Plant Science 157 (2000) 1 – 12
Lipid transfer proteins are encoded by a small multigene family in
Arabidopsis thaliana
Vincent Arondel
1 2, Chantal Vergnolle
2, Catherine Cantrel, Jean-Claude Kader *
Laboratoire de Physiologie Cellulaire et Mole´culaire,CNRS/Uni6ersite´ Pierre et Marie Curie UMR7632,Case 154,4Place Jussieu,
F-75252 Paris cedex 05, France
Received 26 October 1999; received in revised form 28 January 2000; accepted 21 February 2000
Abstract
Lipid transfer proteins (LTPs) are small, basic and abundant proteins in higher plants. They are capable of binding fatty acids and of transferring phospholipids between membranes in vitro. LTPs from this family contain a signal peptide and are secreted in the cell wall. Their biological function is presently unknown. LTPs have been suggested to participate to cutin assembly and to the defense of the plants against pathogens. A genetic approach should prove useful to provide clues on their in vivo functions. Here, the characterization of the LTP gene family inArabidopsis thalianais described. At least 15 genes were identified, their map position determined and the expression pattern characterized for six of them. All the sequences exhibit the typical features of plant LTPs. The molecular weight is close to 9 kDa, the isoelectric point is near 9 (except for three acidic LTPs), and typical amino acid residues such as cysteines are conserved. Genomic DNA blotting hybridization experiments performed using ltp1 to ltp6 as probes indicate that ltps form distinct 1 – 3 gene subfamilies which do not cross hybridize. Expression studies indicate that all the genes tested are expressed in flowers and siliques, but not in roots. Ltp1, ltp5 and ltp2 are expressed significantly in leaves, while ltp6 is detected only in 2 – 4-week-old leaves. In addition, ltp4 and ltp3 are strongly upregulated by abscisic acid (ABA). Tandem repeats can be noted concerning ltp1 and ltp2 on chromosome 2, ltp3 and ltp4 on chromosome 5 and ltp5 and ltp12 on chromosome 3. While ltp7, ltp8 and ltp9 map at the same position on chromosome 2, the other genes are dispersed throughout the genome. The characterization of the Arabidopsis ltp gene family will permit to initiate a genetic approach for determining the in vivo function(s) of these proteins. © 2000 Elsevier Science Ireland Ltd. All rights reserved.
Keywords:Lipid transfer protein;Arabidopsis thaliana; cDNA; Tandemly repeated genes; Abscisic acid
www.elsevier.com/locate/plantsci
1. Introduction
Proteins capable of transferring lipids between membranes in vitro have been purified from a wide range of living organisms [1]. Some of them have been cloned and the amino acid sequence comparisons revealed that these proteins fall into several different classes that are unrelated based on their primary structure. Although all these proteins, called lipid transfer proteins (LTP), were initially supposed to participate to membrane bio-genesis, no clear evidence of such a role has been demonstrated in vivo. Actually, the biological function of some LTPs [2] begins to be investi-gated. For example, the sec14 protein of yeast, which is a phosphatidylinositol transfer protein Abbre6iations: ABA, abscisic acid; BAC, bacterial artificial
chro-mosome; BLAST, basic local alignment search tool; EST, expressed sequence tag; LTP, lipid transfer protein; NMR, nucleic magnetic resonance; PCR, polymerase chain reaction; PITP, phosphatidylinosi-tol transfer protein; RFLP, restriction fragment length polymor-phism; TAIR, The Arabidopsis Information Resource; T-DNA, transferred DNA; TIGR, The Institute for Genomic Research; YAC, yeast artificial chromosome.
3 Accession numbers: ltp1: AF159798; ltp2: AF159799; ltp3: AF159800; ltp4: AF159801; ltp5: AF159802; ltp6: AF159803.
* Corresponding author.
E-mail address:[email protected] (J.-C. Kader).
1Present address: Laboratoire de Lipolyse Enzymatique, UPR
CNRS 9025, Universite´ de la Me´diterrane´e, Marseille, France.
2These authors have contributed equally to this work
(PITP) was shown to be a sensor of the lipid composition of the Golgi membrane, and its ca-pacity to down regulate phosphatidylcholine biosynthesis in this organelle was demonstrated. In addition, mammalian PITPs, which are struc-turally unrelated to sec14p, seem to play a key role in phospholipase C mediated signaling through their binding capacity to phosphoinositides. Therefore, it appears that these two different LTPs do not possess identical physiological functions and that neither of them transfer lipids between intracellular membranes.
In higher plants, LTPs form a very homoge-neous class of protein, if a sec14-like PITP is excluded [3]. They are small (9 kDa), abundant and basic proteins that contain eight cysteine residues [4,5]. They are capable of transferring several different phospholipids, and they can bind fatty acids [6] and acyl-CoA esters. Structural data have been recently published, based on both X-ray diffraction [7] and nucleic magnetic resonance (NMR) [8] techniques. These results indicate that LTPs contain a hydrophobic pocket capable to accommodate a fatty acid or a lysophospholipid molecule.
Numerous LTP cDNAs have been cloned from different plant species [4]. These data have indi-cated the existence of multiple isoforms, that are differently expressed and regulated [9 – 17]. How-ever, most of these genes are preferentially ex-pressed in epidermal cells of leaves and in flowers, and very rarely in roots.
All non-specific plant LTPs characterized so far contain a signal peptide, and immunolocalization data indicate that they locate to the cell wall [18]. These proteins have also been shown to be secreted by cell cultures [15,19]. This localization therefore preclude a priori an intracellular role for these proteins. Possible biological functions have been suggested. LTP might play a role in cutin and wax assembly [20,21]. Another possible role is based on the antifungal properties displayed by some LTP [22]. These proteins might play a role in the defense of the plant against pathogen attack [23 – 25]. Indeed, it has been shown that increasing the level of an LTP in transgenic tobacco enhances the resistance of the plant towards a pathogen [26]. A possible way to find a role for these proteins would consists in obtaining mutants or transgenic plants that express antisense RNA. The phenotypic characterization of these plants would
provide clues with regards to the in vivo function of these proteins. Arabidopsis thalianaseems to be the most appropriate plant material for a genetic approach, since it is very easy to transform [27], that numerous tools are available that allows re-verse genetics (transferred DNA, (T-DNA), [28] or transposons tagged lines) and that the genome programs have yielded a considerable amount of genomic and cDNA sequences [29,30]. Here, the characterization of the Arabidopsis ltp gene family is described.
2. Material and methods
2.1. Plant and DNA materials
A. thaliana (ecotype Columbia:2) plants were
grown at 25°C with a 16 h-photoperiod (150 mE
s−1 m−2) as described [3]. Plant material was rapidly collected and immediately frozen in liquid nitrogen and stored at −80°C prior to nucleic acid isolation. Abscisic acid (ABA) treatments were performed on plants at the rosette stage. The plants were transferred to a nylon mesh floating on a liquid nutrient solution for 4 days. ABA (10−4 M) was then added and the plants were collected 24 or 48 h afterwards.
cDNA clones were obtained from the Arabidop-sis Biological Resource Center at Ohio State Uni-versity and the recombinant inbred lines from the Nothingham Arabidopsis Stock Center. The CIC yeast artificial chromosome (YAC) library [31] was obtained from Dr D. Bouchez (INRA Versailles, France). Rab 18 cDNA and the ribosomal DNA probes were obtained from Dr M. Delseny (CNRS-Perpignan, France).
2.2. Nucleic acids purification
The plant material was ground to a fine powder in liquid nitrogen. RNAs and genomic DNA were extracted as previously described [3].
2.3. Northern and Southern blot hybridization
analyses
RNA was fractionated on 1.5% formaldehyde agarose gels and transferred onto Hybond N membrane (Amersham, UK). Genomic DNA (1
V.Arondel et al./Plant Science157 (2000) 1 – 12 3
agarose gel and transferred onto Positive™ mem-branes (Appligene, France) according to manufac-turer’s instructions.
Hybridizations were carried out as described previously [3] at 65°C with randomly primed cDNA probes or at 50°C with oligonucleotide probes.
2.4. Gene mapping and sequencing
YAC library screening was performed and yeast DNA was prepared according to Ref. [32]. Se-quencing was performed either according to Ref. [33] using the sequenase version 2.0 kit (USB, USA), or by automated sequencing (Company ESGS, France).
Mapping data were processed by D. Bouchez (INRA Versailles, France) with respect to physical mapping and Sean May (Nothingham) with re-spect to restriction fragment length polymorphism (RFLP) mapping. Other routine DNA manipula-tions were as in Ref. [34].
2.5. Bioinformatics
Identification and search for LTP through data-bases (GenBank, The Arabidopsis Information Resource, TAIR) was performed using both key-word searching and the basic local alignment search tool (BLAST) (BLASTX, BLASTN and TBLASTN) softwares [35]. Sequence comparisons and phylogenic analyses were performed using the ClustalW software [36] and Phylip package [37] which is based on the neighbor joining method validated by bootstrap statistical analysis. Mature amino acid sequences were aligned by ClustalW using a PAM matrix. The results from the align-ment were used for constructing the tree using the neighbor joining method. Those programs were accessed from the Infobiogen web server (http:// www.infobiogen.fr), using the default options un-less otherwise indicated.
3. Results
3.1. Identification of ltp-related cDNAs
Expressed sequence tag (EST) database (Univer-sity of Minnesota) was searched for files contain-ing the words lipid and transfer. More than 200
entries were found. The cDNAs were classed into families based on The Institute for Genomic Re-search (TIGR) tentative consensus. The remaining sequences were compared to these consensus and the ESTs that presented more than 93% identity over a 100 nucleotide stretch were considered as being encoded by the same gene. Only ‘typical’ LTPs were retained, that is, proteins that con-tained around 120 amino acid, with the typical LTP amino acid pattern [4]. Larger proteins (150 aa and more), and 7 kDa LTPs [38] were excluded. Six different classes were defined and the largest representative from each class was sequenced and characterized. These cDNAs were designated ltp1 to 6; ltp1 and ltp2 corresponding to the ltp1 and ltp2 genes already described by Ref. [18] and ltp3 is likely to be identical to Clark and Bohnert ltp3 gene [39]. In addition, ltp4 is identical to ltp-a2 which was purified from a crude cell wall prepara-tion [25]. BLAST alignments were carried out using these six genes and gene products as ‘probes’ and nine additional ltp genes were identified. These genes were designated ltp7 to ltp15 (Table 1).
3.2. Sequence analysis
All of the deduced proteins share a similar hydrophobic profile, and contain a typical signal peptide [40]. The characteristics of the proteins coded for by these cDNAs are summarized in Table 1. The number of amino acid ranges from 112 to 123 or from 89 to 98 if the signal peptide is excluded. The isoelectric point of the mature proteins is usually close to 9, with the exceptions of ltp5 (11.4), ltp6 and ltp12 (7.7), ltp15 (7.5). Interestingly, ltp8, ltp9 and ltp14 are acidic, with pI of 4.9, 5.2 and 4.2, respectively. These are the first acidic plant ltps ever reported. These proteins are rich in alanine, glycine and cysteine residues, and devoid of glutamic acid (except for ltp11 to ltp14), tryptophane (except for ltp14) and histidine (except for ltp13), except for ltp9 and ltp15 which contain all 20 amino acids. The Arabidopsis ATA7 protein [41], which appears to be related to ltps, has been added to Table 1. It is slightly larger than ltps (114 amino acids plus 26 residues for the signal peptide).
ltp4/ltp3 which exhibit 79 and 77% homology, respectively. Because the sequence identity is re-stricted to a smaller area, only ltp1 and ltp2 were found to cross hybridize when washed at moder-ately stringent conditions (65°C, 0.3 M NaCl).
All genes for which both the gene and the cDNA sequences are available were found to con-tain an intron 9 – 12 nucleotides upstream from the stop codon. Aside from ltp8 and ltp12 (intron size close to 450 nt), the size of this intron is always close to 100 nucleotides.
When compared to each other, the amino acid sequences deduced from the ltp genes exhibit from 22 to 79% identity and 55 to 90% similarity when conservative replacements are taken into account (Table 2). ATA7 shares 19 – 30% identity and 47 – 56% similarity to Arabidopsis LTPs. Cysteine residues are conserved in all sequences (Fig. 1). The central region of the protein is also strongly
conserved (residues 42 – 57) and the residues val 7, leu 11, tyr 17, gly 21, gly 34, leu 38, asp 48, arg 49, leu 77, pro 78 can be found in more than 80% of the sequences.
The comparison with other plant mature LTP proteins can be done through a phylogenetic tree (Fig. 2). A large homogenous group (group 1) that contains only dicot genes comprises 32 members from Nic.ta1 to Pha.vu. Within group 1, several clades can be distinguished that follows botanical classification. This is obvious for Cruciferae and Solanaceae. A second group (Group 2) appears more heterogeneous: it comprises six Arabidopsis LTP proteins (plus ATA7 [41]), two castor bean, two tobacco, two rice and one pine tree proteins. Monocotyledons can be considered to belong to a third group (Group 3), which includes a barley clade which seems to represent a transition of some sort with group 1.
Table 1
Characteristics of matureArabidopsis ltps deduced from cDNA sequencesa
MW (kDa)
ltp3 86C11T7 AB016890 95 TC8698 (23+) 92 9,2 9,1
ltp4 90B4T7 AB016890 108 TC9488 (23+) 89 8,8 9,1
TC14119
111 TC10173 (25+) 93 9.9 11,4
ltp5 124E14T7 AL133452
TC13850
ltp6 174N16T7 AC012562 88 TC11002 (19+) 94 9,9 7.7
10.5
ltp9 No EST AC007267 No intron? No 5.2
TC53883
ltp13 No EST AB011475 ? No 10.6 8.5
? ATA7 AF037589 AC006439 2 introns
aFor each gene, the expressed sequence tag (EST) sequenced is mentioned and the accession number of the bacterial artificial
chromosome (BAC) indicated. The size of the intron typical of lipid transfer proteins (LTPs) — which is localized at the 3%end
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Table 2
Comparison of amino acid sequences ofArabidopsisltpsa
Ltp5
ltp1 ltp2 ltp3 ltp4 ltp6 ltp7 ltp8 ltp9 ltp10 ltp11 ltp12 ltp13 ltp14 ltp15
25.2 (51.3) 22.6 (44.3) 26.7 (56) 26.3 (47.4) 27.2 (50.8) 29.8 (53.5) 20 (42.6) 23.1 (51.3) 19 (47.4) 22.6 (52.2) 20.2 (49.1) 28.9 (50) 22.4 (47.4) 20.2 (48.2) ATA7 23.9 (52.9)
39 (66) 55.6 (76.8) 33.3 (69.9) 26.3 (58.9) 42.6 (79.8) 37.6 (63.4) 44.8 (76)
64 (81) 25 (55.9)
ltp1 47 (75) 54 (79) 54 (78) 24 (59.1) 19.4 (54.2)
45.4 (72.2) 37.2 (66) 48 (66) 33 (67) 22.3 (55.3) 28.9 (66) 35.2 (57.1) 44.3 (70.1) 21.9 (55.9) 19.8 (61.3) 16.8 (46.9) 47.3(73.9)
ltp2 49.5(74.7)
46.3 (78.7) 48.5 (78.8) 41.3 (79.3) 28.7 (60.6) 46.9 (77.1) 42.4 (67.4) 52.6 (84.2)
54.6(79.4) 24.5 (54.8)
79.3(90.2) 25.3 (62) 22 (53.1)
ltp3
45.7 (75.3) 51.5 (76.8) 40.2 (78.3) 30.8 (60.6) 41.4 (70.7) 41.3 (65.2) 53.7 (76.8)
ltp4 49.5(76.3) 27.1 (57.6) 24.5 (57.1) 22.4 (49)
36.7 (67) 48.5 (69.7) 34.4 (64.5) 28.1 (56.2) 41 (71.6) 36.6 (60.2) 43.8 (70.8) 24.2 (56.4)
ltp5 25 (60.2) 19.4 (53.1)
37 (72) 42.4 (77.2) 27.8 (55.6) 55.3 (81.9) 38.9 (64.2) 41.2 (69.1) 22.9 (48.9)
ltp6 26.8 (62.8) 20.4 (53.1)
32.7 (68.4) 26.5 (57.1) 38.4 (72.7) 36 (60) 42.6 (68.3) 24.2 (51) 24.2 (53.1) 18.4 (48) ltp7
25 (55.4) 35.7 (71.4) 41.3 (70.6) 33.7 (66.3) 24.2 (54.3) 22.6 (60.4) 17.2 (44.8) ltp8
25 (54.2) 31.5 (57.6) 25.8 (54.6) 26.9 (56.5) 18.5 (58.2) 22.5 (51) ltp9
33 (57.4) 35.1 (70.1) 19.8 (48.9)
ltp10 27.8 (60.6) 16.5 (51)
ltp11 38.9 (61) 26.1 (59.8) 23.7 (57.1) 22.3 (55.2)
22.7 (57) 23.5 (62.8) 19.6 (58.3) ltp12
27.7 (56.5)
ltp13 20 (43.8)
25.2 (58.3) ltp14
Fig. 1. Sequence alignment of ltp-deduced proteins of Arabidopsis. The probable cleavage site of the signal peptide was determined according to Ref. [40], and only the mature deduced amino acid sequences were aligned using the ClustalW software [36], followed by processing with the EDITALN software. Amino acids that are strictly conserved are indicated by the corresponding symbol. *, amino acids conserved in 80% of the sequences; +, in 60%; :, in 40%; and ., in 20%.
3.3. Southern blot hybridization analyses
When hybridized and washed at high stringency (68°C, 15 mM NaCl), each probe reveals only one band on DNA restricted with most enzyme chosen (data not shown). At lower stringency (hybridiza-tion at 50°C, 1.2 M NaCl, washes at 50°C, 0.3 M NaCl), several additional bands can be detected, depending on the probe and the restriction enzyme used. Ltp1 and ltp2 exhibit a similar pattern of hybridization. This is due to the strong nucleotide similarities between the two genes combined with the fact that both genes are tandemly repeated on chromosome 2 (see below). The same holds true in the case of ltp3 and ltp4 which locate to chromo-some 5. The ltp1/2 family comprises two strong bands plus up to five additional weak bands de-pending on the enzyme and the ltp3/4 family one strong band plus up to four additional weak bands. Ltp5 exhibits usually one to two bands while ltp6 detects one to two strong bands and up to four additional weak ones. This suggests that ltp1 and ltp2 represent a small subfamily of two to three genes, ltp3 and ltp4 a subfamily of two
genes, ltp5 and ltp6 two subfamilies of one or two genes each. The weakly hybridizing bands (10 – 12) might correspond to other ltps or to unrelated genes (Fig. 3).
3.4. Gene expression studies
Northern analyses where carried out on total RNA isolated from different tissues. All genes were found to be highly expressed in flowers, and the transcript can always be detected in siliques. Virtually no RNA can be detected in roots, while the expression of the genes varies in leaves. Only ltp1, ltp2 and ltp5 are significantly expressed in leaves. While the level of ltp5 mRNA seems to remain constant during the development of the leaf up to its senescence, the expression of ltp1 is maximum in young leaves and decreases with time after bolting. Ltp1 mRNA is barely detected at day 45, when the leaves start senescing. Ltp 6 is slightly expressed during the first 24 days after germination (Fig. 4).
V.Arondel et al./Plant Science157 (2000) 1 – 12 7
while it does not alter significantly the expression of the other ltp genes.
Because there might be some cross reactions between the entire cDNA probes, the same experi-ments were carried out using specific oligonucle-otides, and identical results were obtained (data not shown).
3.5. Mapping experiments
Ltp4 and ltp2 genes were found to detect an RFLP between Columbia and Landsberg using EcoRV as restriction enzyme. They were mapped using 90 recombinant imbred lines, and found to locate on chromosome 5, cosegregating with marker m211A (ltp4) and on chromosome 2, be-tween markers m323 (2.2 cM) and m529 (3.4 cM) (ltp2). Ltp1, ltp5 and ltp6 were found to hybridize to CIC YACs 3G1 and 2G9 for ltp1, 8E1, 7A4, 10B4, 9C9, 9D9, 10A11, 11G7 and 6F4 for ltp5, 1C12, 10B10 and 11D1 for ltp6. These data indi-cate that ltp1 maps to chromosome 2 (close to position 71cM), ltp5 to chromosome 3 (close to
Fig. 2.
Fig. 2. Phylogenetic tree for plant ltps. The phylogenetic tree was built using the protein sequences indexed in GenBank, after removal of the signal peptide [40]. The neighbor joining method/UPGMA version 3.573c from the PHYLIP package [37] was used, as indicated in Section 2. Sequences are men-tioned by six first letters of the Latin name of the plant from which they were obtained followed by a number when there were several lipid transfer protein (LTP) in a same species. Their accession numbers in GenBank are: Bras.na:Brassica napus 1: X60318 2: U22175 3:U22105 4:U22174-Bras.ra:
Brassica rapaL31938-Bras.ole:Brassica oleracea1: L339042: L33905 3: L33906 4: L33907-Hor.vu: Hordeum 6ulgare 1: U181272: Z371143: X686564: X686545: X969796: Z66529, Z66528, U63993 7: U88090 8: X60292, X59253 9
Z3715-Tri.du: Triticum durum X63669-Ory.sa: Oryza sati6a 1: U167212: U29176 3:D15364 4:D22795 5: D16036 6:U29176
7: U29176 8: X83433 9: X83434 10: Z2327111: X83435 12:
D15678-Zea:Zea mays1: U661052: J04176-Sor.bi:Sorghum bicolor 1: X71667 2: X71668 3: X71669-Nic.ta: Nicotiana tabacum1: D13952,2: U14167,3:U14168,4: X62395-Ric. co:
Ricinus communis 1: M86353 2: D11077-All.ce: Allium cepa
S79815-Pha.vu: Phaseolus 6ulgaris: U72765-Pin.ta: Pinus taeda U10432-Pru.du: Prunus dulcis 1: X96714 2
:X96716-Ger.hy: Gerbera hybrida Z31588-Hel. an: Helianthus annuus
X92648-Dau.ca: Daucus carota M64746-Gos.hi: Gossypium hirsutum 1: U64874 2:U15153-Lyc.pe: Lycopersicon pennellii
1: U66466 2: U66465-Lyc.es: Lycopersicon esculentum
U81996-Spi.ol: Spinacia oleracea M58635. Arabidopsis thaliana clones are ATA7 (AF037589), ltp1, ltp4, ltp3, ltp4,
position 72) and ltp6 to chromosome 3. Ltp 1 and 2 appear to be close together on chromosome 2, ltp4 and ltp3 on chromosome 5. Based on genomic sequencing and mapping data available from the TAIR database, ltp7, ltp8 and ltp9 map to the same position (29 cM) on chromosome 2, while ltp12 is clustered with ltp5. Ltp11 and ltp15 lo-cates to chromosome 4 (89 and 31 cM, respec-tively), ltp13 and ltp14 to chromosome 5 (94 and 117 cM, respectively) and the map position of ltp10 remains unknown (Fig. 5).
4. Discussion
Lipid transfer proteins are an ubiquitous protein family in higher plants, whose biological function remains unknown. One of the problems encoun-tered in studying LTPs is the number of isoformes that can be detected. For instance, more than ten genes have been described in rice [17]. Arabidopsis is the most suitable organism for obtaining an exhaustive collection of ltp isoformes, because the small size of its genome suggests that genes families are likely to contain few members. The main reason for using Arabidopsis for such a study is that an important part of its genome has been already sequenced, and that many ESTs are available. ESTs are particularly well suited for looking for LTPs since these proteins are short and almost half of the amino acid sequence is included in an average EST. The other reason is that LTPs are frequently expressed at high levels, and their mRNA represents usually a few percent of a plant total mRNAs. They are therefore well represented in cDNA libraries. This was found to be the case for ltp1 to ltp6, while ltp8, ltp9 to ltp12 are represented by one to three ESTs. No EST could be found for the other genes.
A total of 14 ltp genes could be evidenced in Arabidopsis based on 80% of the genome sequence and ten through EST analysis, for a total of 15 different genes. It is therefore likely that the Ara-bidopsis 9 kDa ltp gene family contains about 15 – 20 genes. However, sequencing errors might hamper the discovery of additional genes. For instance, a couple of nucleotide modifications should be enough to detect another gene on bacte-rial artificial chromosome (BAC) T1N24 (AF149413). The Southern blot hybridization data suggest that ltp genes consist in small subfamilies of one to three genes which may, or may not, weakly cross hybridize at low stringency. Clark and Bohnert [38] have recently characterized three cDNAs (ltp1 to ltp3) InA.thaliana (Wassilewskija ecotype) and the corresponding ltp1 and ltp2 genes [16,39]. These three members of the LTP family are similar to those presented in the study although performed in a different ecotype. These authors have also noted that there was very little crosshybridization, even between ltp1 and ltp2. Therefore, Southern genomic DNA analyses are likely to underestimate the number of ltp genes.
Fig. 3. Southern blot hybridization analysis of ltp gene family in Arabidopsis. Columbia genomic DNA (1mg per lane) was
restricted during 4 h with 5 U of the restriction endonucleases
BamHI (1), BglII (2), ClaI (3), EcoRI (4), EcoRV (5),
HindIII (6),XbaI (7) andXhoI (10). The DNA was fraction-ated on agarose gel, transferred to nylon membrane, and probed with 32P-labeled cDNA probes. The hybridizations
were carried out at 50°C in 0.6 M NaCl, and the washes were performed in 2×SSC, 0.1% SDS at 50°C. Membranes where exposed to X-ray films between intensifying screens. The molecular weight markers were Lambda DNA restricted by
V.Arondel et al./Plant Science157 (2000) 1 – 12 9
Fig. 4. Northern blot hybridization analysis of ltp gene expression. Total RNA (10mg) were fractionated on 1.5%
formaldehyde-agarose gels, transferred to a nylon membrane and probed with cDNA inserts labeled by random priming using [32P]dCTP.
Hybridizations were carried out at 45°C in 50% formamide. Washes were performed at 62°C in 2×SSC 0.1% SDS. A Rab18 probe was used as a positive control for abscisic acid (ABA) induction, and a ribosomal DNA probe (rib) to assess for even loading of the RNA. RNAs were from 40-day-old plants for organ specific expression studies (A), from 28-day-old leaves for ABA induction (C), and from leaves extracted at different stages of development (B). The induction by ABA was carried out in 10−4M for 24 or 48 h. L, leaves; S, siliques; F, flowers; R, roots.
Mapping data indicate that, although ltp genes can be found scattered through four chromosomes of Arabidopsis, four clusters exist that comprise nine genes. Tandem repeats can be noted based on genomic sequences (AC005499, AB016890, AL133452) for ltp5-12, ltp4-3 and ltp1-2 [38]. The two last pairs exhibit more than 75% identity at the nucleic acid level. Taken together, these data strongly suggest that these are duplicated genes. Other pairs of tandemly repeated LTP genes have
been characterized in various plants, such as ltp1 and ltp2 from Sorghum 6ulgare [42], Wax 9D and
The RNA hybridization analysis of all genes tested indicate that transcripts are always present in flower and siliques. The main differ-ences between the genes is their pattern of expres-sion in leaves, which varies especially with regards to development. Clark and Bohnert [39] also noted that ltp1, ltp2 and ltp3 are highly
expressed in flowers and siliques. However, it has been found that ltp2 is also expressed in leaves. All transcripts tested were undetectable in roots, which is also a general feature of plant ltps. It has been observed that ltp4 and ltp3 from Arabidopsis are up-regulated by ABA. A similar result has been obtained for LTP genes from other plants such as rice [17] and rapeseed [14]. Interestingly, the promoter region of the ltp4 gene from barley [43] contains elements which could be linked to ABA responsiveness. In Arabidopsis [16] the pres-ence of several expression-controlling motifs in ltp1 gene, also found in ltp2 gene by [39], similar to those previously reported in plant genes in-duced by various types of stress or by pathogen attack was observed.
The phylogenetic tree is very similar to those already published [17,39]. The group I defined by Ref. [17] corresponds to the groups I and II, while group III corresponds to group II by Vignols et al. [17]. As a control all rice sequences published by these authors have been included and their differ-ent classes can be found at the same position in this tree. Concerning dicots it seems plausible that two different ancestral genes exist: one corre-sponding to group I and the other one to group III. Interestingly, Arabidopsis genes are not far from being equally represented in both groups (nine genes in group I versus six genes in group III). Monocots and Gymnosperms are also represented in group III, so it is likely that the putative ancestral gene to this group has started to differentiate early in higher plant evolution. Con-cerning group II, the barley subgroup appears to be much more closely related to group I than to group III. The situation appears to be different for the other monocots of group II. This discrepancy might be due to the software used, and the fact that this tree is unrooted. In any case, the exis-tence of at least two different groups appears clearly.
It has been suggested by Ref. [39] that the ancestor to Brassicaceae possessed already several copies of ltp genes. The phyletic analysis suggest that this ancestor might have possessed no less than five copies, A (precursor to ltp9, 13 – 15), B (precursor to ltp8, 11), C (precursor to ltp6, 10), D (precursor to ltp1, 2, 5, 7) and E (precursor to ltp3, 4, 12). The closeness of genes such as ltp3 and ltp4 suggests that this family of gene seems to continue to duplicate.
V.Arondel et al./Plant Science157 (2000) 1 – 12 11
5. Conclusion
Fifteen genes have been identified through 45 000 ESTs and 102 megabases of genomic DNA. Although one cannot exclude that other ltp genes exist in Arabidopsis, it is very likely that a large majority of them have been identified [44]. This will permit one to initiate a genetic approach for determining the biological function of LTPs. The important number of genes makes an anti-sense approach difficult to carry out efficiently. Alternatively, it is possible to search for disrupted mutants. The availability of DNA sequence should allow a search by PCR for T-DNA tagged mu-tants. In addition, knowing the map position of these genes will permit to look for transposon-tagged mutants.
Acknowledgements
Part of the work presented in this article has been funded by the GREG program 520 721. We thank Dr D. Bouchez (INRA Versailles) for providing us with the CIC YAC library, Dr M. Anderson (NASC, Nothingham) for Dr Dean’s recombinant inbred lines and the ABRC (OSU, USA) for the Arabidopsis EST clones. We are grateful to Dr D. Bouchez for providing us with the information concerning YAC anchoring and to Dr Sean May (Nothingham) for computing segregation data. We thank Natalie Ferte´ for help with editing figures. We are grateful to Dr F. Grellet for in-depth critical reading of the manuscript and helpful advice concerning se-quence analysis. We are much indebted to Dr A. Zachowski for helpful suggestions concerning the presentation of this manuscript.
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![Fig. 4. Northern blot hybridization analysis of ltp gene expression. Total RNA (10 �agarose gels, transferred to a nylon membrane and probed with cDNA inserts labeled by random priming using [g) were fractionated on 1.5% formaldehyde-32P]dCTP.Hybridization](https://thumb-ap.123doks.com/thumbv2/123dok/1033091.928549/9.612.128.430.44.476/northern-hybridization-expression-transferred-membrane-fractionated-formaldehyde-hybridization.webp)
