3 LNTLGVSLNR 1085.61 1085.65 -0.04 ref|XP_008466206.1| 90
a
The indicated spot numbers were from 2D SDS-PAGE (Fig. 4b)
b
NCBi Reference Sequence

Table 4 Some characteristics of Peruvianin-I and germin/germin-like proteins

Protein (ID)a Plant specie Structure Units Isoelectric Glycosylation Tissue Oxalate References
(Da) point (pI) oxidase
activity

Peruvianin-I Thevetia Hexamer 20,500 4.0 Yes Latex No This study

(C0HJY2) peruviana
Germin (P45850) Hordeum Hexamer 21,203 5.5 Yes Seedling root Yes (Lane et al. 1993;
vulgare Woo et al. 2000)
Germin-like Arabidopsis Hexamer 23,925 6.5 Yes Stems and No (Membré et al.
protein thaliana developing 2000)
(P92996) embryos
Germin-like Citrus Hexamer 23,094 Ndb Yes Fruit No (Crespo et al. 2006)
protein sinensis
(P84159)
a
UniProt knowledgebase
b
Nd not determined

from one mole of oxalate and oxygen (Davidson et al.
2009). Although oxalate may be present in low levels, it
can be formed from L-ascorbic acid, which is found in high
quantities in plants (Keates et al. 2000). Interestingly, some
phytopathogenic fungi may secrete high amounts of oxa-
late, constituting an essential determinant of pathogenicity.
Thus, plants capable of degrading oxalate may be resistant
to these pathogens (Donaldson et al. 2001). The rein-
forcement of cell wall structures and the defense reactions
against pathogens is well known to be highly dependent on
H2O2-generating enzymes (Scheler et al. 2013). Therefore,
germin proteins have been classified as pathogenesis-re-
lated proteins (PR-proteins) belonging to families PR-15
and PR-16 (van Loon et al. 2006).
Three different assays were performed to evaluate the
oxalate oxidase activity of peruvianin-I. Peruvianin-I did
not exhibit oxalate oxidase activity in vitro, in situ or on
nitrocellulose membranes even at 1 mg ml-1. Similar
results were obtained for three germin-like proteins from
Arabidopsis thaliana (Membré et al. 2000). ‘‘True’’ ger-
mins belong to a highly homogeneous group that has only
been found in cereals and usually possess oxalate oxidase
activity (Davidson et al. 2009). On the other hand, germin-
like proteins have been associated with other activities,
such as superoxide dismutase (Guicciardo et al. 2007).
Peruvianin-I (1 mg ml-1) did not exhibit superoxide dis-
mutase activity, in vitro and by zymogram. Some charac-
teristics of peruvianin-I and germin/germin-like proteins,
including structure, isoeletric point (pI) and oxalate oxidase
activity are compared in Table 4.
Antifungal activity of peruvianin-I
The antifungal activity of latex proteins has been described
in the literature, reinforcing the role of latices in plant
defense. This activity was associated mainly with cysteine
peptidases (Souza et al. 2011; Ramos et al. 2014). Latex
proteins from T. peruviana (TpLP) and peruvianin-I did not
exhibit antifungal effects on Fusarium solani, Rhizoctonia
solani and Colletotrichum gloeosporioides, even in pres-
ence of DTT (a cysteine peptidase activator). The lack of
antifungal activity of peruvianin-I may be related to its low
proteolytic activity when compared with other antifungal
cysteine peptidases, such as papain and Calotropis procera
peptidases. This low proteolytic activity may be a conse-
quence its unusual tridimensional structure (homohex-
amer), since, in general, latex cysteine peptidases are
monomers.

123
Planta
Conclusion
Cysteine peptidases frequently are the major constituents in
latex fluids, although the qualitative and quantitative pro-
tein profiles are very different. In this study, besides plant
defense proteins, five storage proteins were identified,
providing evidence of an additional function of latex fluids.
Novelty in our study was the identification of a germin-like
protein exhibiting proteolytic kinetic. Peruvianin-I is the
first cysteine peptidase sharing structural characteristics
with germin-like proteins (GLPs). Peruvianin-I did not
exhibit oxalate oxidase and superoxide dismutase activities
or antifungal effects.
Author contribution statement CDTF designed research,
analyzed some data and wrote the manuscript. WTC,
MZRS, IMV conducted enzymatic assays and analyzed
data. RAM, ACOMM, BAMR and FBMBM designed and
performed all proteomic assays. JSS and LMRA performed
and analyzed the data from atomic force microscopy. MVR
analyzed data and wrote the manuscript. All authors read
and approved the manuscript.
Acknowledgments This study was supported by grants from the
following Brazilian Agencies: Conselho Nacional de Desenvolvi-
mento Cientı́fico e Tecnológico (CNPq) and Coordenação de Aper-
feiçoamento de Pessoal de Nı́vel Superior (CAPES). We thank Dr.
Sandra Vairo Cavalli for critical reading of the manuscript.
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