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A research project to discover isolate and characterize the different bioactive peptides in plants

This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Abstract Bioactive peptides are part of an innate response elicited by most living forms. In plants, they are produced ubiquitously in roots, seeds, flowers, stems, and leaves, highlighting their physiological importance.

While most of the bioactive peptides produced in plants possess microbicide properties, there is evidence that they are also involved in cellular signaling. Structurally, there is an overall similarity when comparing them with those derived from animal or insect sources.

The biological action of bioactive peptides initiates with the binding to the target membrane followed in most cases by membrane permeabilization and rupture. Here we present an overview of what is currently known about bioactive peptides from plants, focusing on their antimicrobial activity and their role in the plant signaling network and offering perspectives on their potential application.

  1. In the likely event of evolutionary changes within the target offender, new forms of peptides naturally emerge to counter the resistant infectious agent. Research is needed to elucidate the strategies adopted by life forms producing AMPs to counter the defensive plots posed by invading germs.
  2. The biological action of bioactive peptides initiates with the binding to the target membrane followed in most cases by membrane permeabilization and rupture.
  3. A similar effect was confirmed with proteinases from papaya Carica papaya , pineapple A.
  4. Based on previous studies and genomic information our major interests are to unravel the importance of different phosphorus sources and salinity gradient on the function and bloom formation of the toxic cyanobacterial genera from the Baltic Sea Nodularia spumigena and Anabaena.

Introduction No doubt proteins were designed to be versatile molecules. The number of functions in which they participate during metabolism supports this affirmation. Proteins act as defense, integrating the immunological system, as part of the enzymatic network required during metabolism, as a nutrient, as storage, contractile, structural, and motile molecules, as transporters, and as signaling and regulatory mediators.

These are well-established functions for which proteins have gained undisputed roles. Aside from these functions other roles are associated with these molecules, such as antifreezers, sweeteners, and antioxidants. A relatively new role involves their ability to interact with cellular membranes in a nonreceptor-ligand type of binding. Antimicrobial peptides AMPs are often the first line of defense against invading pathogens and play an important role in innate immunity [ 1 ].

The list of identified antimicrobial peptides has been growing steadily over the past twenty years. Initially, the skin of frogs and lymph from insects were shown to contain antimicrobial peptides, but now over 1500 antimicrobial peptides have been described, in living organisms including those from microorganisms, insects, amphibians, plants, and mammals [ 2 ].

In 1963, Zeya and Spitznagel described a group of basic proteins in leukocyte lysosomes endowed with antibacterial activity [ 3 ]. Later, Hultmark et al. The vaccinated insects survived a posterior challenge with high doses of the infecting bacteria, indicating the relevance of the bactericidal proteins.

Additional research identified a 35-residue peptide cecropin as responsible for the antibacterial effect. Further investigation by Boman and other groups confirmed that antimicrobial peptides AMPs are distributed ubiquitously in all invertebrates investigated, generating academic and commercial interest [ 15 — 9 ].

Because the rapid increase in drug-resistant infections poses a challenge to conventional antimicrobial therapies, there is a need for alternative microbicides to control infectious diseases [ 210 — 13 ].

A comparative analysis of these molecules reveals that there are no unique structural requirements useful to discriminate these activities and to facilitate their classification. Most bioactive peptides have a high content of cysteine or glycine residues; the disulphide bridges that may be formed between cysteinyl residues increase their stability.

Most of them contain charged amino acids, primarily cationic, and also hydrophobic domains. There is evidence that cationic charged peptides are relevant for antibacterial or antiviral activity but few exemptions of anionic peptides also exist.

This review updates information on plant bioactive peptides. When little or no available information exists on a specific group, we use examples taken from other lifer forms, assuming that upcoming studies may reveal information on peptides whose attributes have not yet been found in plants.

The review does not cover in detail the antimicrobial mechanism underlying the effect of bioactive peptides since two recent reviews on the subject were published [ 4511141525 — 31 ]. Antimicrobial Peptides Isolated from Plants As mentioned above, AMPs are part of important immunological barriers to counter microorganism microbial infections and represent another aspect of the resistance phenomenon known as the hypersensitive response HR.

This phenomenon was described by H. Marshall Ward in cultures of leaf rust Puccinia dispersar or Puccinia triticina and by several plant pathologists 100 years ago [ 1578 ]. The hypersensitive reaction HR is considered the maximum expression of plant resistance to pathogen attack and is defined as a fast death of the plant cells associated with growth restriction and pathogen isolation.

Cell death that happens during HR is considered a lysosomal-type of programmed cell death PCD or autophagy [ 21012 ], unlike mammalian apoptosis.

Also, signaling by resistance gene products RGP triggered during the HR response is not associated with death effectors mammalian caspasesor with the death complex equivalent to the mammalian apoptosome.

BioMed Research International

Therefore, HR is viewed as part of a continuum of effects mediated by defense elicitors [ 45152527 — 29 ]. Although many AMPs are generically active against various kinds of infectious agents, they are generally classified as antibacterial, fungicides, antiviral, and antiparasitic.

The antibacterial activity of peptides results from the amphiphilic character and presence of motifs with high density of positively charged residues within their structure [ 6 — 9 ]. This type of arrangement facilitates peptide attachment and insertion into the bacterial membrane to create transmembrane pores resulting in membrane permeabilization. The amphipathic nature of antimicrobial peptides is required for this process, as hydrophobic motifs directly interact with lipid components of the membrane, while hydrophilic cationic groups interact with phospholipid groups also found in the membrane.

The antifungal activity of AMP was initially attributed to either fungal cell lysis or interference with fungal cell wall synthesis. A comparison of plants antifungal peptides suggests a particular structural-activity arrangement involving polar and neutral amino acids [ 11 — 1332 ]. However, like for antibacterial peptides, there are no obvious conserved structural domains clearly associated with antifungal activity. The antiviral effect of some AMPs depends on their interaction with the membrane by electrostatic association with negative charges of glycosaminoglycans facilitating binding of AMP and competing with viruses [ 11 ].

Such is the case of the mammalian cationic peptide lactoferrin that prevents binding of herpes simplex virus HSV by binding to heparan moieties and blocking virus-cell interactions [ 332 — 34 ]. Alternatively, defensins described below bind to viral glycoproteins making HSV unable to bind to the surface of host cells [ 2527 ]. The antiviral effect of peptides can also be explained by obstruction of viral interaction with specific cellular receptors, as shown during binding of HSV and the putative B5 cell surface membrane protein displaying a heptad repeat alpha-helix fragment.

The effect was demonstrated with the synthetic 30-mer peptide that has the same sequence found in the heptad repeat that inhibits HSV infection of B5-expressing porcine cells and human HEp-2 cells [ 715192022 — 24 ]. A less specific interaction between AMP and viruses causes disruption or destabilization of viral envelope yielding viruses unable to infect host a research project to discover isolate and characterize the different bioactive peptides in plants [ 15171921 — 24 ].

Finally, a peptide mediated activation of intracellular targets induces an antiviral effect as demonstrated with the antiviral peptide NP-1 from rabbit neutrophils that crosses the cell membrane migrating into the cytoplasm and organelles, followed by inhibition of viral gene expression in the infected cell.

The proposed mechanism involves downregulation of VP16 viral protein entry into the nucleus that prevents expression of early viral genes required to propagate viral infection [ 411263031 ]. The initial characterization of molecules displaying AMP activity was followed by isolation of purothionin, the first plant-derived AMP. Purothionin is active against Pseudomonas solanacearum, Xanthomonas phaseoli and X. Since then, several plant peptides have been discovered.

The major groups include thionins types I—Vdefensins, cyclotides, 2S albumin-like proteins, and lipid transfer proteins [ 151922 — 24 ]. Other less common AMPs include knottin-peptides, impatiens, puroindolines, vicilin-like, glycine-rich, shepherins, snakins, and heveins Table 1 [ 35 — 44 ].

Selected plant antimicrobial peptides. Full isolation of plant AMP has been attained in some cases. It is the case of lunatusin a peptide with molecular mass of 7 kDa purified from Chinese lima bean Phaseolus lunatus L. Lunatusin exerted antibacterial action on Bacillus megaterium, Bacillus subtilis, Proteus vulgaris, and Mycobacterium phlei.

The peptide also a research project to discover isolate and characterize the different bioactive peptides in plants antifungal activity towards Fusarium oxysporum, Mycosphaerella arachidicola, and Botrytis cinerea.

Interestingly, the antifungal activity was retained after incubation with trypsin [ 45 ]. Another peptide, named vulgarinin, from seeds of haricot beans Phaseolus vulgariswith a molecular mass of 7 kDa showed antibacterial action against Mycobacterium phlei, Bacillus megaterium, B. Its antifungal activity was also retained after incubation with trypsin. Another example is a peptide from Amaranthus hypochondriacus seeds that displays antifungal activity Table 1 [ 4647 ]. Both lunatusin and vulgarinin inhibited HIV-1 reverse transcriptase and inhibited translation in a cell-free rabbit reticulocyte lysate system, suggesting a similarity of action between these two peptides and that antimicrobial activity might be linked to protein synthesis [ 46 ].

Lunatusin also elicited a mitogenic response in mouse splenocytes [ 45 ] and proliferation of breast cancer MCF-7b cell line while vulgarinin inhibited proliferation of leukemia L1210 and M1 cell lines and breast cancer MCF-7 cell line [ 46 ].

A peptide named hispidulin was purified from seeds of the medicinal plant Benincasa hispida that belongs to the Cucurbitaceae family Table 1. Hispidulin exhibits a molecular mass of 5. Two additional antifungal peptides with novel N-terminal sequences, designated cicerin and arietin, were isolated from seeds of chickpea Cicer arietinumrespectively.

These peptides exhibited molecular masses of approximately 8. Arietin expressed higher translation-inhibitory activity in a rabbit reticulocyte lysate system and higher antifungal potency toward Mycosphaerella arachidicola, Fusarium oxysporum, and Botrytis cinerea than cicerin.

Both lack mitogenic and anti-HIV-1 reverse transcriptase activities [ 24950 ]. There are also some studies on AMP peptides from dry seeds of Phaseolus vulgaris cv. Matador and was active against Gram-positive Clavibacter michiganensis and Gram-negative Ralstonia solanacearum bacterial pathogens, as well as against fungi, such as, Fusarium culmorum, F. Antiparasitic peptides are another group of bioactive peptides.

Following an initial report describing the lethal effect of magainin isolated from Xenopus skin on Paramecium caudatum, another peptide cathelicidin confirmed the antiparasitic activity of AMPs [ 52 — 56 ]. Anthelmintic activity is also a recognized feature attributed to vegetable proteinases Table 1. For instance, bromelain, the stem enzyme of Ananas comosus Bromeliaceaeshows anthelmintic effect against Haemonchus contortus [ 5253 ], similar to the reference drug pyrantel tartrate.

A similar effect was confirmed with proteinases from papaya Carica papayapineapple A. The anthelmintic effect cannot be fully explained by the proteolytic effect of these enzymes, as the inhibited enzymes partially preserve antiparasitic activity. It is suggested that selected domains within the proteinase molecule different from the active site could be responsible for the antiparasitic effect unpublished observations.

  1. Regardless of their size they contain several conserved cysteinyl residues structuring disulphide bridges that contribute to their stability. CLV3 is a 13-residue peptide of this family that plays a fundamental role by promoting stem cell differentiation during meristematic development [ 104 — 106 ].
  2. Another prominent group of toxins are alkaloid neurotoxins, including e.
  3. The largest antimicrobial effect was seen with the 10 kDa fraction and the determined MIC was 0. Aside from these functions other roles are associated with these molecules, such as antifreezers, sweeteners, and antioxidants.
  4. There are no other microbes today that have the capacity to form symbiosis with such a wide range of eukaryotes.

The notion that specific regions within a protein are responsible for the biocide effect is supported by the observation that some AMPs become functional upon protein hydrolysis, like in egg [ 5859 ] and milk proteins hydrolysates [ 5860 — 63 ]. At present, there are not many studies on plant protein hydrolysates with antibiotic properties; this situation encourages the search in protein databases for motifs featuring the signature of AMPs.

Biologically Active and Antimicrobial Peptides from Plants

Plant proteinases also display antifungal activity as demonstrated with latex proteinases from Calotropis procera, Carica candamarcensis, and Cryptostegia grandiflora [ 276061 ]. Using a collection composed of Colletotrichum gloeosporioides, Fusarium oxysporum, F.

The observed IC50 for Rhizoctonia solani with proteinases from C. Chitinases are also chitinolytic enzymes found in different plants that display antifungal activity [ 64 ]. There is no consensus about the size of defensins. According to some authors defensins are AMPs that range from 18 to 48 amino acids, while other groups define them as having 12—54 residues. Regardless of their size they contain several conserved cysteinyl residues structuring disulphide bridges that contribute to their stability.

Defensins are among the best-characterized cysteine-rich AMPs in plants [ 2765 ]. All known members of this family have four disulphide bridges and are folded into a globular structure that includes three L-strands and a K-helix [ 6566 ]. Initially, these proteins were described in human neutrophils [ 6667 ], more specifically in granules of phagocytes and intestinal Paneth cells [ 67 — 71 ].

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Later, they were described in human, chimpanzee, rat, mouse, marine arthropods, plants, and fungi [ 68 — 71 ]. Defensins of group I cause inhibition of Gram-positive bacteria and fungi, and fungal inhibition occurs with marked morphological distortions of hyphae branching ; those of group II are active against fungi, without inducing hyphal branching, and are inactive against bacteria; those of group III are active against Gram-positive and Gram-negative bacteria but are inactive against fungi; while group IV are active against Gram-positive and Gram-negative bacteria, and against fungi, without causing hyphal branching.

The selective action assigned to these four groups of defensins suggests that specific determinants within each group are responsible for targeting different groups of infectious agents.

Several defensins have been purified from plants. The PvD1 defensin from Phaseolus vulgaris cv.