Peptides 55 (2014) 65–78

Contents lists available at ScienceDirect

Peptides
journal homepage: www.elsevier.com/locate/peptides

Review

The use of versatile plant antimicrobial peptides in agribusiness and

human health
Elizabete de Souza Cândido a,b , Marlon Henrique e Silva Cardoso b , Daniel Amaro Sousa b,d ,
Juliane Cançado Viana b,d , Nelson Gomes de Oliveira-Júnior b,c , Vívian Miranda a,b ,
Octávio Luiz Franco a,b,d,∗
a
Programa de Pós-Graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, Brasília, DF, Brazil
b
Centro de Análises Proteômicas e Bioquímicas, Pós-Graduação em Ciências Genômicas e Biotecnologia, Universidade Católica de Brasília, Brasília, DF, Brazil
c
Programa de Pós-Graduação em Biologia Animal, Universidade de Brasília, Brasília, DF, Brazil
d
Programa de Pós-Graduação em Patologia Molecular, Universidade de Brasília, Brasília, DF, Brazil

a r t i c l e i n f o a b s t r a c t

Article history: Plant immune responses involve a wide diversity of physiological reactions that are induced by the
Received 16 January 2014 recognition of pathogens, such as hypersensitive responses, cell wall modifications, and the synthesis of
Received in revised form 5 February 2014 antimicrobial molecules including antimicrobial peptides (AMPs). These proteinaceous molecules have
Accepted 7 February 2014
been widely studied, presenting peculiar characteristics such as conserved domains and a conserved
Available online 16 February 2014
disulfide bond pattern. Currently, many AMP classes with diverse modes of action are known, having been
isolated from a large number of organisms. Plant AMPs comprise an interesting source of studies nowa-
Keywords:
days, and among these there are reports of different classes, including defensins, albumins, cyclotides,
Plant defense
Antimicrobial peptides
snakins and several others. These peptides have been widely used in works that pursue human disease
Biotechnological properties control, including nosocomial infections, as well as for agricultural purposes. In this context, this review
Antibiotics will focus on the relevance of the structural-function relations of AMPs derived from plants and their
proper use in applications for human health and agribusiness.
© 2014 Elsevier Inc. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
1.1. Plant AMP classes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
1.1.1. Defensins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
1.1.2. Knottin-like . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
1.1.3. 2S albumins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
1.1.4. Cyclotides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68
1.2. Lipid transfer proteins (LTPs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69
1.3. Heveins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
1.4. Snakin/GASA peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
1.5. Myrosinase binding protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
1.6. Glycine-rich protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
1.7. ␣-Hairpinins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
1.8. ␣␤-Trumpet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
1.9. Short non-disulfide peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72

∗ Corresponding author at: Universidade Católica de Brasília, Pós-Graduação em Ciências Genômicas e Biotecnologia/Centro de Análises Proteômicas e Bioquímicas – SGAN
916, Av. W5, Módulo C, sala 219, Brasília, DF, CEP 70790-160, Brazil. Tel.: +55 61 34487167/34487220; fax: +55 61 33474797.
E-mail addresses: ocfranco@gmail.com, ocfranco@pos.ucb.br (O.L. Franco).

http://dx.doi.org/10.1016/j.peptides.2014.02.003
0196-9781/© 2014 Elsevier Inc. All rights reserved.
66 E. de Souza Cândido et al. / Peptides 55 (2014) 65–78

2. Plant AMP applications for human health purposes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

3. Plant AMPs: applications for agricultural purposes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

1. Introduction which stabilize an antiparallel ␤-sheet conformation flanked

by an ␣-helical segment generating a compact structure that
Animals and plants share common elements in their defense confers resistance toward extreme pH, temperatures and protease-
processes against pathogens, including direct antimicrobial activ- mediated degradation [7,15,154]. Moreover, plant defensins are
ities by synthesis of hydrolytic enzymes (chitinases, glucanases, mainly small, cationic peptides of 45–54 amino acids in length,
proteinases and oxidases) [109] as well as antimicrobial peptides which have typically been isolated from seeds, but can also be
(AMPs) [13,53]. Furthermore, indirect innate immunity strategies found in other plant tissues such as leaves, flowers, roots and
have also been observed; these include some activities in host stems [80,81]. Commonly, their tridimensional structure consists
organisms, like the production of hydrogen peroxide or synthesis of triple-stranded antiparallel ␤-sheets and one ␣-helix lying in
of host defense peptides (HDPs) that can cause an immunomod- parallel with the ␤-sheet. They are stabilized by four disulfide
ulatory response [145]. AMPs have been isolated from a wide bonds, which incorporate the cystine-stabilized ␣␤ (Cs␣␤) motif
number of sources, including animals, bacteria and plants [73]. In (C1 Xn C2 X3 C3 Xn C4 Xn C5 X1 C6 ) [53,153]. The Cs␣␤ motif can also be
a general perspective, these molecules can be organized into four found in defensins and toxins from other organisms such as insects
large groups: (1) linear ␣-helical peptides; (2) cyclic peptides with and scorpions, respectively [80,82]. Interestingly, two defensins,
␤-sheet structures with two or more disulfide bonds; (3) a com- named PhD1 and PhD2, isolated from Petunia hybrida, showed a
bination of helices and ␤-sheets stabilized by disulfide bonds; (4) different structural pattern, presenting one extra disulfide bond,
peptides with ␤-hairpin or looped arrangement containing disul- which seems to replace a conserved hydrogen bond network
fide bonds; (5) linear peptides with an unusual predisposition for that is present in the classical four-disulfide-bond plant defensins
particular repetition of some amino acid residues, including pro- (C1 Xn C2 Xn C3 Xn C4 X3 C5 Xn C6 Xn C7 X1 C8 ) [120]. Among their func-
line, glycine, tryptophan or histidine and (6) short peptides with tions, plant defensins are known to manifest a wide array of
coil structures or with no defined secondary structures [128]. AMPs biological activities, such as antifungal [104] and antibacterial [38],
have been long described as components of the plant defense arse- and they also act on protein translation [27] and enzyme inhibition
nal, regulating defense barriers or responses mainly activated by [53,98].
direct infections [53,109]. Most AMP families described for plants The first antifungal plant defensins described and characterized
are cysteine-rich (4–8 Cys residues), with their globular structures were two isoforms named Raphanus sativus antifungal protein 1
being stabilized by disulfide bonds (S S) that provide a high struc- (Rs-AFP1) and R. sativus antifungal protein 2 (Rs-AFP2), which were
tural stability; among these are defensins and snakins [13]. AMPs isolated from radish (R. sativus L.) seeds [157] (Table 1). Rs-AFP1
can share similar properties, such as their small size (approximately and Rs-AFP2 are basic peptides stabilized by disulfide bonds with
10 kDa), positive charges and high hydrophobic residues portion. 35–43 amino acid residues length (5 kDa) arranged in oligomeric
These properties provide the possibility to fold into an amphiphilic quaternary organization [157]. When evaluated against different
structure with distinct hydrophobic and positively charged amino fungi, Rs-AFP1 and Rs-AFP2 were able to inhibit Pyricularia oryzae,
acid residues. Despite these general characteristics, some excep- Phoma betae and Cercospora beticola (Table 1) [157]. Since then,
tions can also be observed, such as peptides smaller than 10 kDa several studies have been done in order to purify attractive new
and others with anionic charges [94,109]. Families of plant AMPs proteinaceous compounds from plants for therapeutic or agricul-
have been classified based on their homologies at the primary tural purposes. Years later, the primary structure of the cowpea
structural level [15,109] as well by the pattern mainly provided by (Vigna unguiculata) defensin II (Cp-thionin II) was described [48].
disulfide bond pattern and structural scaffold [109]. In accordance Cp-thionin II is a monomeric peptide of 47 amino acids in length,
with such properties, several classes of plant AMPs have been iden- molecular mass of 5.2 kDa and four disulfide bonds, which presents
tified, including defensins, knottin-like, 2S albumins, cyclotides, high similarities with known defensins. Cp-thionin II was fur-
lipid transfer proteins (LTPs), heveins and snakins. In addition to ther evaluated against both Gram-positive and -negative bacteria,
a vast structural diversity, plant AMPs also have a broad spectrum revealing remarkable bactericidal activities against Staphylococ-
of activity against bacteria, fungi and viruses [109]. The mode of cus aureus, Escherichia coli and Pseudomonas syringae (Table 1)
action of AMPs has been under continuous review, and in general [48]. Additionally, Fujimura et al. [50] purified two antimicrobial
peptide functions vary from membrane permeabilization to a com- defensins, named Fa-AMP1 and Fa-AMP2, from buckwheat (Fagopy-
plex intracellular signaling, also acting with immuno-modulatory rum esculentum Moench.) seeds. Fa-AMP1 and Fa-AMP2 are also
activities [128]. monomeric, cysteine-rich peptides consisting of 40 amino acid
residues and molecular masses of approximately 4 kDa [50]. These
1.1. Plant AMP classes peptides were active also against fungi such as Fusarium oxyspo-
rum and Geotrichum candidum (Table 1) [50]. The same authors
1.1.1. Defensins also showed that two other monomeric defensins, Pp-AMP1 and Pp-
The first plant defensins were isolated from wheat and bar- AMP2, from Japanese bamboo shoot Phyllostachys pubescences, have
ley and were called ␥-thionins, due to their molecular masses antimicrobial activities against several plant-pathogenic bacteria
of 5 kDa and the presence of four disulfide bonds, a pattern and fungi (Table 1) [49].
also found in ␣- and ␤-thionins. However, it was then discov- Furthermore, Lay et al. [82] structurally characterized, for the
ered that ␥-thionins were structurally different from ␣- and first time, a plant defensin from Nicotiana alata flowers named
␤-thionins, being then called defensins [98]. Defensins consti- NaD1. NaD1 is a cysteine-rich, cationic peptide of 47 amino acids
tute an important class of antimicrobial peptides with a highly residues in length, which presents a pattern of conserved residues
conserved structural scaffold, and they have been isolated from such as Ser7 , Glu27 , Gly32 , Phe10 and Gly12 [82]. Electrostatically,
different organisms such as insects, mammals and plants [154]. NaD1 is characterized by a predominant cationic ␤-sheet and a neu-
In general, defensins have three to five disulfide bonds (Fig. 1A), tral ␣-helix. Moreover, it is known that NaD1 acts as a proteinase
 E. de Souza Cândido et al. / Peptides 55 (2014) 65–78 67

Fig. 1. Three dimensional structures of plant antimicrobial peptides from different classes including (a) 2S albumin (pdb code: 1PSY); (b) defensin (pdb code: 1N4N); (c)
hevein (pdb code: 1HEV); (d) a-harpinin (pdb code: 2L2R); (e) cyclotide (pdb code: 1KAL); (f) knottin (pdb code: 2ETI); (g) lipid transfer protein 1 (pdb code: 1SIY) and (h)
myrosinase binding protein 1 (pdb code: 2JZ4). Structures were visualized with Pymol. Disulphide bonds were highlighted as yellow sticks.

inhibitor as well as presenting antifungal and insecticidal activities
(Table 1) [82]. Other defensins and thionins have also been charac-
terized as hydrolase inhibitors. In a study performed by Melo et al.
[97] it was reported that a thionin from cowpea (Vigna unguiculata),
named Cp-thionin, was able to inhibit bovine trypsin [97]. Years
later, Pelegrini et al. [120] purified a defensin from V. unguiculata,
named VuD1, and reported novel insights about its mechanism of
action as an inhibitor of insect-pest ␣-amylases [120]. However,
despite presenting enzyme inhibitor properties these peptides did
not show any antimicrobial activity, suggesting high functional
selectivity in the defensin multifunctional class.
1.1.2. Knottin-like
The knottin peptide was first reported ten years ago by Le
Nguyen and co-workers [83], who recognized a single three-
dimensional fold which featured a cystine-knotted triple-stranded
␤-sheet in Ecballium elaterium seeds. Based on this “knot-like” fea-
ture, they introduced the term “knottins” for this molecular scaffold
[52]. Typically, knottins have fewer than 40 residues in length and
a structure defined by three antiparallel ␤-strands connected by
loops with variable length and sequence, as well as a conserved pat-
tern of six cysteine amino-acid residues (C1 Xn C2 Xn C3 C4 Xn C5 Xn C6 )
(Fig. 1B) [52,74]. The standard arrangement of three disulfide bonds
provides the structure with an extraordinary proteolytic, ther-
mal and chemical stability [74]. These characteristics show a high
potential for drug design.
Cheek and co-workers [25] reported that knottin-like topology
could be found in nearly 40% of the disulfide-rich domain struc-
tures, and it is characterized by two adjacent disulfide bonds (one
bond is formed by the first and third cysteine residues and the
other by the second and fourth cysteine residues in the primary
sequence) and a conserved ␤-hairpin, on which the third and fourth
cysteine residues are located. The two disulfide bonds are roughly
perpendicular, forming an X or cross, and the knottin-like core is
also known as the disulfide ␤-cross. Several unrelated protein fam-
ilies in diverse organisms have the knottin-like structure, such as
toxins from cone snails, spiders, bugs, horseshoe crab and scorpions
[55]. In plants, proteins such as defensins and protease inhibitors
also have the knottin-like cystine motif [55].
Knottins are able to bind to various molecular targets and
perform a number of biological activities, including cytotoxic,
antimicrobial, insecticidal, anti-HIV and hormone-like activities
[30]. Two peptides designated Mj-AMP1 and Mj-AMP2 were iso-
lated from Mirabilis jalapa L. seeds and contain the knottin-like
cystine motif. These peptides exhibit antifungal activity against 13
plant pathogenic fungi including Botrytis cinerea, Alteraria brassi-
cola, Fusarium oxysporum and Colletotrichum lindemuthianum and
two Gram-positive bacteria (Bacillus megaterium and Sarcina lutea)
(Table 2) [18]. Moreover, the antifungal peptide PAFP-S isolated
from Phytolacca americana (pokeweed) seeds had its structure
solved by NMR spectroscopy showing the knottin fold properties
[52] (Table 2) [51,52].

Table 1
Plant defensins active against microorganisms.

Peptides Rs-AFP1 Rs-AFP2 Cp-thionin II Fa-AMP1 Fa-AMP2 NaD1 Pp-AMP1 Pp-AMP2

Isolated from Raphanus sativus Vigna unguiculata Fagopyrum esculentum Nicotiana alata Phyllostachys pubescences
References [19] [20] [21] [14] [22]

IC50 (␮M)
Agrobacterium radiobacter – –
Agrobacterium rhizogenes – –
Cercospora beticola 0.4 0.5
Erwinia carotovora – –
Fusarium oxysporum – –
Phoma betae 0.4 0.3
Pyricularia oryzae 0.1 0.1
– 6.2 4.3 – 3.2 2.6
– 5.1 6.1 – 3.2 3
– – – – – –
– 2.8 3.8 – 4.7 4.1
– – – <1.15 – –
– – – – – –
– – – – – –

MIC (␮M)
Escherichia coli – – 12.2 – – – – –
Pseudomonas aeruginosa – – 8 – – – – –
Staphylococcus aureus – – 24.4 – – – – –

IC50, inhibit cellular proliferation by 50%; MIC, minimum inhibitory concentration; ␮M, micromolar.
68 E. de Souza Cândido et al. / Peptides 55 (2014) 65–78

Table 2 alata seeds, was able to inhibit growth of the filamentous fungus
Plant knottin-like active against microorganisms.
Colletotrichum gloeosporioides (Table 3) [132]. Another 2S albumin
Peptides Mj-AMP1 Mj-AMP2 PAFP-S peptide named Pe-AFP1 previously isolated from Passiflora edulis
was capable of inhibiting the development of filamentous fungi
Isolated from Mirabilis Phytolacca
jalapa americana Trichoderma harzianum, F. oxysporum and A. fumigatus (Table 3)
References [30] [25] [122].
IC50 (␮M) The antibacterial activity of 2S albumin family members has
Alternaria brassicola 3 0.9 – also been reported. An antimicrobial protein (SiAMP2) was isolated
Armillaria mellea – – 7.1 from Sesamum indicum kernels and was able to inhibit development
Ascochyta pisi 30.3 0.9 – of Klebsiella sp. (Table 3). In the same study, in silico analysis demon-
Alternaria tenuis – – 14.7
Botrytis cinerea 9.1 0.3 –
strated that conserved and exposed cationic amino acid residues, as
Cercospora beticola 1.5 0.3 – well as a hydrophobic domain, might be responsible for the antimi-
Colletotrichum lindemuthianum 0.9 0.1 – crobial activity [97]. Moreover, it was also observed that there is
Fusarium culmorum 4.5 0.4 – a wide variation in amino acid residues positions in 2S albumin
Fusarium graminearum – – 14.7
sequences, although the cystine scaffolds have been extremely con-
Fusarium oxysporum – – 14.7
Fusarium oxysporum f. sp. Lycopersici 30.3 1.5 – served, suggesting that the storage function does not depend on a
Fusarium oxysporum f. sp. Pisi 2.3 0.7 – standard position for amino acids in the sequence [96].
Morchella conica – – 7.1
Nectria haematococca 2.3 <0.1 – 1.1.4. Cyclotides
Phoma betae 3 0.9 –
Cyclotides are a family of small, cyclic peptides ranging between
Pyricularia oryzae 0.9 <0.1 –
Pyrenophora tritici-repentis 45.4 2.9 – 28 and 37 amino acid residues, which have been isolated from
Rhizoctonia solani 9.1 2.2 – several plant families such as Rubiaceae, Violaceae, Poaceae
Trichoderma viridae – – 7.1 and Fabaceae [26,66]. Two cyclotide subfamilies have also been
Verticillium dahliae 1.8 <0.1 –
described, named Möbius and bracelet, and their classification is
Venturia inaequalis 1.8 0.1 –
based on the presence, in the Möbius family, or absence, in the
IC50: inhibit cellular proliferation by 50%; ␮M: micromolar. bracelet family, of a cis-proline in loop 5 in the cyclotide backbone
[123]. Cyclotide biosynthesis involves excisions from precursor
polypeptides and cyclization processes that are thought to be
1.1.3. 2S albumins mediated by asparaginyl endopeptidases [123]. Structurally, they
The 2S albumins are storage proteins essential for plant main- present a circular backbone, composed of six loops, which is cross-
tenance and growth, showing an additional function related to linked by six conserved cysteine residues arranged in a knotted
plant defense. 2S albumins represent a water-soluble protein stor- manner, known as a cyclic cystine knot (CCK) and stabilized by
age group, glutamine-rich and present in monocotyledonous and three disulfide bonds (Fig. 1D) [36,123,127]. This cystine knot is
dicotyledonous plants, with a sediment coefficient (S20.w ) of 2S formed when the first two disulfide bonds, Cys1 -Cys4 and Cys2 -
[21,37]. These proteins were isolated for the first time as an aller- Cys5 , with their interconnecting backbone segments, are able to
genic protein fraction of castor bean seeds [37]. Normally, 2S form a ring that is penetrated by a third disulfide bond Cys3 -Cys6
albumins have been encoded as a multigenic family with several [65]. The CCK framework also confers on cyclotides a high resis-
isoforms that are subject to post-translational modification, like tance to thermal and chemical denaturation and to proteolytic
proteolytic processes [21]. degradation, making them potential candidates, for example, for
The amino acid sequence of the first 2S albumin was isolated the development of peptide-based therapeutics with oral bioavail-
from Ricinus communis seeds [143]. As is usual, this 2S albumin ability [54]. In addition to their stability, cyclotides are also well
protein was composed of two different subunits derived from a known for several other biological functions such as anti-tumoral
single precursor molecule. This precursor polypeptide is cleaved [90], anti-HIV [61], nematicidal [33], hemolytic [163], insecti-
by an asparaginyl endopeptidase during its post-translational cidal [67] and antimicrobial [127,151]. All these bioactivities make
processing, generating a heterodimer stabilized by two disulfide cyclotides attractive and promising for therapeutic and diagnostic
bonds (Fig. 1C) and with molecular masses of approximately 4 purposes.
and 9 kDa [63,96]. In general, they are small proteins (12–15 kDa), Pranting et al. [127], using reverse-phase HPLC, isolated cyclovi-
with a characteristic distribution of eight cysteines in a con- olacin O2 (cyO2) from Viola odorata, vaby A and D from Viola
served pattern (C1 Xn C2 Xn C3 C4 Xn C5 X1 C6 Xn C7 Xn C8 ), and different abyssinica and kalata B1 and B2 from Oldenlandia affinis, in order
polypeptide chains linked by two disulfide bridges [115]. Further- to compare the antimicrobial activities of these small cyclic pep-
more, there are ␣-amylase/trypsin inhibitors and nonspecific lipid tides as well as the importance of surface-exposed charged residues
transfer proteins which show structural similarity to 2S albumin for their activities [127]. Firstly, antimicrobial assays evaluated the
by the presence of 4 ␣-helices and 4 disulfide bonds [21]. aforementioned peptides against Salmonella enterica, E. coli and
2S albumins have been isolated from the seeds of a wide num- S. aureus (Table 4) [127]. Secondly, chemical modifications were
ber of plants and have many other biological functions besides applied to cyO2, aiming to understand how charges could affect
being a storage protein. They can act in stabilizing oil-in-water the antimicrobial activity. Three modified peptides were synthe-
emulsions [16,115], inhibiting proteolytic enzymes [92] and also sized, covering their Glu6 , Lys23 , Lys25 and Arg29 residue charges
performing antibacterial or antifungal activities. [2,96,122,131]. A with chemical modifications based on methylation and acetylation
2S albumin, called 2S-1, was purified from peanut (Arachis hypogaea processes. Years before this study, Svangard et al. [34] had proposed
L.) cotyledons, showing antifungal activity against Aspergillus flavus. that cyO2, like other cyclotides, was involved in electrostatic attrac-
This peptide, composed of two subunits, inhibited spore germina- tions with cell membranes due to its positive (+2) charge residues,
tion and hyphal growth of A. flavu. An interesting feature is that acting in cell membrane disruption due to its antimicrobial activ-
SDS improved the inhibitory activity of 2S-1, suggesting that only ities [147]. According to this, it was expected by Pranting et al.
one subunit may be able to show antimicrobial activity [43]. Fur- [127] that when Arg and Lys positive residues were covered, activity
thermore, the Passifloracea family also shows some antifungal 2S should be reduced. It was also expected that Glu residues coverage
albumins. Pa-AFP1, a 2S albumin peptide isolated from Passiflora should improve antimicrobial activity, since their negative charges
 E. de Souza Cândido et al. / Peptides 55 (2014) 65–78 69

Table 3
Plant 2S albumin activities against microorganisms.

Peptides Pa-AFP1 Pe-AFP1 Si-AMP2

Isolated from Passiflora alata Passiflora edulis Sesamum indicum
References [44] [41] [35]

IC50 (␮M)
Aspergillus fumigatus – 13.3 –
Colletotrichum gloeosporioides 6.1 – –
Fusarium oxysporum – 11.3 –
Klebsiella sp. – – 3.1
Trichoderma harzianum – 10.7 –

IC50: inhibit cellular proliferation by 50%; ␮M: micromolar.

Table 4
Plant cyclotide activities against microorganisms.

Peptides Vaby A Vaby D cyO2 Kalata B1 Kalata B2

Isolated from Viola abyssinica Viola odorata Oldenlandia affinis

References [48] [48] [48]

MIC (␮M)
Escherichia coli 32.5 50 2.2 > 100 >35
Salmonella enterica 90 >90 8.7 > 100 >35
Staphylococcus aureus > 90 >90 > 50 > 100 35

MIC: minimum inhibitory concentration; ␮M: micromolar.

were removed, making the peptide more positive [127,147]. How-
ever, the coverage of Glu or Lys caused almost an almost total
loss of bactericidal activity, while the modification of Arg residues
slightly decreased the same activity [127]. To date, it is known that
Glu6 localized at loop 1 is highly conserved among cyclotides and
can play an important role in antimicrobial activities [59,124]. In
addition, Glu residue is also thought to be related with cyclotide
structure stabilization, providing efficient aggregation in mem-
branes [124,127]. These data clearly show that not only the cationic
and hydrophobic properties must be thoroughly observed in plant
antimicrobial peptides, especially in the class of cyclotides.
1.2. Lipid transfer proteins (LTPs)
LTPs are small cationic peptides, with a conserved pattern
of Cys-Cys bonds, molecular masses between 7 and 10 kDa and
lipid transfer activity with low homology between their sequences
(∼30% identity) [169]. LTPs have been subdivided into two fam-
ilies (LTP1 and LTP2), with 70–95 amino acids in length. These
peptides are known for their ability to reversibly bind and trans-
port hydrophobic molecules within in vitro models [24]. Their
tridimensional structures consist of a conserved pattern of eight
cysteines (C1 Xn C2 Xn C3 C4 Xn C5 X1 C6 Xn C7 Xn C8 ) with four disulfide
bonds (Fig. 1E), which stabilize a compact tertiary fold constituted
of four ␣-helices linked by flexible loops with a hydrophobic cavity
that comprises the lipid binding site [24,53]. Furthermore, despite
the conserved pattern of eight cysteines presented by the groups
of LTPs, a variation in the cystine-paring motif to form disulfide
bonds could be observed [24]. In this context, studies performing
NMR and X-ray crystallography methods have demonstrated that,
when comparing the tertiary structure between LTP families, an
exchange in the pairing pattern of cysteines 5 and 6 (CXC) was
observed. In the LTP1 family Cys5 bonds to Cys8 and Cys6 bonds
to Cys1 , but in the LTP2 family it can be observed that Cys5 forms
a disulfide bond with Cys1 and Cys6 with Cys8 [29,169]. Structural
divergences can also be noted between LTP1 and LTP2 hydrophobic
cavities, with the LTP2 cavity being smaller but more flexible than
that of LTP1 [29,62]. In addition to the previous LTP functions cited

Table 5
Plant lipid transfer protein activities against microorganisms.

Peptides Ace-AMP Ha-AP10 Ca-LTP1 Cw18 Cw20 Cw21 Cw22 ns-LTP

Isolated from Allium cepa Helianthus annuus Capsicum annuum Hordeum sp. Raphanus sativus
References [72] [70] [71] [69] [73]

IC50 (␮M)
Bacillus megaterium 0.1 – – – – – – –
Candida tropicalis – – >41.9 – – – – –
Clavibacter michiganensis – – – 0.1 0.2 0.3 <0.1 –
Colletotrichum lindemuthianum – – >41.9 – – – – –
Fusarium solani – 0.5 – 8 9 2.5 12 –
Phoma betae – – – – – – – <0.1
Pseudomonas solanacearum – – – 0.5 0.7 0.8 0.2 –
Pyricularia oryzae – – – – – – – 0.1
Pyricularia oryzae – – – – – – – –
Saccharomyces cerevisiae – – >41.9 – – – – –
Sarcina lutea 0.5 – – – – – – –
Verticillium dahlia – – – – – – – –
Verticillium dahliae – – – – – – – 0.1

MIC (␮M)
Fusarium oxysporum – 3.3 – – – – – –

IC50: inhibit cellular proliferation by 50%; MIC: minimum inhibitory concentration; ␮M: micromolar.
70 E. de Souza Cândido et al. / Peptides 55 (2014) 65–78

above, it is also suggested that LTPs play important roles as food Table 6
Plant hevein activities against microorganisms.
allergens [160], in plant signaling [11] and also in plant defense, in
the form of antimicrobial activities [79,100]. Peptides Pn-AMP1 Pn-AMP2 WAMP1a
Regente et al. [174] demonstrated that an antifungal peptide
Isolated from Pharbitis nil Triticum kiharae
from Helianthus annuus (Ha-AP10), with 10 kDa molecular mass, References [80] [86]
has potent activities against not only against fungal development,
IC50 (␮M)
but also inhibiting Fusarium solani spore germination (Table 5). Bipolaris sorokiniana – – 5
Recently, a similar study performed by Diz et al. [41] character- Botrytis cinerea 3.7 0.5 –
ized and described the antifungal activity of a 9 kDa LTP from chili Colletotrichum langenarium 2.3 0.9 –
pepper (Capsicum annuum) seeds, named Ca-LTP1 . This peptide was Fusarium oxysporum 2.3 0.6 5
Fusarium solani – – 5
evaluated against Saccharomyces cerevisiae, Candida tropicalis and Phytophthora capsici 1.2 0.1 –
Colletotrichum lindemuthianum (Table 5). Ca-LTP1 was especially Phytophthora parasitica 0.7 0.5 –
able to cause morphological damages, with apparent formation of Saccharomyces cerevisiae 3.2 1.9 –
pseudo hyphae [41]. The SYTOX green fluorescence method was Sclerotinia sclerotiorum 2.5 0.7 –
also performed and proved that Ca-LTP1 is able to penetrate the IC50: inhibit cellular proliferation by 50%; ␮M: micromolar.
C. tropicalis plasma membrane. LTPs are also active against F. oxys-
porum, Pythium aphanidermatum and Sclerotium rolfsii as well as
against S. aureus (Table 5). Cammue et al. [19] isolated an antimi- [105]. Several hevein-like AMP variants containing ten cysteines
crobial peptide from onions (Allium cepa L.), named Ace-AMP1, have been described until now [64,110,159], including WAMP-1a
which presented activities against both bacteria and fungi. Ace- and WAMP-1b (recently isolated from Triticum kiharae [110]. The
AMP1 is a monomeric 7.5 kDa cationic peptide with 96 amino effects of cysteine number variations on structure and function of
acid residues in length and four disulfide bonds. When evalu- hevein-like AMPs is still not very well understood.
ated against different microorganisms, Ace-AMP1 presented major Due to its ability to bind to N-acetyl-d-glucosamine, the mode
activities against the fungus C. lindemuthianum, Phoma betae and of action of hevein and hevein-like peptides has been thought
Verticillium dahlia (Table 5), as well as against the Gram-positive to be binding cell wall chitin in growing hyphae and conse-
bacteria Bacillus megaterium and Sarcina lutea (Table 5) [19]. Terras quently inhibiting cell wall biosynthesis [75]. Nevertheless, this
et al. [156] explained that a high basic peptide with pI higher than does not explain WAMP-1a activity against chitin-free pathogens
10.5 and molecular mass around 9 kDa, purified from R. sativus, like oomycetes and bacteria, as observed by Odintsova et al. [110]
also showed antifungal activities against P. betae and V. dahliae (Table 6). Koo [76] demonstrated that hevein-type peptides from
as well as Pyricularia oryzae (Table 5) [156]. Moreover, Molina Pharbitis nil (Pn-AMPs) exhibited inhibitory activity on growth of
et al. [100] showed that four homologous peptides (CW18 , CW19 , several phytopathogens and also toward S. cerevisiae (Table 6). Pn-
CW20 and Cw21 ) were all able to inhibit Clavibacter michiganen- AMP overexpression in transgenic tobacco and tomato conferred
sis, Pseudomonas solanacearum and F. solani (Table 5). It was also enhanced disease resistance to Phytophthora parasitica and Phy-
determined that Cw21 combined with thionins (␣1 + ␤ from wheat) tophthora capsici, respectively [31,84]. Koo [75] showed that the
presented synergistic effects against F. solani [100]. In addition decrease in growth of S. cerevisiae and C. albicans caused by the
to these antifungal and bactericidal responses, Ooi et al. [112] peptide Pn-AMP1 may be associated with actin cytoskeleton depo-
reported that a fetuin-binding non-specific LTP of 9 kDa isolated larization.
from Narcissus tazetta, designated NTP, was also able to significantly
inhibit the proliferation of both cancer cells and viruses such as 1.4. Snakin/GASA peptides
influenza A (H1N1) and respiratory syncytial viruses [112].
Among AMPs families from plants, snakins are the
1.3. Heveins proteins that have the highest number of S S bonds
(C1 X3 C2 X3 C3 Xn C4 X3 C5 X2 C6 C7 X2 C8 Xn C9 X2 C10 ). These proteins
The name hevein was coined in 1960 by Archer [4] who isolated are considered rich in cysteines (∼19%) and have about 63 amino
the first peptide containing this domain from rubber tree (Hevea acid residues and potentially six S S bonds, while the other classes
brasiliensis) latex. This author reported a hevein as a lectin-like of AMPs usually have two to four S S bonds [142,153]. Snakins are
protein with the ability to bind N-acetyl-d-glucosamine (Glc- known to have certain patterns in a sequence of amino acids found
NAc), the monomeric unit of the polymer chitin present in cell in snake venom hemotoxic proteins like disintegrins [10,142].
walls of fungi. The hevein domain is extremely common among Snakins StSN1 [142] and StSN2 [10] were described in potato
plant lectins, found in chimerolectins, hololectins and merolectins tubers (S. tuberosum). Both StSN1 and StSN21 caused inhibition
[126]. Hevein is a cysteine-rich single-chain protein of 43 amino of bacterial growth of C. michiganensis, Rhizobium meliloti (Gram-
acids that present similar structures to chitin-binding domains positives) and Ralstonia solanacearum (Gram-negative), and also
from other lectins and enzymes [5]. The hevein domain struc- against multiple fungi, including F. solani and B. cinerea (Table 7)
ture is composed of an anti-parallel ␤-sheet and occasional short [99]. Recently Kovalskaya et al. [77] reported the expression of a
␣-helices with the scaffold stabilized by three to five disul- fusion gene (SAP) for production of two AMPs (snakin-1 (SN1) and
fide bonds (C1 Xn C2 Xn C3 C4 Xn C5 Xn C6 Xn C7 Xn C8 ) (Fig. 1F) [126]. a defensin-1 (PTH1)) using a recombinant bacterial expression
Although hevein-like AMPs share sequence homology, different system and purification. The authors demonstrated that SN1/PTH1
numbers of disulfide bridges have been found with similarity to has antimicrobial activity equivalent to snakins purified from
the chitin-binding domains of class I/IV chitinases [9,129]. Most plant tissues, and confirmed its activity against the bacteria C.
of them have eight cysteine residues forming four disulfide bonds michiganensis subspecies sepedonicus and more weakly against
such as Pn-AMP1 and Pn-AMP2 isolated from Pharbitis nil (Table 6) Pseudomonas syringae pathovar syringae and Pseudomonas syringae
[76] and oat avesin [87]. Truncated forms with only six cysteine pathovar tabaci. SN1/PTH1 also showed growth inhibition of the
residues have also been reported, such as the peptides Ac-AMP1 fungi Colletotrichum coccoides and B. cinerea [77].
and Ac-AMP2 isolated from Amaranthus caudatus seeds [14], Ay- The snakin family has no three-dimensional structures eluci-
AMP isolated from Amaranthus hypochondriacus [135], Ar-AMP dated until now, but a molecular model was recently proposed
from Amaranthus retroflexus [91] and IWF4 from sugar beet leaves by Porto et al. [125] for this family. These authors described
 E. de Souza Cândido et al. / Peptides 55 (2014) 65–78 71

Table 7 Table 8
Plant snakin activities against microorganisms. Plant myrosinase binding protein activities against microorganisms.

Peptides StN1 StN2 Peptides Ib-AMPs1 Ib-AMPs2 Ib-AMPs3 Ib-AMPs4

Isolated from Solanum tuberosum Isolated from Impatiens balsamina

References [93] References [101]

IC50 (␮M) MIC (␮M)

Aspergillus flavus 20 20 Alternaria longipes 1.2 4.7 2.3 1.2
Bipolaris maydis 20 20 Bacillus subtilis 4.1 – – 2
Botrytis cinerea 0.8 2 Botrytis cinerea 4.9 9.8 2.3 2.4
Clavibacter michiganensis 4 1 Cladosporium sphaerospermum 0.4 2.4 1.2 0.4
Colletotrichum graminicola 20 10 Fusarium culmorum 0.4 2.4 2.3 0.4
Colletotrichum lagenarium 20 10 Micrococcus luteus 4.1 – – 2
Fusarium culmorum 2 2 Penicillium digitatum 1.2 2.4 1.2 1.2
Fusarium oxysporum f. sp. Conglutinans 13 10 Staphylococcus aureus 12.2 – – 7.9
Fusarium oxysporum f. sp. lycopersici 13 20 Streptococcus faecalis 2.4 – – 2
Fusarium solani 2 3 Trichoderma viride 2.4 4.7 4.7 2.4
Plectosphaerella cucumerina 10 10 Verticillium alboatrum 1.2 4.7 2.3 2.4
Ralstonia solanacearum 15 30
MIC: minimum inhibitory concentration; ␮M: micromolar.
Rhizobium meliloti – 8

IC50: inhibit cellular proliferation by 50%; ␮M: micromolar.

the structure of snakin-1, combining in silico strategies like
ab initio and comparative molecular modeling with disulfide
bond predictor. The final disulfide bond of snakin was rep-
resented by twelve cysteine residues forming six disulfide
bonds (C1 X3 C2 X3 C3 Xn C4 X3 C5 X2 C6 C7 X2 C8 X3 C9 Xn C10 X2 C11 Xn C12 )
[125]. The model showed two ␣-helices composed of
2 SSFCDSKCKLRCSKA16 and 20 DRCLKYCGICCEE32 residues and
a small short helix composed of 43 NKH45 [125]. Predicted snakins
can be important in understanding plant systems and can also be
applied to other non-predicted peptides. However, more studies
seem to be necessary in order to better elucidate the mechanisms
of action for this class of peptide.
1.5. Myrosinase binding protein
Takeda et al. [150] elucidated the structure of myrosinase bind-
ing proteins (MBP) by NMR, which showed only two S S bonds
(C1 C2 Xn C3 X3 C4 ) and a molecular mass of 32 kDa. MBPs have 299
amino acid residues, which consist of a lectin-like domain tan-
dem of two homologous sequences (residues 1–144, 153–299),
each being composed of three ␤-sheets (Fig. 1G). The MBPs are
extremely cationic and known to be related to plant development
as well as defense [153]. Moreover, the defense function could be
intrinsically associated with the participation of such proteins in
the myrosinase-glycosylate system [22]. MBPs have been described
as hydrolases and components of high molecular mass complexes
in myrosinases (corresponding to 250–1000 kDa) in some groups
[22,45,150]. MBPs were first isolated from Impatiens balsamina
seeds by Tailor et al. [149]. These were named Ib-AMPs and pre-
sented significant results against Gram-negatives, Gram-positives
and also fungi (Table 8). The Ib-AMPs have 20 amino acid residues
that are highly cationic and show two disulfide bonds. The sen-
sitivity of Ib-AMPs against fungus was affected when the in vitro
culture was previously supplemented with 1 mM CaCl2 and 50 mM
KCl. In the presence of such ions the deleterious activity of Ib-AMPs
has about 70% glycine residues in the primary structure, the sec-
ond has fewer glycine residues and the third has a higher number
of Gly residues, but without a glycine domain [102,133]. The GRPs
are usually more hydrophobic, and this characteristic has been
commonly associated with phenylalanine and tyrosine residues
[133]. Pelegrini et al. [121] demonstrated for the first time the
activity of plant GRPs against Gram-negative bacteria. The peptide
Pg-AMP1 was isolated from Psidium guajava and, after being char-
acterized as a homodimer with molecular mass of approximately
6 kDa, showed activity against Klebsiella sp. and Proteus sp. (Table 9)
[121]. Pg-AMP1 presents two ␣-helices, one at the N-terminus and
another at the C-terminus, with a loop connecting them. Further-
more, arginine residues provide a positive charge at the extreme
of the ␣-helix, which can facilitate peptide/pathogen interactions.
Several hydrophobic residues are available in the structure, which
may be important for the dimerization. Additionally, Pg-AMP1
was expressed in E. coli Bl21 (DE3) using a recombinant plasmid
pBSKPg-AMP1. Other glycine peptides have been isolated from
different sources. In order to evaluate antimicrobial activity from
GRPs, Zottich et al. [173] isolated GRPs from Coffea canephora seed,
naming them Cc-GRP. Cc-GRP, with a molecular mass of 7 kDa, has
glycine residues at positions 4, 5, 9, 11, 12, 35, 36, 44, 45 and also
a tyrosine residue at position 39. The peptide causes morpholog-
ical changes in the membrane permeability of F. oxysporum and
Colletotrichum lindemuthianum fungus and also prevents colony
formation by yeasts (Table 9). This peptide was further found in
the cell wall, cell surface and nucleus of F. oxysporum, suggesting
that these three structures could be possible targets [173].
1.7. ˛-Hairpinins
The ␣-hairpinins (also known as hairpin-like peptides) have a
length of 31–35 amino acids. They are characterized by having two
Table 9
Plant glycine-rich peptides activities against microorganisms.

was severely reduced, suggesting a weakening of the electrostatic Peptides Pg-AMP1 Cc-GRP
interactions between the AMPs and their target [149].
Isolated from Psidium guajava Coffea canephora
References [108] [109]
1.6. Glycine-rich protein
MIC (␮M)
Candida albicans – 29.2
Glycine-rich proteins (GRPs) are storage proteins that are com- Candida tropicalis – 58.5
monly found in Brassicaceae, Solanaceae and Panicoideae families Colletotrichum lindemuthianum – 29.2
[21]. Previous studies revealed that GRPs are found in xylem, Escherichia coli 11.9 –
Fusarium oxysporium – 29.2
hypocotyls, stems and petioles [71,72,137,168]. Glycine-rich pro-
Klebsiella pneumoniae 5.3 –
teins are divided into three groups and characterized by their
MIC: minimum inhibitory concentration; ␮M: micromolar.
abundant sequences of glycines on their primary structure. The first
72 E. de Souza Cândido et al. / Peptides 55 (2014) 65–78

Table 10
Plant ␣-hairpinins and ␣␤-trumpet activities against microorganisms.

Peptides MiAMP2c Ec-AMP1 Ec-AMP2 MBP-1 Ps-AFP1 Tk-AMP1 Tk-AMP2

Isolated from Macadamia integrifolia Echinoa cruss-galli Zea mays Pisum sativum Triticum kiharae
References [111] [115] [110] [6] [117]

IC50 (␮M)
Actinotus helianthi 0.8 – – – – – –
Alternaria alternata – 3.7 – – – – –
Alternaria solani – 3.3 – – – – –
Aspergillus niger – – – – 15.6 – –
Aspergillus terreus – – – – 7.8 – –
Bipolaris sorokiniana – 3.1 – – – – –
Candida albicans – – – – 3.9 – –
Cercospora nicotianae 0.8 – – – – – –
Colletotrichum gloeosporioides – – – – 7.8 – –
Cyanophora paradoxa 3.4 – – – – – –
Diplodia maydis – – – – – 7.8 4.8
Fusarium graminearum – – – – – 1.9 2.1
Fusarium oxysporum 1.7 – – – – – –
Fusarium solani – 0.9 – – – – –
Fusarium verticillioides – – – – – 3.9 2.8
Glomerella sp. – – – – 7.8 – –
Leptosphaeria maculans 4.2 – – – – – –
Phytophthora betae – 3.2 24 – – – –
Phytophthora cryptogea 0.8 – – – – – –
Phytophthora debaryanum – 2.8 – – – – –
Phytophthora infestans – 1.4 – – – – –
Phytophthora parasitica 1.7 – – – – – –
Phytophthora ultimum – 3.4 – – – – –
Pythium sp. 15.6 – – – – – –
Saccharomyces cerevisiae 3.4 – – – – – –
Sclerotinia sclerotiorum 3.4 – – – – – –
Verticillium dahliae 0.8 – – – – – –

MIC (␮M)
Fusarium oxysporum – – – 14.5 – – –

IC50: inhibit cellular proliferation by 50%; MIC: minimum inhibitory concentration; ␮M: micromolar.

helices connected by two disulfide bonds (Fig. 1H), as observed in
the pattern C1 X3 C2 Xn C3 X3 C4 . ␣-Hairpinins display antibacterial,
antifungal and trypsin inhibitory activities. The first ␣-hairpinin
described was MBP-1 (maize basic peptide 1), isolated in 1992 by
Duvic and coworkers from corn seeds (Zea mays) [44]. This peptide
showed in vitro bactericidal activity against E. coli and Clavibac-
ter michiganensis, and also antifungal activity against Fusarium,
Alternaria, Sclerotinia and Aspergillus (Table 10). Another member of
this family is MiAMP2, isolated from macadamia seeds (Macadamia
integrifolia) [95], which seems to be a fragment of a protein homol-
ogous to a 7S globulin. It showed inhibitory activity against fungi
from the genera Alternaria, Fusarium and Chalara and also against
the bacteria C. michiganensis (Table 10). Despite not being classi-
fied as AMPs, some trypsin inhibitors have been discovered in this
family, like BWI-2b [119], isolated from buckwheat (Fagopyrum
esculentum); the C2 peptide [167] from pumpkin seeds (Cucurbita
maxima) and the peptide VhTI [35], isolated from Veronica hederi-
folia seeds. Furthermore, EcAMP1 [106] and EcAMP2 were isolated
from barnyard grass (Echinochloa crus-galli). These peptides were
evaluated for their trypsin-inhibitory activity as well their activity
toward bacteria and fungus. In both cases only antifungal activ-
ity was observed (Table 10) [136]. Utkina and collaborators [158]
isolated two peptides named Tk-AMP-X1 and Tk-AMP-X2 from
Triticum kiharae Dorof. et Migush with activity against the fungi
Fusarium and Diploidia maydis (Table 10). Since Tk-AMP-X2 showed
higher antifungal activity, the authors suggested that a few varia-
tions in the amino acid sequence could have a measurable influence
on the peptide’s inhibitory activity. It is also possible that ␣-
hairpinins can present multiple activities, since the same structural
motif has been found in different peptides pertaining to this family.
1.8. ˛ˇ-Trumpet
In addition to the above cited classes, Mandal and colleagues
[94] reported a novel AMP isolated from P. sativum radicle, Ps-
AFP1, which presents an unusual structural fold and disulfide
bond pattern. Ps-AFP1 presents a length of 38 amino acids,
showing a tertiary conformation with an ␣-helix and an antipar-
allel ␤-sheet, with two ␤-strands, which are stabilized by two
disulfide bonds (C1 Xn C2 Xn C3 Xn C4 ), as demonstrated by in silico
experiments. Due to this peculiar display, the authors named
this novel peptide the ␣␤-trumpet. Ps-AFP1 was able to inhibit
germination of fungal spores in in vitro experiments with A.
niger, A. terreus, Glomerella sp., and Pythium sp., among others,
showing IC50 varying from 3.91 to 15.62 ␮M, as demonstrated
in Table 10. Moreover, it seems that this peptide was able to
bind to carbohydrates as determined by microcalorimetry and
microscopy.
1.9. Short non-disulfide peptides
Antimicrobial peptides from plants are usually characterized
as cysteine-rich peptides, but a small number of AMPs isolated
from plants are cysteine-free or present very low cysteine content.
Despite this peculiar characteristic, they are not usually reported as
an AMP class. One of these peptides, JCpep7, isolated from Jatropha
curcas seeds, shows 7 amino acids residues length and displays
activity against the bacteria S. typhimurium, Shigella dysenteriae, P.
aeruginosa, S. aureus, Bacillus subtilis and Streptococcus pneumoniae
(Table 11). This peptide has no homology with any other previously
described AMPs [166].
 E. de Souza Cândido et al. / Peptides 55 (2014) 65–78 73

Table 11
Plant short non-disulfide peptides activities against microorganisms.

Peptides JCpep7 Shepherin I Shepherin II Ginkbilobin Cn-AMP1 Cn-AMP2 Cn-AMP3

Isolated from Jatropha curcas Capsella bursa-pastoris Ginkgo biloba Cocos nucifera
References [118] [120] [119] [121]

IC50 (␮M)
Alternaria alternata – 3 >30.7 – – – –
Aspergillus flavus – 27.5 18.4 – – – –
Aspergillus fumigatus – >42.3 >30.7 – – – –
Bacillus subtilis – – – – – – –
Botrytis cinerea – – – 0.25 – – –
Candida albicans – – – – – – –
Coprinus comatus – – – 3.4 – – –
Cryptococcus neoformans – – – – – – –
Erwinia herbicola – 26.2 <0.8 – – – –
Escherichia coli – <1.1 <0.8 – – – –
Fusarium culmorum – – – – – – –
Fusarium oxysporum – – – 3.6 – – –
Mycosphaerella arachidicola – – – 6.5 – – –
Pseudomonas putida – <1.1 <0.8 – – – –
Pseudomonas syringae – <1.1 <0.8 – – – –
Rhizoctonia solani – – – 8.7 – – –
Saccharomyces cerevisiae – – – – – – –
Salmonella typhimurium – <1.1 19.9 – – – –
Serratia sp. – 3.4 <0.8 – – – –
Staphylococcus aureus – – – – – – –
Streptococcus mutans – – – – – – –

MIC (␮M)
Bacillus subtilis 29.9 – – – 86.8 118.4 265.8
Escherichia coli 59.7 – – – 93.6 134.2 312.3
Pseudomonas aeruginosa 79.6 – – – 90.2 133.4 267.8
Salmonella typhimurium 59.7 – – – – – –
Staphylococcus aureus 49.8 – – – 91.3 134.2 283.4
Streptococcus pneumoniae 69.7 – – – – – –

IC50: inhibit cellular proliferation by 50%; MIC: minimum inhibitory concentration; ␮M: micromolar.

The peptide ginkbilobin, isolated from Ginkgo biloba seeds,
presents 40 amino acids in length and demonstrated activity
against the fungi B. cinerea, Mycosphaerella arachidicola, F. oxyspo-
rum, R. solani and Coprinus comatus (Table 11). This peptide also
inhibits development of the bacteria S. aureus, P. aeruginosa and
E. coli (Table 11). Ginkbilobin shows clear identity to an embryo-
abundant protein from white spruce (Picea glauca), but shares a
small portion of its sequence with GAFP, an antifungal peptide
from Ginkgo biloba leaves [164]. Another two cysteine-free AMPs
were isolated from shepherd’s purse (Capsella bursa-pastoris) and
were named shepherin I, with 28 amino acid residues, and shep-
herin II, with 38 amino acid residues, both originating from one
single polypeptide chain (shep-GRP). They are glycine-histidine-
rich peptides, active against the bacteria E. coli, Pseudomonas
putida, Pseudomonas syringae and Serratia sp. (Table 11) and against
the fungi C. albicans, Cryptococcus neoformans and S. cerevisiae
(Table 11) [117].
Finally, the AMPs Cn-AMP1 (9 amino acids), Cn-AMP2 (12 amino
acids), and Cn-AMP3 (8 amino acids), isolated from Cocos nucifera
water, were also described as antimicrobial peptides, revealing
bactericidal activities against E. coli, B. subtilis, P. aeruginosa and
S. aureus (Table 11). Low homology with the AMPs temporin G
and uperin was observed [145]. In addition, immunomodulatory
activity has been also reported for Cn-AMP1. Its three-dimensional
structure has been predicted by homology modeling, exhibiting a
poorly defined conformation in aqueous solution, with a possible
3–10 helix in hydrophobic environments [145].
2. Plant AMP applications for human health purposes
Plant AMPs are being increasingly considered as potential
therapeutic agents, due mainly to multiple-drug resistance and
hospital infections. These molecules exhibit a broad spectrum of
action against pathogenic fungi and bacteria, being extended to
viruses, parasites, in addition to applications as insecticides [8,141],
analgesics [140], immunomodulators [3] or in the treatment of neu-
rological disorders [139,141]. Some peptides have shown promise
as anticancer agents due to their activities against target cells
and/or the fact that they elicit an immune response that may be
effective in controlling infections and decreasing tumor progress
[58,93,114]. Several studies have employed AMPs that have antitu-
mor and/or antiviral activities combined with traditional treatment
and have found excellent outcomes [32,162].
Plant peptides, like other peptides from different sources, are
mostly present naturally at very low concentrations [86]. For use
in pharmaceutical or biological applications, a reasonable amount
of purified and active molecules is needed. Thus, these compounds
need to be produced in larger quantities. Different methods are
described in the literature for producing AMPs, such as the direct
isolation of the organism that produces the peptide of interest,
chemical synthesis or recombinant expression via heterologous
systems [40,57,116]. Both strategies can be applied to plant AMPs.
The isolation of peptides from a natural source is a strategy that is
not economically sustainable for pharmaceutical companies, being
restricted to universities and bio prospection labs. These techniques
are important in the discovery of novel peptides, but take a long
time, and the purified peptide is sometimes inefficient [88,172].
Furthermore, this technique can also have an adverse environmen-
tal impact, especially for peptides found in rare species and/or in
low population levels [86,116]. On the other hand, the problem that
arises from the chemical synthesis of peptides is economic. This
technique allows the production of synthetic and natural peptides,
but it involves a high cost for synthesis of sequences with more than
ten amino acid residues. Moreover, these costs can be considerably
higher in the synthesis of sequences that have disulfide bridges,
such as the defensins, making peptide production difficult [88].
74 E. de Souza Cândido et al. / Peptides 55 (2014) 65–78
For these reasons, chemical synthesis of plant AMPs with complex
folds can be considered ineffective for large-scale production, and
is consequently limited to smaller AMPs lacking post-translational
modifications and presenting low cysteine content.
Advances in recombinant DNA technology have enabled AMPs
to be expressed in larger quantities for several applications
[103,116]. These techniques permit the cloning of genes encoding
exogenous proteins in specific vectors for expression in prokaryo-
tic and eukaryotic host cells [118,171], facilitating the production
of these molecules on a large scale. Nevertheless, one enormous
challenge in AMP production by a heterologous system involves
reducing intrinsic AMP toxicity to the vector. At high concentra-
tions, AMPs are able to kill the expression vector, mainly E. coli
or Pichia, impeding expression at high concentrations. In this con-
text, the use of E. coli is considered the most efficient method
in terms of time and cost [88]. As an example, Pg-AMP1, the
antimicrobial peptide isolated from guava seeds and free of disul-
fide bridges, was expressed and purified from E. coli [152]. The
recombinant peptide that was produced showed inhibitory activity
against Gram-negative and -positive bacteria [152]. Other pep-
tides from plants have also been expressed in E. coli [20,23,38,70]
and P. pastoris [17,28,70,146]. Although heterologous expression
in plants presents some bottlenecks, fusion proteins, tags and tan-
dem repeats can be used for the production of these molecules
[161]. The non-fused antimicrobial peptide WT1 was expressed in
N. benthamiana leaves and reached 0.40 g of purified peptide per
gram of leaf [138]. However, the antimicrobial peptides SMAP-29,
RC101, PG and sarcotoxin A were expressed by the fusion pro-
teins as intein, GFP and GUS [85,101,111]. The production of these
peptides for biotechnological techniques can broaden their use in
different applications.
Efforts to bring antimicrobial peptides into new stages of devel-
opment are ongoing. Some peptides are currently undergoing
clinical trial design despite unsuccessful forays a decade ago. For
antimicrobial peptides to enter production, some bottlenecks have
to be eliminated. Among these, some systemically administered
peptides can lead to toxicity, and more tests are required before
starting preclinical and clinical tests, since these molecules have
different sources and most studies only perform a simple test
for activity in bacterial cultures [47]. There is an urgent need for
antimicrobials that target resistant bacterial pathogens and for
oral alternatives to treat resistant diseases. Unfortunately, many
antimicrobial peptides lack potency against resistant and infectious
bacteria [47].
Despite the many problems that have arisen in clinical trials of
some peptides, authorities in the USA and Europe have made an
effort to facilitate antibiotic development and incorporate innova-
tive trial designs and measures of clinical effectiveness. In the USA,
the Food and Drug Administration has set up the Antibacterial Drug
Development task Force [46], while in Europe, new guidelines have
been issued by the European Medicines Agency, establishing direct
clinical criteria for evaluating antibiotics and allowing patients who
received other antibiotics to enroll in clinical trials [47].
Thus, overcoming a range of impediments, peptides from
different sources are at advanced stages of pharmaceutical devel-
opment. For example, magainin, an antimicrobial peptide isolated
from the skin of the African clawed frog Xenopus laevis, is at phase 3
of development for treatment of diabetic foot ulcers. Another two
synthetic peptides, omiganan and OP-145, are at phase 2 of treat-
ment against rosacea and chronic bacterial middle-ear infection,
respectively. There are other antimicrobial peptides at phase 1
and preclinical stages of development, but no peptides from plants
are undergoing clinical trials yet [47]. However, it is important to
emphasize that these clinical studies can be extended to plant pep-
tides, although most of them until now have been applied to crop
improvement. The expense of clinical evaluations, dependence on
federal programs for financial support and the need for corporate
partners has meant that few peptides are in clinical trials.
As can be seen, antimicrobial peptides for human purposes are
on the scientific agenda and these molecules have arisen as a possi-
ble answer to antimicrobial resistance issues. However, the issues
surrounding financial support or partners for clinical trials and cur-
rent dependence on federal programs for research support still
need to be solved.
3. Plant AMPs: applications for agricultural purposes
Screenings of novel plant defense peptides with antimicrobial
activity have been conducted with a view to developing resis-
tant cultures against phytopathogenic microorganisms. The in vitro
activity of many plant AMPs is already known, demonstrating clear
potential utility for agribusiness. In fact, many works involving the
heterologous expression of plant AMPs, especially defensins, have
been reported for many crops of economic importance, with the
aim of increasing resistance to phytopathogens.
The radish defensin Rs-AFP2 was the first heterologous plant
peptide expressed in another plant, conferring protection against
the fungus A. longipes in lineages of tobacco and tomato [155]. The
peptide Rs-AFP2 was also expressed in wheat, protecting this plant
against the attack of different fungi [89]. After this successful appli-
cation, a number of plant peptides were used to confer resistance
to distinct pathogens, like the peptide ␤-purothionin, a thionin iso-
lated from wheat, which was used to make transgenic lines of A.
thaliana less susceptible to bacteria and fungi [108]. Unfortunately,
most thionins can be toxic to animal and plant cells, restricting
the application of this class of plant peptides, or demanding co-
expression with fusion proteins [130].
Other plant antimicrobial peptides with higher potential use
have also been used to transform different plants, like the MsDefI
alfafa defensin, expressed in vivo in tomato, conferring protec-
tion against the fungus F. oxysporum [1]. PhDef1 and PhDef2,
defensins from petunia, have been expressed in banana, providing
this plant with significant resistance to infection by F. oxyspo-
rum [56]. Indeed, a long list of plant peptides has been used to
transform plants. Among them, the heterologous expression of
chili defensin has enhanced the resistance of tomatoes to different
fungi [170]. Additionally, a peptide from mustard was expressed
in tobacco, conferring resistance against F. moniliforme and P.
parasitica. This peptide was also expressed in peanuts, confer-
ring resistance against late leaf spot disease [148]. Recently, the
same AMP was expressed in rice, providing resistance against var-
ious fungal pathogens [68]. Other peptides that confer resistance
to pathogens, such as AP24 osmotin from N. tobacum, have been
expressed in potatoes [134]; the hevein-like peptides pro-SmAMP1
and pro-SmAMP2 from Stellaria media were expressed in Arabdop-
sis [144]; the hordothionin from H. vulgare was expressed in apples
[78] and Wasabi defensin from Wasabia japonica was produced in
Egusi melon [107].
Another two different strategies have been employed for use of
plant antimicrobial peptides. The first is the overexpression of an
endogenous peptide, as seen in the overexpression of snakin-2 in
transgenic tomato, which increases the resistance of this plant to
bacteria [6]. The second is the double in vivo expression in a single
rice plant of two different AMPs, as demonstrated with Dm-AMP1
from the seeds of Dahlia and Rs-AFP2, which confers more resis-
tance to fungi when compared to plants transformed with control
single-gene constructs of these peptides [69].
Current and future research in transformation of plants with
AMPs focuses on three areas: first on the control of expression of
AMPs, so that this only occurs in the presence of pathogens, prefer-
ably at attacked sites [39]; second in the improvement of desirable
 E. de Souza Cândido et al. / Peptides 55 (2014) 65–78
characteristics such as high activity and low cytotoxicity by modifi-
cations in sequence via bioinformatics tools [113]; and thirdly, the
synergic expression of different AMPs, with diverse mechanisms of
action [42,69].
Until now, there have been no reports of commercial cultivars
of these transgenic plants, since they were not released on the mar-
ket due to a great number of remaining questions. The first doubt is
related to whether the amount of peptide observed in the plant dur-
ing exposure to the pathogen will be effective, since some factors
like degradation by plant host proteases and genic silencing can
greatly diminish the efficiency of protection by these molecules
[42]. Heterologous expression of antimicrobial peptides can also
alter plant development, as demonstrated in potato expressing
the heterologous AMP msrA3, which conferred protection against
fungi, but also delayed development of floral buds, prolonged the
vegetative phase and altered the pattern of host defense [60].
Another issue is if the transgene can be epigenetically silenced by a
process called RNA-directed DNA methylation, which can be seen as
a heritable loss of gene expression. This phenomenon was demon-
strated in transgenic wild tobacco plants, where the production of
a heterologous AMP was down-regulated by more than 200 fold
after only one plant generation [165].
As an alternative to production of transgenic plants, a new
strategy of production of AMPs in plants has recently been devel-
oped, based on the discovery, by bioinformatics tools, of putative
antimicrobial peptides encrypted in protein sequences of the host,
followed by the expression of these molecules alone. A fragment
from a larger protein of soybean plant (Glycine max) that showed
antimicrobial activity was transformed in this plant, conferring
resistance against Phakopsora pachyrhizi. The AMPs resulting from
this procedure were named intragenic antimicrobial peptides [12].
A huge number of crops have been improved by advances in
genetic engineering, increasing nutritional quality and total pro-
duction per planted area. The antimicrobial peptides from plants
represent an alternative for enhancing resistance to pathogens,
contributing to an increase in crop yields and reducing pesticide
use. The remaining issues surrounding the use of these peptides are
being answered, indicating a promising future for these molecules.
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