Material and methods Directory UMM :Data Elmu:jurnal:P:PlantScience:PlantScience_Elsevier:Vol157.Issue1.2000:

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 thaliana seems 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 m g per lane was restricted, fractionated on 0.8 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