peptides 28 (2007) 11441153

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Review

Role of plant lipid transfer proteins in plant cell physiologyA concise review
Andre de Oliveira Carvalho, Valdirene Moreira Gomes *
Laboratorio de Fisiologia e Bioqumica de Microrganismos, Centro de Biociencias e Biotecnologia, Universidade Estadual do Norte Fluminense, Darcy Ribeiro, Av. Alberto Lamego, 2000 Campos dos Goytacazes, RJ CEP: 28013-600, Brazil

article info
Article history: Received 23 January 2007 Received in revised form 7 March 2007 Accepted 7 March 2007 Published on line 13 March 2007 Keywords: Antimicrobial activity Antimicrobial peptides Gene expression Lipid transfer protein Plant signaling

abstract
Plant lipid transfer proteins (LTP) are cationic peptides, subdivided into two families, which present molecular masses of around 7 and 10 kDa. The peptides were, thus, denominated due to their ability to reversibly bind and transport hydrophobic molecules in vitro. Both subfamilies possess conserved patterns of eight cysteine residues and the three-dimensional structure reveals an internal hydrophobic cavity that comprises the lipid binding site. Based on the growing knowledge regarding structure, gene expression and regulation and in vitro activity, LTPs are likely to play a role in key processes of plant physiology. Although the roles of plant LTPs have not yet been fully determined. This review aims to present comprehensive information of recent topics, cover new additional data, and present new perspectives on these families of peptides. # 2007 Elsevier Inc. All rights reserved.

Contents
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. The structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.1. The relationship between the structure and 1.2. Localization and gene expression . . . . . . . . . . . . . . . 1.3. Biological activities . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3.1. Plant signalling . . . . . . . . . . . . . . . . . . . . . . . 1.3.2. Antimicrobial activity . . . . . . . . . . . . . . . . . . 1.3.3. As food allergens . . . . . . . . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ......................... ......................... the capacity of transfer lipids . ......................... ......................... ......................... ......................... ......................... ......................... ......................... ......................... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1145 1145 1147 1148 1149 1149 1150 1150 1151 1151 1151

2.

* Corresponding author. Tel.: +55 22 2726 1689; fax: +55 22 2726 1520. E-mail address: valmg@uenf.br (V.M. Gomes). 0196-9781/$ see front matter # 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.peptides.2007.03.004

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1.

Introduction

In living plant cells, lipids in the plasma membrane and in the different membranes of organelles undergo anabolism, catabolism and renewal. The glyoxysome membranes, for example, possess phosphatidylcholine, phosphatidylglycerol and phosphatidylethanolamine; however this organelle does not have the biosynthetic enzymes to produce these phospholipids. Thus, glyoxysomes must import these phospholipids from the organelle that synthesizes them, the endoplasmatic reticulum. A similar situation is also found for the phospholipids that constitute the membranes of other organelles, such as chloroplasts, mitochondria and the plasma membrane [44] where a transport system for the intracellular movement of lipids is necessary, due to the poor solubility of lipids inside the aqueous milieu of cell cytoplasm. Approximately 30 years ago, the lipid transfer proteins (LTPs) were discovered [30] and thus denominated due to their ability to facilitate the transfer of phospholipids between a donor and an acceptor membrane, in vitro [31]. LTPs are small peptides that comprise two families. The LTPs that form the rst family, namely LTP1, have molecular masses of approximately 10 kDa and are basic, presenting isoeletric points (pI) of between 9 and 10. These LTPs have 9095 amino acid residues, of which eight are cysteines conserved in similar positions along the primary structure of the already characterized LTP1 family. These eight cysteines, bound to each other, form four disulde bridges that help the stabilization of the peptide tertiary structure [31]. The LTP2 family is formed of peptides that have molecular masses of approximately 7 kDa, possessing on average 70 amino acids; their other characteristics, such as a high pI, lipid transfer activity and another pattern of four conserved disulde bridges, are shared with the LTP1 family [14,18,39]. Both families present a signal peptide at the amino terminal region, which in general varies between 21 and 27 amino acids, for the LTP1 family [1,67,79], and from 27 to 35 amino acids, for the LTP2 family [20,32] (Fig. 1A and B). This signal peptide is excised, rendering the mature peptide and targeting the LTPs to cell secretory pathway where they are exported to the apoplast. In keeping with these discoveries and corroborating with the extracellular location, LTP1 of various plants species are localized at the cell wall, as demonstrated in Arabidopsis thaliana [69], in Brassica oleracea var. italica leaves [56] and in Ricinus communis and Vigna unguiculata seeds [12,72]. These ndings were inconsistent with the hypothesis of a biological role for the LTPs in the plant cell cytoplasm.

Taking together these localizations with other ndings, different functions are suggested for the role of LTP in the physiology of plants, such roles may include cutin synthesis [24,56], b-oxidation [72], somatic embryogenesis [65], alergenics [75], plant signaling [4,7,40] and plant defense against phytopathogens [33,42,57,61,77] (Table 1), but the true physiological roles fullled by the LTPs have yet to be determined. For the correct denomination of these peptides, biochemical assays have been used to determine the transfer of radioactivity or uorescence labeled phospholipids from a donor membrane to an aceptor membrane, in vitro [18,78,82]. These assays have the purpose of determining whether one given protein belongs to the LTP class. In these assays, a bidirectional movement of phospholipids between the donor and aceptor membranes has been observed. Due to these discoveries, these peptides were initially called phospholipid exchange proteins; however, since this exchange does not occur at a rate of 1:1 (donor:acceptor), these peptides were renamed as phospholipid transfer proteins. Later the name was changed once again, this time to the lipid transfer proteins, due to these peptides can transport lipid molecules other than phospholipids. Since the activity of the lipid transfer is not specic, these peptides are also called nonspecic lipid transfer proteins [31,82].

1.1.

The structure

Table 1 Proposed biological activities of plant lipid transfer proteins Biological activity
Cutin synthesis b-Oxidation Somatic embryogenesis Alergenics Plant signaling Plant defense against phytopathogens Pollen adherence

References
[24,56] [72] [65] [75] [4,7,40] [33,42,57,61,77] [48]

The primary structures of the mature LTPs of both groups comprise a unique polypeptide chain containing 9095 amino acid residues, in the case of the LTP1 family, and approximately 70 residues in the case of the LTP2 family [18,31,60]. Among these, there are eight strictly conserved cysteine residues that form four intrachain disulde bridges. Although the eight cysteine residues are conserved between the two groups, there is a mismatch in the cysteine paring motif. In the LTP1 family, the Cys3 pares with Cys50 and Cys48 pares with Cys87 and in the case of LTP2 family, the Cys3 pares with Cys35 and Cys37 pares with Cys68 [18,39,60] (Fig. 1A and B). The secondary structure of the LTP1 family is composed of four a-helices (helices H1 from Cys3 to Ala17, H2 from Ala25 to Ala37, H3 from Thr41 to Ala56 and H4 from Ala63 to Cys73) and a long carboxy terminal tail that is devoid of a dened secondary structure, except for the presence of one turn of the 310 helix (Fig. 2) [34,62]. The LTP2 family follows the same secondary structural pattern as the LTP1 family, but presents three ahelices (H1, from Cys3 to Ala16, H2 from Thr22 to Ala31 and H3 from Gln33 to Ala40) and a region containing two single-turn helices (Tyr45 to Tyr48 and Ala54 to Val58). The carboxy terminal tail also does not present any dened secondary structure (Fig. 2) [18,60]. LTPs are abundant in charged residues, among the LTPs1 there are 12 in the Oryza sativa LTP [34], 11 in the Vigna radiata var. radiate LTP [36], 11 in LTP1 and 13 in LTP2 of Sorghum vulgare [54], 11 in the LTP1 of Brassica napus [34] and 11 in the EP2 of Daucus carota [65]. The presence of such amino acids endows the LTPs1 family with a high pI, varying between 9 and 10 [2,36,56]. Among those residues Asp43, Arg44 and Lys52 are conserved in the LTPs1 that have been characterized (Fig. 1A). Of the LTPs2, 10 amino acids are charged in the LTP(P) and LTP(G) of Triticum aestivum [18], 9 in the LTP2 of Oryza sativa [39]

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Fig. 1 (A) Comparison of the complete amino acid sequences of various plant LTPs from family 1 obtained from SWISS-PROT and aligned with the Clustal W [71]. Gaps, indicated by dashes, are included to optimize alignment and the numbers at the bottom of the sequences indicate the peptide size in amino acids with numbering based on maize sequence [34]. In bold, the amino acids that compose the signal peptide are shown. The lines over the sequences indicate the pattern of the disulfide bounds connectivity, according to [34] and the cysteine residues are boxed. Other conserved residues are shaded in light gray, namely Val6, aromatic13, Gly30, hydrophobic34, Leu/ Ile51, Lys52, Ala66, Val72, hydrophobic77, aromatic79, Ile81 and Ser82. The two consensus pentapeptides (T/S-X-X-D-R/K and P-Y-X-I-S, where X is any amino acid) are indicated with asterisks. The names of the species and data bank accession numbers are shown as follows: Cicer arietinum (O23758), Phaselus vulgaris (O24440), Prunus dulcis (Q43017), Prunus avium (Q43017), Prunus domestica (P82534), Malus domestica (Q9M5X7), Prunus armeniaca (P81651), Prunus persica (Q8H2B3), Gossypium hirsutum (Q9FVA5), Gerbera hybrida (Q39794), Nicotiana tabacum (Q42952), Helianthus annuus (Q39950), Solanum chacoense (Q1PCI0), Lycopersicon esculentum (P93224), Brassica napus (Q42614), Brassica oleracia var. italica (Q43304), Arabidopsis thaliana (Q42589), Sorghum bicolor (Q43193), Zea mays (P19656), Oryza sativa (P23096), Triticum aestivum (Q8GZB0), Hordeum vulgare (Q43766), Pinus taeda (Q41073), Daucus carota (P27631), Ricinus communis (Q43119) and Allium cepa (Q41258); (B) Comparison of the complete amino acid sequences of various plant LTPs from family 2 obtained from SWISS-PROT and aligned with the Clustal W [71]. Gaps, indicated by dashes, are included to optimize alignment and the numbers at the botton of the sequence indicate the peptide size in amino acids with numbering based on rice sequence [60]. In bold the amino acids that compose the signal peptide are shown. The lines over the sequences indicate the pattern of the disulfide bounds connectivity, according to [60] and the cysteine residues are boxed. Other conserved residues are shaded in light gray, namely Gln7, Leu8, Ile/Leu14, Gly17, Arg30, Gln32, aromatic39, Tyr48, Pro52, and Pro66. The names of the species and data bank accession numbers are shown as follows: Triticum aestivum (P82900 and P82901), Hordeum vulgare (P20145), Oryza sativa (P83210), Zea mays (P83506) and Prunus armeniaca (P82353).

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Fig. 2 The side-chain orientations of LTP1 and LTP2 at the CXC motifs are shown with the ball-and-stick model. The hydrophilic Asn49 present in nsLTP1 is projected to the periphery of the protein, whereas the hydrophobic Phe36 of LTP2 is buried inside the molecule. Figure reproduced from reference [60].

and 6 in the LTP2 of Zea mays [14] (Fig. 1B). The pIs of some members of this family have been calculated as being approximately 9 [14]. The three-dimensional structure of the plant LTP1 family, determined either by X-ray crystallography or NMR, has revealed a compact and globular structure that is stabilized by four disulde bridges [34,62]. These bridges are formed by the Cys3 from H1 with the Cys50 from H3, Cys13 from H1 and Cys27 from H2, Cys28 from H2 and Cys73 from H4 and by the Cys48 from H3 and Cys87 from the carboxy terminal region, as exemplied by the O. sativa LTP [34,62]. The three-dimensional structure of the LTP2 family was determined by NMR for an O. sativa LTP and demonstrated a similar tertiary structure, even with the superimposition of some elements of the secondary structure. The four bridges are formed by the Cys3 with the Cys35, Cys11 and Cys25, Cys27 and Cys61 and by the Cys37 and Cys68 [60]. The most important structural feature of the LTP1 family is the presence of a exible hydrophobic cavity in a form of a tunnel that runs through the molecules axes. The tunnel-like cavity is covered with a lateral chain of amino acids, such as Ala, Arg, Ile, Leu, Lys, Pro, Ser, Thr, Tyr and Val, which confers a hydrophobic character to the cavity. The cavity has two entrances, one small and the other large [34,62] and possesses two charged amino acids, an Agr44 and a Lys35, which are strategically localized on the larger entrance of the hydrophobic cavity, indicating a possible role in the interaction with the lipids. The lipid molecules interact with the protein at the larger entrance and their hydrophobic portions stay buried inside the cavity, while the carboxylate portion remains turned towards or exposed to the solvent. This structure in the LTP2 family is a triangular hollow box, instead of a tunnel, and is covered by amino acids such as Ala, Cys, Ile, Leu, Phe and Val [60]. Computational studies have revealed that this box is more exible than the cavities of the LTP1 family, as conrmed by the association of LTP2 with sterols that are not able to bind to LTP1 [7,60]. The volume of the cavity of both groups can increase or contract in order to better accommodate the hydrophobic molecule and this plasticity is responsible for the lack of specicity in the transport ability [24,60].

1.1.1. The relationship between the structure and the capacity of transfer lipids
For the LTP1 family, computational studies [22] and the structural characterization of the complex between the Z. mays LTP and the palmitic acid (C16:0) [62] has demonstrated that the lipid molecule interacts with the hydrophobic cavity. In order to characterize the binding of lipids to the cavity of the LTPs, studies were conducted with lipids marked with uorescent molecules. Sodamo et al. [63] demonstrated that LTP, obtained from T. aestivum, was able to accommodate two acyl chains of the 1,2-dimyristoylphosphatidylglycerol. Further studies conducted with LTP, obtained from Z. mays, showed that saturated molecules of 1618 carbons best interact with this LTP. Similarly, saturated molecules of 12 14 carbons are not able to compete with lipids that contain fatty acids of 1618 carbons, due to the low level of interaction that they have with the peptide. Lipids of 2022 carbons also do not efciently compete with lipids that contain fatty acids of 1618 carbons, due to their long chains that are not properly accommodated by the hydrophobic cavity of the LTP1 family [84]. The ability of the cavity to accommodate hydrophobic molecules is determinant for the activity of binding and transport of lipids [63]. In fact, one LTP obtained from Allium cepa seeds, named Ace-AMP1, closely resembles the LTP1 family in the secondary and three-dimensional structure, being more divergent at the primary structure level. Despite these differences at the primary structure, the Cys are strictly conserved these proteins, as well as other consensual amino acids such as a Val6, an aromatic residue at position 16, a Thr40, an Arg45, a Leu51, a Pro70, a Pro78 and an aromatic residue at position 79 (Fig. 1A) [68]. However, the Ace-AMP1 has a characteristic in sharp contrast with the other LTPs. Its hydrophobic cavity is obstructed by bulk side chains of aromatic amino acids, such as Phe and Try. The number of these amino acids also differs greatly from the number found in the other LTPs1. The primary structure of the Ace-AMP1 contains seven aromatic residues, four just at the carboxy terminal portion and 19 Arg, in discrepancy with the other LTPs1 that in general present one or two aromatic residues and

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three or ve Arg, respectively (Fig. 1A). This obstruction explains why this peptide is not able to bind and transport free lipids [10], but it is noteworthy that this peptide is still able to interact with lipids in membranes [68]. The primary structures of LTP1 present some amino acids that are relatively conserved. Of these an aromatic residue, a Try at the carboxyl terminal region at approximately position 79, is of particular note (Fig. 1A). In the three-dimensional structure, this residue is positioned at the larger entrance of the hydrophobic cavity and it has been shown that it interacts with fatty acids and stabilizes the binding between the peptide and the hydrophobic molecule by a hydrogen bond that is formed between the hydroxyl of the Try and the carboxyl group of the polar head of the lipid [24]. The fatty acids must have between 16 and 18 carbons to reach this Try residue, possibly explaining the preference of binding in relation to the stability of the molecules of such size and also why capric acid (C10:0) binds to maize LTP in two orientations, with the carboxyl group towards the interior of the protein or towards the exterior. Since the molecule is small and remains extremely concealed inside the cavity, it does not reach the Try and, thus, does not form the hydrogen bond that stabilizes the larger molecules with their polar head always turned towards the exterior [24]. These specicities also explain why fatty acids larger than 20 and 22 carbons do not interact so strongly with LTPs1. Another residue of note is the Ala that is relatively conserved at position 66 in the primary structure of LTPs1 (Fig. 1A). A hydrogen bond between this Ala and the hydroxyl group of fatty acids, such as ricinoleate (C18:1, 9, 12-OH) stabilizes the binding of hydroxy-fatty acids. The relevance of this fact is that it indicates that these proteins might be really involved with the cutin synthesis that is composed of fatty acids with hydroxyl and cetoxyl groups [24,25]. In the case of the LTP2 family, these characterizations have not yet been performed.

1.2.

Localization and gene expression

Different possible functions have been proposed for plant LTPs. The genetic structure of LTP1 indicates the presence of different genes codifying LTPs that possess different expression patterns and possibly different functions as well. Assays performed on O. sativa, for example, demonstrate that at least three genes codify LTPs [79]. Similarly, in S. vulgare, the presence of at least ve genes codifying these peptides has also been demonstrated; two of these genes have been characterized and denominated as ltp1 and ltp2 [54]. In C. annuum, another three genes have been identied [27], indeed LTP is codied by several genes that belong to a multigene family, as demonstrated in A. thaliana and O. sativa [2,80]. The analysis of where, when and how the LTP genes are expressed maybe of paramount importance to the understanding of their function in vivo. The extracellular location of LTP is not a general rule; advances in the study of LTPs1 have revealed atypical localizations, such as in R. communis seeds where a LTP isoform has been found inside an organelle, which was characterized as the glyoxosome. This LTP seems to increase the activity of the acetyl-CoA oxidase enzyme in in vitro tests,

indicating a presumed involvement in b-oxidation, possibly in the regulation of the catabolism of lipid storage [72]. In T. aestivum seeds, the presence of a LTP has also been demonstrated inside the alleurone granules that are rich in proteins, and differently from other plants, LTP was not detected in the seeds cell walls [19]. The presence of the LTP was also demonstrated inside protein storage vacuoles in V. unguiculata seeds [11,12] and the physiological role of these peptides in these organelles requires further investigation. In B. oleracea var. italica, LTP was found associated with the waxy surface of the leaves. The pattern of expression of these peptides demonstrated that they are expressed at high levels in young leaves, constituting 50% of leafy proteins, and as the leaves become older, the level of expression drops to 4%. This expression pattern suggests a role of the LTP in the transport of monomers of cutin, necessary during the expansion of the leaf and the formation of cutin [56]. The ltp1 gene of A. thaliana was shown to be highly expressed in young developing tissues and its expression diminished in fully expanded tissues, this pattern is consistent with a role in deposition of cuticular material and reinforced by the observation of the this gene expression in the petal and sepal abscission zone, where additional structural materials are expected to be deposited to seal off the abscission zone [70]. In embryonic cells of D. carota, involvement of a LTP was also suggested in the deposition of monomers of cutin, necessary for the formation of a lipophilic layer around the embryo [65]. The expression of LTPs1 in owers or ower organs of different plant species is noteworthy, as demonstrated by Pyee ` et al. [56], Soueri et al. [64], Suelves and Puigdomenech [67], Botton et al. [5], Jung et al. [27] and Yubero-Serrano et al. [83], especially since the expression of ower LTP is related to a possible facilitation in association with another uncharacterized protein, of pollen adherence to the stigma during pollen elongation in the Lilium longiorum [48]. Despite these reports, some exceptions to LTP1 expression in owers have been demonstrated by Vignols et al. [79], Liu et al. [38] and Carvalho et al. [13] and further analyses will evaluate the relevance of such a negative expression. Differential transcription levels of LTP genes have been shown in a variety of plants and plant tissues during diverse developmental stages and physiological conditions [2,64,80]. LTP genes are also responsive to environmental changes such as drought, cold, salt stress and also infection with bacterial and fungal pathogen [26,27]. Signal molecules such as abscisic acid, salicylic acid, ethylene and methyl jasmonate are involved in the signaling pathway responsible for the expression of LTP genes [20,2729]. The role of LTPs in the defense mechanisms of plants has been investigated either by studying the activity of puried proteins [10,42,21,61], or through the expression pattern of LTPs genes following the response to pathogen infection [27,47]. Transgenic A. thaliana and Nicotiana tabacum plants, expressing a barley LTP, demonstrate enhanced tolerance to pathogen infection. In tobacco, the growth of the bacterium Pseudomonas syringae pv. tabaci was retarded in comparison to the non-transformed control plants and the percentage of infection points that became necrotic lesions was reduced to 38%. The average size of those lesions was also reduced to 61 81% in regard to the control. In A. thaliana, the transformed

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plant demonstrated a reduction of 2238% in the number of infection points that became necrotic lesions in comparison to the non-transformed plants and that the average lesion sizes were 5367% smaller than the control plants when infected with the bacterium, P. syringae pv. tomato [43]. Transgenic A. thaliana, over-expressing a CALTP1 from C. annuum, had an enhanced resistance to P. syringae pv. tomato and the fungus Botrytis cinerea, both with smaller lesions in comparison with control plants. This transgenic plant also exhibited high levels of tolerance to NaCl and drought stresses [28]. Transgenic wheat plants, over-expressing Ace-AMP1, showed disease resistance towards Blumeria graminis f. sp. tritici and Neovossia indica [59]. Inspired on the work of Maldonado et al. [40], these authors accessed the effect of Ace-AMP1 over-expression on the induction of defense-related genes such as phenylalanine ammonia lyase (PAL), PR-2 and PR-3. These genes were induced, as well as salicylic acid, a product of the phenylpropanoid pathway in which PAL is a key enzyme [59]. These data strongly corroborate the defense function of LTPs against biotic and abiotic stresses. Studies performed with in situ hybridization, promoter fusion with b-glucoronidase (GUS) and Northern blotting with different plant tissues, demonstrate that the genes that codify LTPs present complex temporal and spatial control. In A. thaliana, the localization of the expression of the ltp1 gene was studied by promoter fusion with the GUS. This promoter was shown to be active in protoderm cells of embryos at the heart stage, in vascular tissues, shoot meristems and stipules during early development. In emerged seedlings, its activity was observed in cotyledons and hypocotyls near the root. In adult plants, GUS activity was determined in epidermal cells of young leaves, stem, inorescence, in external layers of the ovule, stigma, petals and sepals, demonstrating that the ltp1 gene is under tissue-specic developmental regulation. Analysis of the promoter sequence showed that it contains regions with high homology with conserved regions of genes of the phenylpropanoid pathway, such as PAL and chalcone synthase genes. This study indicates that the ltp1 gene might be regulated by similar mechanisms involved in biotic or abiotic stress stimuli [70]. Three cDNAs clones that codify LTPs in B. napus were isolated and their expressions were detected only in cotyledons and hypocotyl of seedlings. It has also been demonstrated that the level of expression of these genes increases in response to treatment with abscisic acid and sodium chlorate [64]. Three LTP cDNAs, CALTPI, CALTPII and CALTPIII, were identied from a pepper (C. annuum) cDNA library prepared from hypersensitive lesions of leaves infected with the bacterium Xanthomonas campestris pv. vesicatoria. These are differentially expressed in leaves, stem and fruit tissues in response to X. campestris pv. vesicatoria, Phytophthora capsici and Colletotrichum gloeosporioides infection. CALTPI and CALTPIII had a similar pattern of induction with different pathogens and were also induced in a similar manner by drought, high salinity, low temperature and wounding, as well as by the hormones involved in biotic and abiotic stresses, such as ethylene, methyl jasmonate and abscisic acid. In contrast, CALTPII was not induced by P. capsici and C. gloeosporioides and only high salinity induced its expression [27].

Jung et al. [28] reported the presence of a regulatory elements binding site in the promoter of the CALTPI gene from C. annuum, among them were LTRE-1 (low temperature responsive element), DPBF (drought responsive element), Ebox (involved in the response of plantpathogen interaction), W-box (pathogen-responsive element) and ERE box (ethyleneresponsive element). Jung et al. [29] also reported the presence of an ERE box, a W box and MYB core elements (involved in water stress) in the promoter region of the CALTPIII gene from C. annuum. Both these studies corroborate with the initial report of Jung et al. [27]. Yubero-Serrano et al. [83] also found cis-regulatory elements in the promoter region of a LTP (Fxaltp) gene in Fragaria ananassa, these include the ABRE, E-box, LTRE and MYB-MYC responsive elements. In the case of the LTP2 family, ltp2 gene transcripts were demonstrated to be accumulated strongly in the dry seeds of rice plants; however no transcripts were detected in roots or shoots of seedlings. This gene also reacted to treatment with abscisic acid and agents that provoke osmotic stress, such as sodium chlorate and mannitol. The seedlings that received treatment with these substances demonstrated increased ltp2 gene expression in roots compared to shoots. The analysis of the genes promoter revealed the presence of cis-regulatory elements, such as MYB, MYC, RY repeats and ABRE [20]. These reports also demonstrated the functionality of cisregulatory elements. Taken together, these results indicate that LTPs may be responsible for the plants adaptation to stress conditions, especially to those provoked by water stress.

1.3. 1.3.1.

Biological activities Plant signalling

Recently, the hypothesis that LTPs could have a role, or at least be involved, in plant defense signaling emerged. Maldonado et al. [40], working with an A. thaliana T-DNA tagged mutant, screened one especially compromised mutant for the development of systemic acquired resistance (SAR), denominated dir1-1, for defective in induced resistance. This mutant exhibited unaffected local resistance against virulent or avirulent strains of Pseudomonas syringae, but was unable to express the PR-1 gene in uninfected distant leaves and also to develop SAR against virulent P. syringae and Perenospora parasitica. The failure of this A. thaliana mutant to express the PR-1 gene in uninoculated distant leaves, as well as SAR, indicates that the product of the dir1-1 gene appears to be required for long-distance signaling during SAR for signal generation or transmission or even for signal perception level. To try to differentiate between these hypotheses, this group performed petiole exudate experiments and demonstrated that the mutant is defective in the generation or transmission of the signal. In addition, super-expression of dir1-1 did not induce SAR, implying that dir1-1 is probably not the mobile signal itself. The dir1-1 gene encodes a sequence of 102 amino acids, which shows homology with a putative LTP1. The authors proposed that this putative LTP interacts with a lipid-derived molecule to function as a long distance signal complex [40]. Providing support to the involvement of LTPs in plant signaling, it has been shown that a LTP from T. aestivum is able to bind with a high afnity rate to the tobacco plasma

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membrane [7]. The binding site was the same as that used by elicitins, as demonstrated by binding and in vivo competition experiments. Elicitins are peptides of approximately 10 kDa, secreted by oomycetes and belonging to the gender Phytophthora or Pythium; these peptides induce a hypersensitive reaction and SAR in tobacco plants [55] and resemble plant LTPs in some biochemical characteristics, such as small size (98 amino acids and 10 kDa), are basic, possess three disulde bounds and have an a-helix secondary structure and also have a hydrophobic pocket that endows them with the capacity to bind hydrophobic molecules, mainly sterols. Despite these similarities, the primary structure homology is low between the two peptides, but at the tertiary level there are superimpositions of some helixes [4,7,41]. Buhot et al. [8] revealed that a recombinant LTP1 from N. tabacum is able to bind jasmonic acid (JA), and the complex between LTP1 and JA is able to bind the elicitin receptor. The authors also suggested that the formation of the complex provokes a conformational change on LTP that facilitates its recognition by the receptor. This LTP1-JA complex, when applied to the N. tabacum plant, induces long distance protection against P. parasitica. However, it has not been demonstrated whether the complex is the mobile signal or whether the binding of the complex on the plasma membrane receptor is the requirement for the production of the mobile signal [8].

1.3.2.

Antimicrobial activity

The antimicrobial activity of the LTPs1 was discovered by the screening of proteic extracts of plants, in order to nd proteins that could inhibit the growth of phytopathogens, in vitro [42]. Among the phytopathogens inhibited were bacteria and fungi, however the activity was stronger against fungi [31]. Molina et al. [42] isolated four LTPs from barley leaves and one from Z. mays leaves and all of them presented biological activity against the bacteria, Clavibacter michiganensis subsp. sepedonicus and Rhalstonia (Pseudomonas) sonanacearum, and the Fusarium solani fungus. Two other peptides that are homologues of the LTPs, obtained from A. thaliana leaves, and two others from Spinacia oleracea leaves, also demonstrated antimicrobial activity against the aforementioned pathogens [61]. Wang et al. [81] demonstrated the antimicrobial activity of a LTP isolated from mung bean seeds against the fungi, F. solani, F. oxysporum, Pythium aphanidermathum and Sclerotium rolfsii and also against the Gram positive bacterium, Staphylococcus aureus. In regard to human pathogens, two LTPs isolated from Pandanus amaryllifolius did not inhibited S. aureus as other Gram negative enteric bacteria, namely Escherichia coli, Enterobacter aerugenes, Proteus vulgaris, Vibrio cholera, V. parahaemolyticus and Salmonella typhimurium. The only Gram negative bacterium inhibited was Pseudomonas aeruginosa [45]. The human infectious yeast Candida albicans was also not inhibited by LTPs isolated from P. amaryllifolius [45] and Hordeum vulgare [23]. Albeit plant LTPs have been considered an antimicrobial peptide it had been reported that some LTPs1 present low or did not present antifungal activity, among them are examples from T. aestivum [19] and Z. mays [10]. The antimicrobial activity, sequence similarities and induction upon pathogen attack have led to the inclusion of these peptides in the family of pathogenesis-related proteins

that compose the family 14 [74]. The activity of the LTPs seems to depend on the microorganism tested, for example, one LTP from O. sativa leaves, expressed in E. coli, presented activity against the Pyricularia oryzae fungus at concentrations of 27 mg mL1. This LTP also inhibited the bacteria, Pseudomonas syringae, at the same concentrations, but did not present inhibitory activity agaisnt Xanthomonas oryzae, except a delay in its growth [21]. The most potent peptide belonging to the LTP class was obtained from onion seeds, the above mentioned Ace-AMP1 [10]. This peptide was able to inhibit all of the 12 fungi tested and the Gram positives bacteria, Bacillus megateruim and Sarcina lutea, at concentrations below of 10 mg mL1. As already demonstrated with other LTPs, this peptide did not present activity against Gram-negative tested bacteria [10]. Despite its strong antimicrobial activity, Ace-AMP did not presented toxicity against mammal cells (broblasts) or cause hemolysis of erythrocytes until concentrations of 200 mg mL1 were reached, the same lack of cytotoxicity against mammals was demonstrated for LTPs from other plant species [10,21]. Differently to the other LTPs, this peptide was not able to bind and transport hydrophobic molecules as mentioned above [10]. The example of Ace-AMP1 demonstrates that the binding and transport activities of lipids may not be directly associated or correlated with the ability of interaction with membranes and, in this case, with the antimicrobial activity. This example also reects that the interaction of LTPs with membranes is not as well understood as the activity of binding and transport hydrophobic molecules. Since the discovery of the LTPs as peptides with the capacity to inhibit phytopathogens, it has been speculated that this effect could result from the interaction of the LTPs with biological membranes, possibly leading to the permeabilization due to loss of membrane integrity [31]. Indeed LTPs1 have been shown to interact with model membranes, such as monolayers composed of dipalmitoilphosphatidylglycerol [66] and large unilamellar vesicles lled with uorescent dyes [9]. It has been recently demonstrated that a fraction enriched on LTP, obtained from chilli pepper seeds, inhibited the growth of Saccharomyces cerevisiae, C. albicans and Schysosaccharomyces pombe at concentrations of 9150 mg mL1. This same report demonstrated that the fraction containing the chilli pepper LTP is able to permeabilize yeast plasma membrane and allow the entrance of the small dye (900 Da), SYTOX Green (Molecular Probes), a high afnity nucleic acid stain that uoresces upon binding to nucleic acids and that only penetrates cells with compromised plasma membranes [17]. Another LTP, isolated form sunower seeds [57], completely abrogated the growth of F. solani spores at 40 mg mL1, decreasing the viability of these cells to a lethal condition at this concentration. The Helianthus annuus LTP is able to permeabilize the membranes of F. solani spores, as also demonstrated by the SYTOX Green permeabilization assay [58]. Nevertheless, these results validate the information obtained from articial membranes and liposomes, the mechanism of action on these target organisms as well the antimicrobial properties of the LTP2 family have not yet been elucidated.

1.3.3.

As food allergens

Several reports have unambiguously suggested that the major allergens of diverse plant species are proteins members of

peptides 28 (2007) 11441153

1151

LTPs1 family, such peptides have been reported in fruits of Rosaceae [15,49,51] in fruits of Vitaceae [52] as well as in other plant species such as Aspargus ofcinalis, B. oleracea var. capitata and Z. mays [16,46,50,75]. It has been demonstrated that LTPs are relatively stable, resisting thermal and chemical denaturation and enzymatic digestion [3,37,53,76]. These stable physicalchemical features allow these peptides to reach the intestine of mammals in an immunenic form. Although the ability of LTPs to sensibilize via the gastrointestinal tract is not fully understood yet, it is supposed that once they are present in the gastrointestinal tract and in an immunogenic form they are free to interact with the intestinal immune system of sensitizing the individuals. Allergenic proteins share some characteristics, such as the ability to bind to ligands, as related in parvalbumin from sh and casein from milk, both bind Ca+2 ions and seem to remain more stable after this binding [6,73]. In the case of LTP, peptides bind to phosphatidylcholine, a physiological surfactant that is secreted by gastric mucosa and also occurs in bile. It has also shown that this binding results in an additional enzymatic protection, slowing down the breakdown of the grape LTP [76]. The understanding of the mechanism by which LTPs cause allergy may allow the possibility understand the mechanisms of other allergenic proteins, especially for assessing how allergenic a given protein in new foods could be or when used in the development of allergen variants with reduced side effects that could then be used as vaccines and also for the development of a reliable method for diagnosis [35].

[2]

[3]

[4]

[5]

[6] [7]

[8]

[9]

[10]

2.

Conclusion

[11]

Finally, LTPs are peptides that still do not possess a single consensus in relation to their physiological role, in vivo. Despite all the information related, herein, a denitive biological function has not been conclusively provided for these peptides. Investigations studying biological functions in plants that bear antisense transcripts to the LTP genes should yield further insights; however, studies to date have proved to be particularly complicated, since these peptides comprehend a multigenic family with different genes that are expressed in different tissues, in different development stages of plants and that also react differently to an array of stimuli [20,31,64].

[12]

[13]

[14]

Acknowledgements
This project was supported by the Brazilian agency CNPq, FAPERJ, FENORTE/TECNORTE and International Foundation for Science, Stockholm, Sweden, through a grant to C/28063F. This work is part of fellowship of Andre O. Carvalho carried out at the Universidade Estadual do Norte Fluminense through a fellowship to FAPERJ (E-26/150-015/2006).

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