Plant storage proteins with antimicrobial activity: novel insights into

The FASEB Journal • Review

Plant storage proteins with antimicrobial activity: novelinsights into plant defense mechanisms

Elizabete de Souza Candido,* Michelle Flaviane Soares Pinto,*Patrícia Barbosa Pelegrini,† Thais Bergamin Lima,* Osmar Nascimento Silva,*,‡

Robert Pogue,* Maria Fatima Grossi-de-Sa,*,† and Octavio Luiz Franco*,‡,1

*Centro de Analises Proteomicas e Bioquímicas, Pos-Graduacao em Ciencias Genomicas eBiotecnologia, Universidade Catolica de Brasília, Campus Avancado Asa Norte, Brasilia, Brazil;†Laboratorio de Interacao Molecular Planta-Praga, Empresa Brasileira de Pesquisa AgropecuariaRecursos Geneticos e Biotecnologia, Parque Estacao Biologica, Brasilia, Brazil; and ‡Programa dePos-Graduacao em Imunologia/Genetica e Biotecnologia, Universidade Federal de Juiz de Fora, Juizde Fora, Brazil

ABSTRACT Storage proteins perform essential rolesin plant survival, acting as molecular reserves importantfor plant growth and maintenance, as well as beinginvolved in defense mechanisms by virtue of theirproperties as insecticidal and antimicrobial proteins.These proteins accumulate in storage vacuoles insideplant cells, and, in response to determined signals, theymay be used by the different plant tissues in responseto pathogen attack. To shed some light on these re-markable proteins with dual functions, storage proteinsfound in germinative tissues, such as seeds and kernels,and in vegetative tissues, such as tubercles and leaves,are extensively discussed here, along with the relatedmechanisms of protein expression. Among these pro-teins, we focus on 2S albumins, Kunitz proteinaseinhibitors, plant lectins, glycine-rich proteins, vicilins,patatins, tarins, and ocatins. Finally, the potential use ofthese molecules in development of drugs to combathuman and plant pathogens, contributing to the devel-opment of new biotechnology-based medications andproducts for agribusiness, is also presented.—De SouzaCandido, E., Pinto, M. F. S., Pelegrini, P. B., Lima,T. B., Silva, O. N., Pogue, R., Grossi-de-Sa, M. F.,Franco, O. L. Plant storage proteins with antimicrobialactivity: novel insights into plant defense mechanisms.FASEB J. 25, 3290–3305 (2011). www.fasebj.org

Key Words: biotechnological potential � pathogen attack � drugdevelopment

Plant storage proteins are associated with specificplant components, such as seeds, nuts, and kernels;stem parenchyma of trees; grains and legumes; andsome roots and tubers. These organs are responsiblefor protein synthesis and storage, presenting high pro-tein content. The storage proteins comprise an excel-lent source of amino acids (1) and can be mobilizedand utilized for maintenance, defense, and growth ofplants, as well as in the embryonic and developmentalstages (1).

Some of these molecules present in plants and

responsible for defense have known functions as anti-microbial proteins or peptides (AMPs). AMP efficiencydepends on several characteristics of the protein orpeptide, including molecular mass, sequence, charge,conformation, secondary and tertiary structures, pres-ence or absence of disulfide bonds, and hydrophobicity(2). AMPs are small, structurally diverse, cationic pro-teins that are able to destroy microbes through a rangeof effector mechanisms, including cellular membranerupture (2). Within the context of storage proteins, itcan be asserted that some of these proteins exhibitcharacteristics as antimicrobial agents, acting on plantdefense, and are also related to defense against unre-lated pathogens, such as human pathogens (3). Thesemolecules can be described as showing antimicrobialactivity, and here we designate them as antimicrobialproteins or peptides, depending on their molecularsize. In this sense, we discuss multiple storage proteinclasses with antimicrobial activity, as shown in Table 1,and also the biotechnological perspectives for healthimprovement and agribusiness.

STORAGE MECHANISMS OF PLANT PROTEINS

Each protein acts as a reserve of amino acids during thedevelopmental process of organisms, since the aminoacids generated by the degradation of these proteinsare recycled for the de novo protein biosynthesis process(1). As reviewed in Shewry et al. (4), seed proteins canbe grouped based on their extraction and solubility: inwater (albumins), dilute saline (globulins), alcohol/water mixtures (prolamins), and dilute acid or alkali(glutelins). However, it is necessary to highlight thatthis classification system is not immutable, since some

1 Correspondence: Centro de Analises Proteomicas e Bio-químicas, Universidade Catolica de Brasília, Campus Avan-cado Asa Norte, SGAN 916 Avenida W5, CEP: 70790-160,Brasilia, DF, Brazil. E-mail: [email protected]

doi: 10.1096/fj.11-184291

3290 0892-6638/11/0025-3290 © FASEB

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proteins can transition between one or more groupsdepending on their functions (4).

Plant storage proteins can be classified into seedstorage proteins (SSPs) and vegetative storage proteins(VSPs). The protein amount in seeds (cereal, oil seeds,and legumes) varies from 10 to 40% of the dry weight,providing a major source of protein for absorption byhumans (4). In contrast to SSPs, the VSPs are less wellcharacterized, the most studied vegetative organismsbeing potato (patatins), sweet potato (sporamins), andOxalis tuberosum (ocatins) (5).

During the developmental process, plant storageproteins are protected against premature breakdown bydiverse mechanisms. The most evident is the sequestra-tion of these proteins from the cytoplasm and packag-ing into protein bodies (PBs). Storage proteins areformed at the cytoplasmic surface of the rough endo-plasmic reticulum, and then transported to the storagecompartment, as shown in Fig. 1A. These proteins arestored in vacuoles, or PBs that are embedded in thevacuole, and remain there during the maturation of thetissue, protected from premature degradation. Whenseed germination starts or the tissue development iscompleted, the storage proteins are rapidly degraded,thereby providing necessary nutrients for survival. Inseeds, the polypeptides are protected from degradationduring the translocation event. Storage proteins havespecific targeting information that permits them to besecreted and transported to PBs; usually, this signalresides in the primary structure of the polypeptides;some of them, such as that found in the Brazilian nut2S albumin, can be located at the C terminus. Onarrival in the vacuole, the protein undergoes limitedproteolysis, removing the signal peptide to produce themature form (1). PBs are formed either from theendoplasmic reticulum or from the vacuoles. In tissues,such as developing seeds, there occurs a specializationof the vacuolar system for the purpose of proteinstorage (for storage and defense), and other metabo-lites such as minerals (phytin), sucrose, and otheroligosaccharides. Even though the SSPs and VSPs ex-hibit several similarities in their amino acid composi-tion, the vegetative proteins cannot be included in theseed-protein classes. The VSPs are deposited in vacuole-derived membrane-bound PBs, as are SSPs, but theyhave a different mobilization mechanism, and this

occurs at a different time to formation and deposition(1). Several VSPs have been shown to have enzymaticactivities; for example, the patatins of potato tubers,which show an esterase activity, and soybean VSP, whichpresents a weak acid phosphatase activity (1, 4).

At least two trafficking pathways have been identifiedfor the protein storage vacuole (PSV): the Golgi-depen-dent and Golgi-independent pathways. Storage pro-teins, such as those classified as 7S and 11S, and defenseproteins, such as lectins, are transported through theGolgi apparatus, and afterward, they are deposited intothe PSV by dense vesicles. On the other hand, thestorage proteins in wheat are, in part, delivered to thePSV through a Golgi-independent route (6). Somesignals, such as those required for the targeting ofsoluble plant proteins to lytic vacuoles, and the recep-tors involved are probably unique to plants; however,despite these significant differences, the protein traf-ficking involves certain compartments, vesicle coats,and accessory proteins, which are very similar to thoseinvolved in trafficking to yeast vacuoles and animallysosomes, for example (6).

REGULATION OF STORAGE PROTEIN GENEEXPRESSION

Current models of eukaryotic gene expression show thecomplex and dynamic mechanisms and pathways thatdetermine whether a gene is expressed and at whatlevel (Fig. 2). The mechanisms that control geneexpression and activity include transcriptional regula-tion, post-transcriptional regulation, chromatin remod-eling and modification, DNA methylation, binding ofmultiple factors to DNA cis elements, alternative splic-ing of mRNA, mRNA stability, translational control,post-translational control, and protein degradation.

The regulation of storage proteins occurs at differentlevels by a combination of hormonal, genetic, andmetabolic factors. Both up-regulators and down-regula-tors of storage proteins have been described. Levels ofnitrogen compounds, sugars, abscisic acid (ABA), gib-berellic acid (GA), and auxin, among other molecules,are involved in control of synthesis of storage proteins(Fig. 2 and ref. 7). In Glicine max (soybean), forexample, the physiological levels of nitrogen, methyl

TABLE 1. Storage protein classification by differential storage compartment and plantrequirements

Protein family Classification Size (kDa) Requirement

2S albumins SSP 10–20 ConstitutiveKunitz proteinase inhibitors SSP/VSP 18–26 Constitutive/defense induciblePlant lectins SSP/VSP 30–60 ConstitutiveGlycine-rich proteins SSP 6–30 ConstitutiveVicilins SSP 150–170 Constitutive/defense induciblePatatins VSP 40–45 ConstitutiveOcatins VSP 18 ConstitutiveTarins VSP 12.5 Constitutive

SSP: seed-storage protein; VSP: vegetative storage protein.

3291PLANT STORAGE PROTEINS WITH ANTIMICROBIAL ACTIVITY

3292 Vol. 25 October 2011 DE SOUZA CANDIDO ET AL.The FASEB Journal � www.fasebj.org

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jasmonate (MeJA) and soluble sugars synergisticallystimulate accumulation of VSP mRNAs (8). Besidesplaying an important role in the expression of the vspgenes, jasmonate is also important in the expression ofvsp genes with insecticidal activity, as was demonstratedby applying methyl jasmonate to Arabidopsis, which ledto the differential expression of the gene AtVSP, whichshowed insecticide activity (9). Auxin, on the otherhand, represses soybean vsp gene expression in suspen-sion-cultured cells and in leaves and petioles of excisedtrifoliates (10).

In addition, the levels of certain amino acids areimportant for the synthesis of storage proteins. Thelevel of asparagine, for example, in soybean cotyledonsis crucial to the synthesis of SSPs (11). The embryo,however, is symplasmically isolated from maternal tis-sues; hence, this process involves active transport, withthe participation of amino acid/H� symporters of theamino acid permease family (11). Moreover, levels ofsucrose also regulate the expression of storage proteins;for example, high levels of sucrose (3%) are necessaryfor the expression of sporamins in tubers (12). Further-more, high levels of sucrose (1–6%) restrict the expres-sion of tarin to the tuber. The expression of tarin inpotatoes follows the pattern for patatin synthesis (5). Ina recent study, Bolon et al. (13) identified 11 genes thatare believed to be involved in the regulation of seedprotein; among these candidates are a regulatory pro-tein of the Mov34–1 family; a heat-shock protein,Hsp22.5; and an ATP synthase.

A number of transcription factors (TFs) that act asregulators of SSP expression have been identified. Oneof the most extensively studied TFs involved in thisregulation in plants is the bZIP protein from maize,OPAQUE2 (O2; ref. 14). Vicente-Carbajosa et al. (15)have demonstrated that the O2 gene encodes a bZIP TFthat binds to a promoter element in the 22-kDa class ofzein protein genes to activate their expression. InArabidopsis, 8 TFs have been detected that are expressedpreferentially in seeds during maturation: the fourmaster regulators LEC1 (Leafy cotyledon 1), LEC2(Leafy cotyledon 2; (single, double, and triple mu-tants), ABI3 (abscisic acid-insensitive 3) and FUS3(Fusca 3), as well as EEL (enhanced EM level), ABI5(abscisic acid-insensitive 5), PEI1 (putative zinc-fingerprotein transcription factor), and HSFA9 (a heat stresstranscription factor) (7). LEC1 and LEC2 central reg-ulators are required for many aspects of Arabidopsisembryogenesis; these two regulators are sufficient toconfer embryonic characteristics in vegetative organswhen expressed ectopically (14). LEC1 is homologousto the subunit HAP3 CCAAT-binding factor CCAAT

(CBF-CCAAT binding factor), also known as NF-Y (16).Ectopic expression of LEC1 induces the expression ofABI3 and Fus3, resulting in a hierarchical activationnetwork; on the other hand, activation of LEC2, in invitro assays, induces expression of LEC1, Fus3, andABI3 (17).

ABI3, FUS3, and LEC2 (AFL) are TFs belonging tothe family of proteins with B3 DNA-binding domains(17, 18). The B3 domains of AFL and an ortholog ofABI3 in maize (VP1) bind directly to the RY element(CATGCAT), and the bZIP factors ABI5, bZIP10, andbZIP25 bind to the ACGT motif, activating ssp genes(19). ABI3 in in vitro assays does not bind to the DNA ofa yeast hybrid (Y1H), however, in vivo assays suggest thatABI3 interacts with the RY element indirectly throughprotein-protein interaction with Fus3/LEC2 (18).

The RY/Sph (purine-pyrimidine, CATGCAT sequence)DNA motif is a cis-regulatory element target of several TFB3 domains (17). ABI3, together with Fus3 and LEC2,form a well-conserved cluster, designated ABI3/VP1-like(20). The ABI3/VP1-like domain functions as an activatoror repressor, depending on the promoter context, bybinding the sequence CATGCA, (an RY/SphDNA motif)through the B3 domain (21). This gene family (65 genemodels in Arabidopsis) acts as an intermediary in theregulation of ABA-responsive genes during seed develop-ment by recruiting additional DNA-binding proteins topromoters, acting on �353 genes (21).

The major factor-binding sites are two regulatoryelement complexes: the RY/G motif and the B box,which act in synergy, based on studies with the napin Apromoter from Brassica napus and legumin B4 pro-moter from Vicia faba (7). The RY/G motif is formed bytwo RY elements (CATGCA, as previously mentioned;ref. 22) and a G box (CACGTG) that are binding sitesfor bZIP (basic leucine zipper proteins) or bHLH(basic helix-loop-helix proteins) (23). The B box is anABA-responsive element (ABRE) formed by two ele-ments, a distB element (GCCACTTGTC) and a proxBelement (TCAAACACC), which mediates ABA re-sponse in seeds (24); however, these elements requireadditional bZIP transcription factors, such as ABI5 andAREB/ABF in Arabidopsis and TRAB1 in rice. Theinteraction between these bZIP proteins and ABRE inthe promoter region of the ABA-inducible genes in-duces their transcription (25).

Although the genetic mechanisms regulating theexpression of storage proteins are well known, recentfindings reveal that epigenetic processes are an integralpart of the system regulating the expression of storageproteins. Epigenetic mechanisms, namely, histonemodifications and cytosine-DNA methylation-induced

Figure 1. A) Storage proteins with antimicrobial potential present in storage compartments in germinative and vegetative planttissues. Proteins are translocated to storage vacuoles and stored in the protein bodies (PB), or remain free in the vacuolar space.Depending on plant requirements, these proteins will be used as an amino acid source or as defense molecules. Protein DataBank (PDB) identities are indicated for each protein 3-dimensional structure. B) Tridimensional structures of storage proteinswith antimicrobial activity. a) 2S albumin (PDB 1S6D). b) Kunitz inhibitor (PDB 1TIE). c) Lectin (PDB 2ZR1). d) Glycine-richprotein (PDB 3A2E). e) Vicilin (PDB 2EA7). f) Patatin (PDB 10XW). Disulfide bonds are represented in yellow. Structures weredesigned in PyMOL (http://www.pymol.org). Ocatins and tarins have no structure deposited at PDB.


modification of the genome, give rise to epigenomes.For example, the structure of chromatin is directlyinvolved in seed-specific expression of the phaseolingene in Phaseolus vulgaris. Furthermore, the positioningof the nucleosome is altered in seeds to allow access bytranscriptional regulators (26). The ectopic expressionof the transcription factor PvALF, a P. vulgaris orthologABI3/VP1, is involved in positioning of nucleosomesand other nonhistone proteins and in the expression ofelements of the ABA signaling cascade.

Finally, and no less important, some enzymes areclearly involved in plant defense as a secondary func-tion. It is well known that the cupin superfamily encom-passes a large range of proteins responsible for theactivation of plant defense, such as plant oxalate oxi-dase (OXO). These proteins can be important in theresistance response due to their ability to cause degra-dation of oxalate, which is a virulence factor synthe-sized by fungal pathogens. As a result of oxalate degra-dation, the H2O2 produced can act in the promotion ofoxidative cross-linking between lignin and proteinsinvolved in cell wall formation. This event is capable ofactivating the transcription of defensive genes and hasbeen clearly correlated with antimicrobial defense (27).

In rice blast fungus-resistant varieties, it was demon-strated that among the 4 promoters of OXO (OsOXO1,OsOXO2, OsOXO3, and OsOXO4), only OsOXO4contains an extra element (a QTL at chromosome 3),suggesting differential regulation of the defense re-sponse genes. Furthermore, this nonspecific promotercould be induced by the exposure to fungal, bacterial,and insect pests (27).

Storage proteins show a clear example of how differ-ent proteins found in different tissues may play thesame biological role. An interesting feature of storageproteins is that most of them have a protective roleagainst pathogens. Finally, as noted earlier, many fac-tors are involved in the expression of proteins, and themodulation of these factors is of crucial importance forthe maintenance of such activities.

2S ALBUMINS

2S albumins are a water-soluble storage protein groupwith low molecular weight, rich in glutamine, and withphysicochemical properties shared within the group.They are widely present in monocotyledonous and

Figure 2. Simplified network of gene regulation involving the biosynthesis of storage proteins. Levels of sucrose, abscisic acid(ABA), gibberellic acid (GA), and auxin are involved in control of the level of expression of 4 master regulatory genes (LEC1,LEC2, ABI3, and FUS3). Ectopic expression of LEC1 induces the expression of ABI3 and FUS3, resulting in a hierarchicalactivation network.


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dicotyledonous seeds (26). Usually, this group of glob-ular storage proteins is encoded by a multigene family,leading to several isoforms that are subjected to post-translational modifications, mainly related to proteo-lytic processes. Despite the presence of different iso-forms, it is possible to define a typical 2S albumin.These molecules are synthesized as a single largerprecursor polypeptide of 18–21 kDa. Subsequently, themolecule is transported into the vacuole, and thereoccurs the processing of the molecule, giving rise to twosubunits; the large subunit of 8–14 kDa and the smallone of 3–10 kDa (28). Structurally, they have as char-acteristics 4 � helices and 4 disulfide bonds (Fig. 1Ba),as found in the �-amylase/trypsin inhibitors and non-specific lipid transfer proteins. This protein class pres-ents a large amount of nitrogen- and sulfur-containingamino acids, and typically shows a high level of cysteineresidues; in some cases, these can represent 6 to 13% ofthe total size of molecule (28).

Some of these molecules can play a physiological rolein plant defense, as is the case with 2S albumins fromradish seeds that have shown an interesting inhibitionactivity against fungi and bacteria, displaying synergisticactivity with defensins of either wheat or barley origin,to cause the permeabilization of the plasma membraneof phytopathogens (3). Furthermore, Ngai and Ng (29)evaluated extracts from seeds of Brassica chinensis, a napin-like albumin, which showed activity against gram-positivebacteria, such as Pseudomonas aeruginosa, responsible forcases of respiratory infections; Bacillus subtilis and Bacilluscereus, responsible for cases of food poisoning; and Bacillusmegaterium. It was found that this polypeptide could beused as the active ingredient in the biotechnologicalproduction of antibiotics (29). Costa et al. (30), perform-ing a study with sesame kernels, showed severe inhibitoryactivity against the human pathogenic bacteria Klebisiellasp., but not against fungi. The 2S albumins from B. napus(napins) present significant antimycotic and antibacterialproperties. The synergistic interaction between these na-pin-like albumins is capable of producing significantdamage to the fungal plasmalemma, causing permeabili-zation through strong interactions between napin and thephospholipid bilayer (31).

The antifungal activity of Passiflora sp. seed proteinshas been studied and shows encouraging results. Agiz-zio et al. (3) found a 2S albumin-like protein in Passi-flora edulis f. flavicarpa with activity against the phyto-pathogens Fusarium oxysporum, Colletotrichum musae, andColletotrichum lindemuthianum and against the yeast Sac-charomyces cerevisiae. They also observed hyphal morpho-logical modifications in these fungi, induced by this 2Salbumin-like protein. Ribeiro et al. (32), performing astudy with the Passiflora alata Curtis, showed a relevantantifungal activity of a peptide with similarity to 2Salbumin. They found activity only against the phyto-pathogenic fungi Colletotrichum gloeosporioides; however,no activity was observed against pathogenic bacteria orhuman pathogenic fungi.

In 2007, Yang et al. (33) conducted a study of seedextracts from motherwort (Leonurus japonicus), a plant

widely used in popular Chinese medicine to treatvarious diseases. They discovered LJAMP1, a peptide of7.8 kDa, with a sequence similar to the napins, and amember of the 2S albumin class. The LJAMP1 in vitrotest showed activity against the fungi Alternaria alternata,Cercospora personata, and Aspergillus niger. In the samestudy, the researchers conducted an in vivo test; theyinduced the overexpression of LJAMP1 in tobaccoleaves and observed that this peptide was very effectiveagainst the fungus A. alternata and the bacteriumRalstonia solanacearum, showing that LJAMP1 can beused in the production of transgenic plants able toresist these pathogens. Furthermore, peptides fromseed extracts of chili pepper, with a similar sequence tomembers of the 2S albumin family, were identified(34). The researchers found an inhibitory activityagainst a wide variety of yeasts, including Candidaalbicans, which accounts for oral and vaginal infection,as well as Candida parapsilosis, Candida tropicalis, andCandida guilliermondii, opportunistic pathogens respon-sible for deaths among patients with immunocompro-mising diseases, associated with use of catheters orpostsurgical infections (34). The peptide also showedactivity against Pichia membranifaciens, Kluyveromycesmarxiannus and S. cerevisiae (34). This peptide can beused to treat or help control infectious diseases causedby the pathogens mentioned above, as well as toproduce more effective antibiotics that do not havetoxic effects and have minimal side effects, either localor systemic (34). In 1999, Marcus et al. (35) extractedprotein from seeds of Macadamia integrifolia, the Mi-AMP2 protein that showed antimicrobial activityagainst a wide variety of species of phytopathogenicfungi, such as F. oxysporum, Alternaria helianthi, Ceratocys-tis paradoxa, Cercospora nicotianae, Chalara elegans, Lepto-sphaeria maculans, Sclerotinia sclerotiorum, Verticilliumdahliae, Phytophthora cryptogea, and Phytophthora parasiticanicotianae; against the yeast S. cerevisiae; against thephytopathogenic bacteria Clavibacter michiganensis andR. solanacearum; and against the human pathogenicbacterium Escherichia coli.

In general, the peptides or proteins could be promis-cuous, showing multiple mechanisms of action. Despitetheir storage and defensive characteristics, the 2S albu-mins are known for their allergenic properties, whichcan be observed with the oily seeds, such as castor, andother plants, such as mustard, sesame, Brazil nuts, andEnglish walnuts.

KUNITZ PROTEINASE INHIBITORS (PIs)

PIs are molecules capable of inhibiting the action ofproteolytic enzymes. They are present in seeds andtubers of plants and act as storage proteins, carrying outimportant functions in the development of seeds andsprouts. It is known that by virtue of their capacity toinhibit enzymes, they can act as defense mechanismsagainst predators and pathogens. These inhibitors dif-fer in terms of mass, cysteine content, and number of


reactive sites. In general, they are single-chain polypep-tides with molecular masses of 18 to 26 kDa, with twoCys-Cys bonds in a single or double-chain polypeptide(Fig. 1Bb and ref. 36). The members of this inhibitorfamily are especially active against serine proteinases,such as trypsin, chymotrypsin, elastase, and subtilisin.However, they also inhibit other proteinase classes,including aspartic and cysteine proteinases, such ascatepsin D and papain, respectively. It is important tomention that the members of the Kunitz family have intheir structure a � trefoil formed by 12 interconnectedantiparallel � strands that are stabilized by numeroushydrogen and disulfide bonds (37). They perform theirfunction through an arginine or lysine amino acidresidue in position P1, which is responsible for forma-tion of a site ligation complex between enzyme andinhibitor (37). These direct interactions between resi-dues from the active site of the inhibitor and thecatalytic site of the enzyme are responsible for a mech-anism of competitive inhibition (36). On the otherhand, the direct interaction between a specific residueof an active loop from an inhibitor and a region of theenzyme different from the catalytic site characterizes anoncompetitive mechanism. In general, these reactionsare reversible, but some Kunitz trypsin inhibitors (KPIs)have such strong affinities for their target protease thatthey form stable complexes that influence catalytic activ-ities and cause irreversible interactions (38).

In general, KPIs are stored in diverse plant tissues,including seeds, tubers, leaves, rhizomes, and fruits (39).The Kunitz-type inhibitor has been isolated from a widevariety of plants, including the Leguminosae, Solanaceae,and Graminae superfamilies (40). They are abundant inseeds of such Leguminosae subfamilies as Mimosoideae,Caesalpinoideae, and Papilionoideae. In general, the KPIsof Caesalpinoideae have one chain containing disulfidebridges and are effective inhibitors of trypsin. However,Bauhinia bauhinoides cruzipain inhibitors and B. bauhin-ioides kallikrein inhibitors lack disulfide bridges and alsohave the capacity to inhibit two distinct classes of enzymes:the serine and cysteine proteases; this emphasizes theexistence of a new subclass of these inhibitors (41).

In Solanaceae, the KPIs also are found in other plantstorage tissues, such as in tubers from Solanum tubero-sum, which contain 19- and 22-kDa species (42). An-other family is characterized by the existence of 5classesof KPIs from S. tuberosum of the Kuras cultivar, includ-ing KPI A, KPI B, KPI C, and KPI K, these beingobserved only in Kuras. Proteins of each group arecharacterized by an N-terminal conservative amino acidsequence consisting of the first 10 residues. Accordingto Bauw et al. (43), these KPIs present 17 residuesignatures of the form [LIVM]-x-D-{EK}-[EDHNTY]-[DG]-[RKHDENQ]-x-[LIVM]-x-{E}-x-x-x-Y-x-[LIVM], asis observed in some potato miraculin-like sequences([], residues allowed; {}, residues excluded from theposition; X, any residue).

The same researchers report that KPIs can accumu-late in different tissues and at different levels. Inpotatoes, they accumulate in leaves and tubers in

response to mechanical wounding, UV-radiation, andlesion by insects or phytopathogenic microorganisms.Some gymnosperms can also accumulate Kunitz-typeserine PIs in reproductive organs, as occurs in Cycasrumphii and Ginko biloba, or in vegetative areas, as occursin Picea and Pinus species (44). In addition, KPIs areencoded in the latex of Carica papaya, being namedpapaya PIs (PPIs), and presenting with a molecularmass of 23 kDa, 2 disulfide bonds, and �-sheet struc-tures, with high stability over a wide pH (1.5–11.0) andtemperature (up to 80°C) range, thus showing strongresistance to proteolytic degradation (45).

The process of subexpression and localization ofthese inhibitors in certain tissues is governed by thegenes that encode them. These inhibitory proteins maybe expressed constitutively or be induced by plantpathogens (44). According to Speransky et al. (46),KPIs are induced during pathogen infection, suggest-ing that these inhibitors play a role in defense againstlytic enzymes involved in insect and pathogen attacks.In Cicer arietinum, screening of an epicotyl cDNA libraryrevealed the presence of at least two different cDNAsencoding KPIs, CaTPI-1 and CaTPI-2, that expressTPI-1 and TPI-2, respectively, with TPI-2 reported to beinvolved in the defense against mechanical damage(47). Speranskaya et al. (48) described the presence of4 genes that express KPIs in S. tuberosum L: PKPI-B1,PKPIB2, PKPI-B9, and PKPI-B10. According to theseauthors, the PKPI-B10 protein has the ability to inhibitthe activity of trypsin and chymotrypsin, as well as tosuppress the growth and development of the phyto-pathogenic fungus Fusarium culmorum, supporting thehypothesis that these inhibitors are expressed as part ofthe defense mechanism. Recently, there has been anincreasing effort to search for KPIs with potentialantimicrobial activity against fungi and gram-positiveand gram-negative bacteria. From Acacia plumosa seed,a Leguminosae–Mimosoideae plant, 3 KPIs have beenisolated, designated ApTIA, ApTIB, and ApTIC, thatexhibit antifungal activity against A. niger, Thielaviopsisparadoxa, and Colletotrichum sp., which demonstratesthat these isoforms can act as specific inhibitors regu-lating proteolytic processes (49).

The isolation and characterization of the proteinaseinhibitors present in many plants of the Solanaceafamily have been reported, especially in tubers of thepotato (S. tuberosum L.). Among these inhibitors de-scribed in tubers are potamin-1 (PT-1), a serine pro-tease inhibitor of 5.6 kDa, with the capacity to inhibittrypsin and chymotrypsin (50). The researchers alsodemonstrated the thermostable antimicrobial activityagainst C. albicans, Rhizoctonia solani, and Clavibactermichiganense subsp. michiganinse exerted by PT-1 (50).However, after characterization of antimicrobial activ-ity, it was demonstrated that PT-1 also presents hemo-lytic activity (51). Furthermore, PT-1 has also beendescribed as potide-G, a peptide of 5578.9 Da with thecapacity to inhibit growth of diverse bacteria, includingS. aureus, L. monocytogenes, C. michiganense, E. coli, andfungi, such as C. albicans and R. solani (52).


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PLANT LECTINS

Lectins were initially classed as carbohydrate-bindingproteins with the ability to agglutinate erythrocytes.Later, different lectins were discovered, but without theagglutination property. Consequently, a new nomencla-ture was created, giving the name “agglutinins” to theproteins that agglutinate cells, and leaving the name“lectins” to a wider group of proteins. Therefore,nowadays, lectins include all proteins containing anoncatalytic domain that binds to a specific carbohy-drate. They are involved in several biological processes,such as antimicrobial/antiviral action, and in the inhi-bition of growth of tumor cells. The molecular mass ofplant lectins is very diverse, varying from 11 to 110 kDa.Recently, lectins have been classified according to theirsecondary and tertiary structure, as well as by themolecular interactions between amino acid residues.Although lectins present a wide specificity for variouscarbohydrate molecules, the secondary and tertiarystructure variation is very small. Specifically, the base oflectin configuration is an arrangement of � sheets (Fig.1Bb, c and ref. 53).

Plant lectins are considered storage proteins that canalso exhibit biological properties related to the protec-tion of plant tissue against pests and pathogens, as wellas during abiotic stresses. This characterization occursmainly because of their property of interacting withglycoconjugates or at the surface of pathogenic cells insolution. Currently, it is suggested that lectins acquiredtheir storage role as a secondary function, with thedefense activity being the main characteristic for thisprotein group (54). The presence of lectins was ob-served in tubers of Colocasia and Xanthosoma sagittifolia,where they act as storage molecules, in addition tohaving secondary functions, such as agglutination activ-ity with rabbit erythrocytes. Mannose-binding lectinswere found in tubers of Alocasia, also demonstratingboth defensive and storage roles (55).

Furthermore, plant lectins have been reported tohave an important role against fungi, bacteria, andinsects (48, 52). The cell membranes of these organ-isms normally present glycoconjugates of different na-tures, such as glycoproteins, glycolipids, and polysaccha-rides, which can have a structure similar to carbohydratesthat interact with lectins. Therefore, it seems that lectinsare able to work as defense proteins by binding toglycoconjugates from these microorganisms. Plant lectinsalso have the advantage of remaining stable under a widepH and temperature range, as well as being resistant toanimal and insect proteases. These features make them apotential tool for the inhibition of pathogenic microor-ganisms.

However, as fungi present a thick and rigid cell wall,lectins cannot interact with glycoconjugates from thecell wall, or insert in the cytoplasm of the cell, therebycausing structural or permeability alterations. Hence,the mechanism of action for lectins is based on theirbinding to the carbohydrates located on the surface ofthe fungal cell wall. Thus, a polypeptide from Amaran-

thus caudatus seeds was able to inhibit fungal growth butcould not present a fungicidal activity (56). A chitin-free lectin from Urtica dioica also showed a fungalgrowth inhibition activity against Botrytis cinerea, Tricho-derma hamatum, and Phycomyces blakesleeanus (57). Fur-ther experiments showed that a hevein from the rubberHevea brasiliensis had similar antifungal features to U.dioica lectin (58).

Bacterial cell walls offer similar features for targetingby lectins. Glycoconjugates in bacteria are located inthe membrane, thus impeding lectins from interactingwith them and from penetrating to the cytoplasm,causing structural alteration or increasing permeability.Consequently, plant lectins may play an indirect role asantibacterial agents, by interacting with extracellularglycans or cell wall glycoconjugates. One interestingmechanism of action is the blocking of bacterial move-ments, causing loss of motility and preventing invasionof seedling roots by the pathogens. This mechanismwas demonstrated by a lectin from thorn apple (Daturastramonium), confirming that this group of proteinspossesses different modes of action for defendingplants against pathogenic microorganisms (59). On theother hand, not many plant lectins have been reportedto have antiviral activity. As plant viruses lack glycocon-jugates in their membrane, lectins may act in anindirect way in order to cause toxicity to these micro-organisms. However, lectins are able to reduce viralinfection by animal and human viruses, as these presentglycoproteins in their virions. Until now, only type-2RIP has been described as showing antiviral activity, butit is not well known whether the plant protein inhibitsviral infection or replication, or is a systemic toxin (60).

In summary, considering that lectins act as storageproteins in plant organs and because of their role innitrogen accumulation in the form of carbohydrate-binding molecules, it is possible to suggest that thisgroup of proteins is not only involved in plant defense,but also presents a wide variety of mechanisms ofaction, including direct binding to glycoconjugates andindirect activities outside the pathogen cell wall.

GLYCINE-RICH PROTEINS (GRPs)

GRPs belong to a group of storage proteins from plants,with molecular mass varying from 6 to 30 kDa. Theywere previously described as an essential source ofamino acid residues. Earlier studies revealed that GRPgene expression can be found in cells from the vasculartissue, especially in xylem. Nevertheless, this peptideclass can also be found in the cotyledon, and membershave been identified in chloroplasts and in plant cellwalls (58).

GRPs are found commonly in Brassicaceae, So-lanaceae, and Panicoideae (Fig. 1Bd) and can be char-acterized mainly by their high glycine residue content.However, glycine concentration varies among differentplant species (61). As such, GRPs can be classified into3 main groups, according to glycine content and the


presence of some conserved domains. The first groupconsists of proteins with �70% glycine residues in theirprimary sequence and also with specific RNA-bindingdomains called RNA recognition motifs (RRMs; ref.62). This group includes GRPs from Arabidopsis thalianaand B. napus (58, 61). Proteins with a lower content ofglycine residues, also containing the RNA-binding do-mains, are classified into the second group of GRPs,which can be observed in some species of tomato,saltbush, and Arabidopsis (63, 64). The third groupincludes proteins with a high percentage of glycineresidues in their primary sequence but no conserveddomains (61), these being found in several species.such as Psidium guajava (62). More recent findings haveadded one more group for GRP classification: group IVcontains mainly RNA-binding proteins with a glycine-rich domain and either a cold shock domain (CSD) oran RRM. Some members of this group can also presentCCHC (Cys-x8-Cys-x5-Cys-x3-His) zinc fingers in theirstructure (65). In addition, the fact that some GRPspresent mixed patterns of repeats is leading to theintroduction of a fifth group, especially to includeproteins with these patterns found in Arabidopsis, rice,and eucalyptus genomes (unpublished results).

However, there is still a lot to be learned about thephysiological mechanism of action of GRPs and, untilnow, no direct relationship between RNA-binding mo-tifs of GRPs and antimicrobial activities has been ob-served. Despite several studies on GRP primary se-quences, there are still no reports on crystal structureor NMR studies for this group of proteins. Further-more, there are no reports relating GRP groups withtheir functions, although most GRPs from group 1 haveshown a remarkable expression enhancement duringdifferent stress-induction experiments (66). Neverthe-less, GRPs at the cell wall, playing a role in water andnutrient transportation or working as signaling mole-cules, were observed in all groups. The same wasdemonstrated with proteins acting as storage mole-cules, suggesting that these functions were extremelywell conserved throughout evolution (58).

Furthermore, more than just storage functions havebeen observed for GRPs. Among the 4 groups reportedhere, several functions have been noted. GRPs can actin plant protection against biotic stresses. Some reportshave described the antifungal activity of GRPs againstphytopathogens. Earlier studies showed that 8 proteinsisolated from Triticum kiharae seeds, including oneGRP, did not present activity against bacteria, butexhibited the ability to inhibit filamentous fungusdevelopment, acting against organisms such as Helmon-trhosporium sativum and F. culmorum (67). The resultsshowed that GRPs could also act as an elemental pieceof the complex defense mechanism puzzle in plants(67). Analyses of the primary sequences revealed thepresence of a conserved CXG motif, which seems to beimportant for antifungal activity, but no experimentalprocedures have yet been done to confirm this hypoth-esis (67). Furthermore, because of the fact that 8proteins were isolated from different groups in a single

species (67), it was suggested that there might be asynergy between AMPs that could lead to the defensefunction.

Despite the previous description, there are few datain the literature relating GRPs to plant defense pro-cesses. It was observed that an RNA-binding GRP iso-lated from Nicotiana glutinosa is probably related toplant-pathogen interaction, since the gene expressingGRPs from the plant increases its expression wheninfected by tomato mosaic virus (TMV; ref. 68). Themechanism of action for the RNA-binding regions fromGRPs and their relationship to plant defense againstpathogens and pests is still not clear. However, there isa hypothesis indicating that GRPs are able to bind todifferent proteins and RNA molecules, poly(G)-,poly(U)-, and/or poly(A)-homopolymers, or even tosingle-stranded DNAs (69). Some studies reported thatthe bound RNA molecules can also be from the hostplant (70). Therefore, this interaction seems to inducethe regulation of several plant pathways, depending onthe environmental stress that has occurred (68). Thisalso indicates that the presence of the pathogen is impor-tant for enhancing the expression of proteins from thisgroup of plants, causing a cascade of pathways that willlead to plant defense against the pathogenic attack.Another GRP isolated from pumpkin, known as cucur-moschin, showed antifungal activity against B. cinerea, F.oxysporum, and Mycosphaerella oxysporum (71). Moreover,some GRPs, such as GRPs from oats (Avena sativa) and G.biloba, contain a chitin-binding domain, which gives themthe capacity to inhibit filamentous fungal growth (72).

In addition to the antifungal activities observed, onlya few reports have shown activity toward pathogenicbacteria. Recently, a novel GRP was isolated from guavaseeds and showed activity toward two gram-negativebacteria; Klebsiella sp. and Proteus sp., which causenosocomial and urinary infections (73). Pg-AMP1, asthe peptide was called, was able to inhibit bacterialgrowth at micromolar concentrations, revealing thatGRPs can act against diverse pathogens (73). Its pri-mary sequence classified the guava seed peptide intothe third group of GRPs, as it does not present anyRRM motif, although it has many glycine residues. A3-dimensional structure of Pg-AMP1 was recently ob-tained by molecular modeling, revealing 2 � helicesand 2 spiral loops, one at the N terminus and other atthe C terminus of the peptide (73). This was the firststructure of an antimicrobial GRP proposed so far.Glycine-rich residues were mainly placed at these loops,conferring flexibility to the peptide. Since several pep-tides described earlier in the literature presented adimeric formation for their activity against pathogens(74), the construction of a homodimer of Pg-AMP1 andanalysis of molecular dynamics were performed. Hence,this structure could lead to the development of hypothe-ses regarding the mechanism of action of antimicrobialGRPs. The most acceptable hypothesis, however, is thatthe exposed cationic residues of the dimer interact withnegatively charged regions from the phospholipids on thepathogen’s membrane surface, leading to a pore forma-


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tion, increase in membrane permeabilization and, subse-quently, to bacterial cell death (73).

VICILINS (7S GLOBULINS)

The vicilins constitute a class of proteins found inabundance as reserves in seeds of leguminous andnonleguminous plants, representing as much as 70 to80% of total protein in the seeds of these plants (35).Their structure consists of a trimeric organization, witha molecular mass ranging from 150 to 170 kDa. How-ever each subunit has an average molecular mass ofbetween 40 and 70 kDa, with no disulfide bonds, due tothe low abundance of cysteine residues in their se-quences (Fig. 1Be and ref. 35). Vicilins may have a largenumber of small polypeptides associated with theirmajority subunits, produced by proteolytic changesduring post-translational processing, which may alsoresult from the addition of incomplete or partial deg-radation of the binding of oligosaccharide side chainsof protein (35). The post-translational proteolysis andglycosylation result in subunits with molecular massesbetween 12.5 and 33 kDa. Furthermore, the presenceof oligosaccharides covalently attached to protein sidechains further interferes with the process of digestionby insects and fungi. The vicilin tertiary structureusually presents a � barrel, followed by 2 antiparallel �helices (75).

In plants, specifically in seeds, this class of proteinhas proven to be multifunctional, acting as an energysource and providing amino acids during the germina-tion process, while also being involved in the defense ofmany plant species, with activity against fungi andinsects (76). Examples include the vicilins extractedfrom Vigna unguiculata (string bean), Vigna radiata(Chinese bean), P. vulgaris (bean), G. max (soybean)and Canavalia ensiformis (jack bean) (74). The mecha-nism of action may be through binding to the fungalcell wall, thus interfering with the germination ofspores or conidia of a variety of fungi (77). Besides theantifungal activity, vicilins show insecticidal activity bybinding to chitinous structures of the midgut of insects,thus interfering in their development (35, 78).

The potency of the antifungal activity displayed byvicilins may vary according to the extracted material.Gomes et al. (74) extracted a vicilin from V. unguiculata,showing inhibitory activity between 90 and 100%against the yeast S. cerevisiae, in addition to interferingin the development of the fungi Fusarium solani, F.oxysporum, C. musae, Phytophthora capsici, Neurosporacrassa, and Ustilago maydis sporidia, acting on the sporegermination processes of these fungi. Seed vicilin ex-tracted from V. radiata showed 65% inhibitory activityagainst C. albicans (78), whereas vicilin found in Malvaparviflora showed inhibitory activity against Phytophthorainfestans(79).

TUBER STORAGE PROTEINS: PATATINS,TARINS (G1 GLOBULINS), AND OCATINS

Patatin (Fig. 1Bf) is the common name given to a familyof plant storage glycoproteins that make up 40% of thetotal soluble protein in potato (S. tuberosum) tubers(80). These proteins can be N-glycolsylated, and whilethis process does not affect their enzymatic properties,the molecule charge heterogeneity is probably affectedby its glycosylation pattern (81). The isoforms of pat-atins have molecular masses of 40 to 45 kDa (82). Theseisoforms have homologous sequences (85–98%) andshow biochemically (83) and immunologically similarproperties (81, 83). They are accumulated in largeamounts in tubers and in the vacuoles and leaves andare mainly found in parenchymal cells (82) but can beobserved in such other plant organs as stems, petioles,and more rarely in roots (84), the latter showing onlysmall amounts (85). Patatin gene expression is highlystimulated following tuber initiation but can be stimu-lated in other nontuber organs by sucrose treatment.Therefore, sucrose might be associated with patatingene regulation, but not as a direct regulator of thisprocess (83). These proteins show clear differences atthe N-terminal region, allowing the differentiation intotwo different classes, according to the presence (classII) or absence (class I) of a short sequence of aminoacids (54). Patatins and patatin-like proteins (86) canact as esterase enzymes, as lipid acyl hydrolases, acyltransferases, and phospholipases, and also present an-tioxidant activities (82, 87). It was known that theseenzymatic activities increase when the cell is disrupted,and this suggests participation in the defense mecha-nism (77). However, their physiological role remainsundetermined (88). The observation of the acyl hydro-lase activity of these proteins suggests a function in thetuber transition from dormancy to vegetative growth orin protection against growth of microbial pathogens(89). Patatin lipolytic activity can provide some defenseagainst insect pests, such as Diabrotica larvae (84) andcan work in plant signal transduction (87). Patatinswere described as showing insecticidal activity againstthe corn rootworm (81). Comparing patatins withother lipases, it is possible to visualize a conservedamino acid motif (Gly-X-Ser-X-Gly), which is character-istic of esterases (81, 86). Tonon et al. (90), in studieswith potato tuber, found a patatin isoform with �-1,3-glucanase activity, Glu-40, which showed a potentialantifungal activity for this storage protein. Since the�-1,3-glucanase proteins are involved in the defensemechanisms of plants against fungal pathogens, thiscan improve the tuber defense responses through thegeneration of response elicitors on contact with thepathogen surface. It has also been reported that pat-atins in tobacco leaves show activity against TMV. Afterthe infection was detected, 3 patatin-like genes werehighly induced, and one of the proteins expressed wasa phospholipase A2, reinforcing the hypothesis that thisprotein is able to engage in antimicrobial activity, aswell as the already described storage function (91).


TABLE 2. Storage proteins with experimentally antimicrobial activities observed

Peptide class and designation Source Activity against MIC (�M) Ref.

2S albuminsNapin Brassica rapa Pseudomonas fluorescens 66 101

Bacillus subtilis 236Bacillus cereus 222Bacillus megaterium 215Mycobacterium phlei 146

Napin-like polypeptide Brassica alboglabra P. fluorescens 89 29B. subtilis 278B. cereus 145B. megaterium 156M. phlei 104

Napin Momordica charantia Trichoderma hamatum 0.18 102Pf-AFP1 Passiflora edulis Trichoderma harzianum 11 103

Fusarium oxysporum 11Aspergillus fumigatus 13

Kunitz proteinase inhibitorsAntifungal protein J (AFP-J) Solanum tuberosum Candida albicans ND 104

Saccharomyces cerevisiae NDTrichosporon beigelii ND

PKPI-B10 S. tuberosum Fusarium culmorum ND 48ApTIA Acacia plumosa Aspergillus niger ND 49ApTIB A. plumosa Thielaviopsis paradoxa ND 49ApTIC A. plumosa Colletotrichum sp. ND 49Potamin-1 (PT-1) S. tuberosum C. albicans ND 50, 52

Rhizoctonia solani NDClavibacter michiganensis NDStaphylococcus aureus NDListeria monocytogenes ND

Plant lectinsHevein Urtica dioica Botrytis cinerea 106 58

F. culmorum 128F. oxysporum 266Phycomyces blakesleeanus 64Pyrenophora tritici-repentis 74Pyricularia oryzae 106Stagonospora nodorum 106T. hamatum 19

Glycine-rich proteinsGRP Triticum kiharae Helminthosporium sativum ND 67

F. culmorum NDPg-AMP1 Psidium guajava Klebsiella sp. ND 73

Proteus sp. NDEscherichia coli NDKlebsiella pneumonia ND

Vicilins (7S)Mi-AMP2c Macadamia integrifolia Alternaria helianthi 0.0016 35

Ceratocystis paradoxa 0.0084Cercospora nicotianae 0.0016Chalara elegans 0.0033F. oxysporum 0.0084Leptosphaeria maculans 0.0042Sclerotinia sclerotiorum 0.0084Verticillium dahliae 0.0016S. cerevisae 0.0084Phytophthora cryptogea 0.0042Phytophthora parasitica 0.0033C. michiganensis 0.0084

Vicilin Vigna unguiculata Fusarium solani ND 74, 78F. oxysporum NDColletotrichum musae NDC. albicans NDS. cerevisae ND

(continued on next page)


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Ocatins are the major storage proteins purified andcharacterized from tubers of Oxalis tuberosa Mol. Theyhave �153 amino acid residues, a molecular mass of�18 kDa, and isoelectric point of 4.8. Like otherstorage proteins, ocatins constitute 40–60% of totalsoluble proteins present in tubers of O. tuberosa Mol(92). In addition to their core function, which is tostore nutrients for the plant, ocatins also exhibit anti-microbial activity, acting against various species ofphytopathogenic bacteria, such as Pseudomonas aureofa-ciens, Serratia marcescens, Agrobacterium tumefaciens, andAgrobacterium radiobacter, and fungi, such as Nectria he-matococcus, F. oxysporum, Phytopthora cinnamomi, and R.solani, which attack many crops, causing problems inagriculture (93). The expression of ocatins seems to behighly regulated; they are found only in the marrowcells, epidermis, and subepidermal regions of the tuberand are absent in the cortical tuber. In certain regions,ocatin expression reaches peak levels when the tuber isnear maturity and will enter the period of sprouting(93). Studies conducted by Flores et al. (93) have shownthat ocatins have little homology with other proteinsperforming the same functions of storage, and theiramino acid residue sequences have similarity to the Betv1/PR-10/MLP family proteins. This family is responsiblefor cases of allergy to pollen and is found in a variety ofplant species. However, ocatin is the first molecule be-longing to the Bet v1/PR-10 family with proven antimi-crobial activity. These proteins are responsible for form-ing a protective barrier in the plant against pathogenattack (93–95). The expression of members of the PR-10family in plants can be triggered in two ways. The firstmechanism is through direct attack by a pathogen, caus-ing an infection, thus making the plant express theseproteins. The other mechanism of induction is through“abiotic stress,” which does not require direct contactbetween the pathogen and the plant for expression (94).The other family to which ocatin can be assigned is themajor latex protein (MLP) family; this family is involved inprocesses of defense against pathogens, as well as otherfunctions (95).

Continuing with tuber proteins, tarins are the majorglobulins found in taro. Taro, a tuber crop from Asiaand tropical countries (5), has an enlarged, starchyunderground stem, which is designed as a corm and iscultivated in tropical and subtropical climates (89). It

includes 4 related species in the Araceae family: Coloca-sia esculenta (taro), Cyrtosperma chamissonis (giant swaptaro), and Xanthossoma sagittifolium, which are eaten,and Alocasia macrorhiza (giant taro), which has thethickened stem as the edible part (54). Guimaraes et al.(5) showed that the promoter region of the tarin genepresents homology with the promoters of the patatingene in potato, the TarI site showing 65% identity withthe pgT12 of patatin genes. The researchers suggestedthat when transformed with a genomic fragment of 5.7kb, the expression of the entire TarI gene in potatoesfollowed the pattern of patatin synthesis. The TarI genecontains an open reading frame of 765 bp. As withpatatins, tarins accumulate in the vacuolar space of theparenchyma cells (5), and in this space, the proteolyticcleavage of the proprotein occurs, giving origin to twomature tarins, analogous to albumin and globulinstorage in seeds (54). The two tarins formed by theproprotein cleavage have two cysteine residues, but thedisulfide bond pattern formed has not yet been deter-mined. The total amount of these proteins in tarocorresponds to 1.5–3.0% of the total corm weight, themajority of which consists of albumins and globulins(89). Among these, tarin or G1 globulin represents 40%of total soluble proteins (54). Tarins present at least 10isoforms, with masses around 12.5 kDa and pI rangingfrom �5.5 to 9.5. Among the isoforms, it is possible todetect 25% identity in amino acid sequence (54). Bezerraet al. (96) showed that Colocasia tarins present at least 40%homology with snowdrop (Galanthus nivalis) lectins, andShewry (54) determined that sequence comparisonsshowed homology between tarins and mannose-bindinglectins. Furthermore, the group of G1 globulins (taringroup) presents similarities with circulins from Curculigolatifolia (89), which leads to the suggestion, althoughthere are no data to testify yet, that this class of proteinshas storage and defensive roles.

BIOTECHNOLOGY POTENTIAL ANDCONCLUSIONS

Plants possess an exceptional range of broad-spec-trum antimicrobial properties encompassing activitiesagainst bacteria and fungi, and in this sense, plant storageproteins may have fundamental roles in defense (97).

TABLE 2. (continued)

Peptide class and designation Source Activity against MIC (�M) Ref.

OcatinsOcatin Oxalis tuberosa Agrobacterium radiobacter ND 93

Agrobacterium tumefaciens NDPseudomonas aureofaciens NDSerratia marscescens NDNectria hematococcus NDF. oxysporum NDPhytopthora cinnamomi NDR. solani ND

MIC, minimum inhibitory concentration; ND, not determined (but protein showed antimicrobial activity).


Storage is a major plant function, and in this function, wecan include reserve roles and elements of the defensivemechanism, growth, and accumulation. It is extremelyimportant to construct a detailed understanding regard-ing storage protein structure and diversity, as this is aprerequisite for manipulating and improving the use ofthese molecules (4), as well as attempting to explain thehuge multiplicity of functions for these proteins.

In addition to their function as a nutritional reserve forplants, and in many cases plant protection, this group ofproteins represents a powerful tool, especially for thepharmaceutical industry, which aims to produce moreeffective drugs at lower cost. Table 2 distinguishes eachantifungal and antibacterial activity associated with thestorage proteins covered in this review. However, thepatatin and tarin classes are not included, since the dataabout them are mainly based on gene homology compar-isons and do not specify target pathogens. Despite theabsence of in vitro and in vivo experiments, these proteinclasses are broadly known by their performance in plantdefense mechanisms. For example, GRPs display a widevariety of functions in the plant kingdom. The discoveryof GRPs with antifungal and antibacterial activities canprovide novel insights into their mechanisms of action.Nonetheless, GRPs can also be exploited as new biotech-nological tools for control of biopathogens in agroeco-nomics and also in health sciences, through geneticengineering advances. Moreover, the increasing knowl-edge of GRPs in plant stress response can help in identi-fying the expression mechanism observed at the post-transcriptional level due to cold acclimatization. As such,plant germination and development can be improvedusing biotechnological tools in order to enhance GRPexpression in transgenic plants. In the same way, themechanics of ocatin synthesis are still unknown but maybecome an important target for future research becauseof the great potential for new product generation, as wellas the fact that the cloning and expression of ocatinscould lead to the production of transgenic plants withgreater resistance to phythopathogenic microorganisms.

The characterization of plant storage proteins couldadvance the development and production of new drugs.Action may be through topical treatments for dermatitisor fungal infections, or systemic use of antibiotics incombating infectious diseases. However, they may alsopresent a specific mechanism of action against a particu-lar type of pathogen responsible for diseases in humans,thus reducing the side effects in patients, especially thosewho are immunocompromised (97).

In addition to this application in the pharmaceuticalarea, these molecules can be used as a tool in geneticimprovement, as in the development of transgenicplants that could express storage proteins at high levelsin the presence of the pathogen, thus potentiallyincreasing plant resistance to attacks, reducing the lossin crops, and also reducing the need for pesticideapplication (98, 99). One possibility that is beingstudied and could be used in the future, helping toadvance developments both in health and in agricul-ture, would be to integrate the use of these molecules

with new biotechnological tools that are currently be-ing elaborated (12). This would include nanotechnol-ogy, which is being used intensively in research todevelop new products (100). Its application comesthrough the embodiment of these proteins in nanocap-sules, thus providing medicines and pesticides withcontrolled release, as well as release at specific sites.Furthermore, this technology can be used in conjunc-tion with early disease detection, thereby assisting inearly treatment of microbially induced diseases (100).The technology can also serve to improve the levels ofnutrients in plants, as well as controlling the presenceof microorganisms in water treatment centers (100).The use of biotechnology tools is being increasinglyintroduced in the development of new treatments withmore effective natural or synthesized compounds; thesemay be capable of inhibiting growth of microorganismsand also reducing their resistance.

Today, there are several papers in the literature bygroups of researchers around the world working in thefield of biotechnology, as well as in other areas ofresearch, that employ various methods of purificationand characterization in order to identify storage pro-teins, with the aim of generating energy through thestorage of essential nutrients for the survival and pro-liferation of plants. They are also showing promisingantimicrobial activity against many kinds of pathogenicand phytopathogenic microorganisms (98).

This work was supported by the Conselho Nacional deDesenvolvimento Científico e Tecnologico, Coordenacao deAperfeicoamento de Pessoal de Nível Superior, UniversidadeCatolica de Brasilia, and the Fundacao de Apoio a Pesquisado Distrito Federal.

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Received for publication May 2, 2011.Accepted for publication June 23, 2011.


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Plant storage proteins with antimicrobial activity: novel insights into