Review

Role of plant lipid transfer proteins in plant cellphysiology—A concise review

Andre de Oliveira Carvalho, Valdirene Moreira Gomes *

Laboratorio de Fisiologia e Bioquımica 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

Contents

1. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1145

1.1. The structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1145

1.1.1. The relationship between the structure and the capacity of transfer lipids . . . . . . . . . . . . . . . . . . . . 1147

1.2. Localization and gene expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1148

1.3. Biological activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1149

1.3.1. Plant signalling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1149

1.3.2. Antimicrobial activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1150

1.3.3. As food allergens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1150

2. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1151

Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1151

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1151

p e p t i d e s 2 8 ( 2 0 0 7 ) 1 1 4 4 – 1 1 5 3

a r t i c l e i n f o

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

a b s t r a c t

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-dimen-

sional 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.

avai lable at www.sc iencedi rec t .com

journal homepage: www.e lsev ier .com/ locate /pept ides

* 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

p e p t i d e s 2 8 ( 2 0 0 7 ) 1 1 4 4 – 1 1 5 3 1145

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 phos-

pholipids. Thus, glyoxysomes must import these phospholi-

pids 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 first 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

90–95 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 disulfide 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 disulfide 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 findings were

inconsistent with the hypothesis of a biological role for the

LTPs in the plant cell cytoplasm.

Table 1 – Proposed biological activities of plant lipidtransfer proteins

Biological activity References

Cutin synthesis [24,56]

b-Oxidation [72]

Somatic embryogenesis [65]

Alergenics [75]

Plant signaling [4,7,40]

Plant defense against phytopathogens [33,42,57,61,77]

Pollen adherence [48]

Taking together these localizations with other findings,

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], aler-

genics [75], plant signaling [4,7,40] and plant defense against

phytopathogens [33,42,57,61,77] (Table 1), but the true phy-

siological roles fulfilled by the LTPs have yet to be determined.

For the correct denomination of these peptides, biochem-

ical assays have been used to determine the transfer of

radioactivity or fluorescence 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 specific, these peptides are also called non-

specific lipid transfer proteins [31,82].

1.1. The structure

The primary structures of the mature LTPs of both groups

comprise a unique polypeptide chain containing 90–95 amino

acid residues, in the case of the LTP1 family, and approxi-

mately 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 disulfide 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 defined 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 a-

helices (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 defined 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 Sorghumvulgare

[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]

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).

pe

pt

id

es

28

(2

00

7)

11

44

–1

15

31

14

6

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].

p e p t i d e s 2 8 ( 2 0 0 7 ) 1 1 4 4 – 1 1 5 3 1147

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 disulfide 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

exemplified 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 flexible hydrophobic cavity in a form of a

tunnel that runs through the molecule’s 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 hydro-

phobic 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 flexible than the cavities of the LTP1 family, as confirmed

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 specificity in the transport ability [24,60].

1.1.1. The relationship between the structure and the capacityof transfer lipidsFor 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

fluorescent 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 16–18 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 16–18 carbons, due to the low level of interaction

that they have with the peptide. Lipids of 20–22 carbons also do

not efficiently compete with lipids that contain fatty acids of

16–18 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

p e p t i d e s 2 8 ( 2 0 0 7 ) 1 1 4 4 – 1 1 5 31148

three or five 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 specificities 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 expres-

sion 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 five 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 identified [27], indeed

LTP is codified 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 demon-

strated 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 flowers or flower organs of

different plant species is noteworthy, as demonstrated by Pyee

et al. [56], Soufleri 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 flower LTP is related to a

possible facilitation in association with another uncharacter-

ized protein, of pollen adherence to the stigma during pollen

elongation in the Lilium longiflorum [48]. Despite these reports,

some exceptions to LTP1 expression in flowers 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 [20,27–29].

The role of LTPs in the defense mechanisms of plants has

been investigated either by studying the activity of purified

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

p e p t i d e s 2 8 ( 2 0 0 7 ) 1 1 4 4 – 1 1 5 3 1149

plant demonstrated a reduction of 22–38% in the number of

infection points that became necrotic lesions in comparison to

the non-transformed plants and that the average lesion sizes

were 53–67% 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 phenylpro-

panoid 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, inflorescence, in external layers of the

ovule, stigma, petals and sepals, demonstrating that the ltp1

gene is under tissue-specific 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 identified 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 capsiciandColletotrichumgloeosporioides 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 byP. 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), E-

box (involved in the response of plant–pathogen interaction),

W-box (pathogen-responsive element) and ERE box (ethylene-

responsive 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 gene’s promoter revealed the presence of cis-regulatory

elements, such as MYB, MYC, RY repeats and ABRE [20].

These reports also demonstrated the functionality of cis-

regulatory elements. Taken together, these results indicate

that LTPs may be responsible for the plant’s adaptation to

stress conditions, especially to those provoked by water stress.

1.3. Biological activities

1.3.1. Plant signallingRecently, 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 devel-

opment 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 demon-

strated 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 affinity rate to the tobacco plasma

p e p t i d e s 2 8 ( 2 0 0 7 ) 1 1 4 4 – 1 1 5 31150

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 Phy-

tophthora 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 disulfide

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 super-

impositions 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 activityThe antimicrobial activity of the LTPs1 was discovered by the

screening of proteic extracts of plants, in order to find 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. sepedoni-

cus and Rhalstonia (Pseudomonas) sonanacearum, and the

Fusarium solani fungus. Two other peptides that are homo-

logues 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, Staphy-

lococcus 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. para-

haemolyticus 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 mL�1. 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 mL�1. 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 (fibroblasts) or cause hemolysis

of erythrocytes until concentrations of 200 mg mL�1 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 reflects 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 filled with fluorescent dyes [9]. It has been

recently demonstratedthata fraction enriched onLTP,obtained

from chilli pepper seeds, inhibited the growth of Saccharomyces

cerevisiae, C. albicans and Schysosaccharomyces pombe at concen-

trations of 9–150 mg mL�1. 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 affinity nucleic acid stain that fluoresces upon binding to

nucleic acids and that only penetrates cells with compromised

plasma membranes [17]. Another LTP, isolated form sunflower

seeds [57], completely abrogated the growth of F. solani spores at

40 mg mL�1, 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 artificial 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 allergensSeveral reports have unambiguously suggested that the major

allergens of diverse plant species are proteins members of

p e p t i d e s 2 8 ( 2 0 0 7 ) 1 1 4 4 – 1 1 5 3 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 officinalis, B. oleracea var. capitata

and Z. mays [16,46,50,75]. It has been demonstrated that LTPs

are relatively stable, resisting thermal and chemical dena-

turation and enzymatic digestion [3,37,53,76].

These stable physical–chemical features allow these pep-

tides to reach the intestine of mammals in an immunenic form.

Although the ability of LTPs to sensibilize via the gastrointest-

inal 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 fish 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. Conclusion

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 definitive

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].

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/2806–3F.

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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