J. Plant Physiol. 160. 201 – 203 (2003) Urban & Fischer Verlag http://www.urbanfischer.de/journals/jpp
Short Communication A cDNA encoding a putative lipid transfer protein expressed in sunflower seeds Mariana Regente, Laura de la Canal* Instituto de Investigaciones Biológicas, Facultad de Ciencias Exactas y Naturales, Universidad Nacional de Mar del Plata, Casilla de Correo 1245, 7600 Mar del Plata, Argentina
Received April 4, 2002 · Accepted August 12, 2002
Summary Based on the N-terminal sequence of a sunflower antifungal protein, a full length cDNA (Ha-LTP5) encoding a putative lipid transfer protein from sunflower seeds was cloned using a RT-PCR-based strategy. However, the sequence of the deduced protein is not identical to that of the antifungal protein previously isolated. The nucleotide sequence presents an ORF of 116 amino acids with a putative signal peptide, thus encoding a mature protein of 90 amino acids that is basic and hydrophobic. In contrast to the pattern of expression described for most LTP-like genes from dicots, Northern blot analyses detected constitutive expression of Ha-LTP5 in seeds, but not in aerial parts of sunflower plants. Key words: antifungal protein – antimicrobial protein – Helianthus annuus – LTP Abbreviations: Ha-AP10 = Helianthus annuus antifungal protein 10 KDa. – Ha-LTP5 = H. annuus LTP5 clone. – LTP = lipid transfer protein. – RACE = rapid amplification of cDNA ends. – RT-PCR = reverse transcriptase polymerase chain reaction. – UTR = untranslated region
Introduction Non-specific lipid transfer proteins (LTPs) are largely distributed in plants and are potential allergens. They are small proteins of about 10 kDa and basic properties that show a conserved pattern of cysteine residues, which confer a remarkable stability to the molecule (Kader 1996). Although their function in vivo is still controversial, accumulated evidence suggest that LTPs can perform diverse roles (see * E-mail corresponding author:
[email protected]
Kader 1997 for review). Among them are the plant defence against pathogens and the transport of acyl monomers to form cutin layers. Also, their participation in embryogenesis and in abiotic stress has been reported. The role of LTPs in plant defence has been considered, taking into account that some members of the family show antimicrobial properties in vitro and, moreover, they are frequently induced by microbial infection. In this sense, LTPs were recently included as a novel class of pathogenesis related proteins and named PR-14 (Van Loon and van Strein 1999). This hypothesis was further supported by the observa0176-1617/03/160/02-201 $ 15.00/0
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Mariana Regente, Laura de la Canal
tion of enhanced resistance towards a bacterial pathogen in transgenic plants over-expressing a LTP gene from barley (Molina and García Olmedo 1997). We have previously characterised Ha-AP10, a potent sunflower antifungal protein homologous to members of the LTP family (Regente and de la Canal 2000). For the purpose of analysing the function and operational mechanisms of HaAP10 using molecular approaches, we decided to clone its cDNA. Here we report the molecular cloning of a putative LTP from sunflower, describing its primary structure and showing its pattern of expression.
Materials and Methods Mature dried seeds of Helianthus annuus (line AR10018, Advanta) were stored at room temperature. Plants were grown under standard greenhouse conditions and samples from different plant parts were frozen in liquid nitrogen and stored at –70 ˚C until used. Partial cDNA clones were obtained from reverse-transcribed RNA using oligo (dT) and primer LTP3 [5′-C(A/C/G/T)CC(A/C/G/T)TG (C/T)(C/T)T(A/C/G/T)CC(A/C/G/T)TA(C/T)(C/T)T(A/C/G/T)(A/C)G-3′] in standard RT-PCR reactions. The clones obtained served to deduce a specific antisense primer corresponding to the 3′ UTR (primer LTP5: 5′-TGCCAACTGCATACTACATAC-3′) that was further used in 5′ RACE reactions to obtain the full-length clone. 0.3 µg of mRNA from seeds were reverse-transcribed using the SMART RACE cDNA amplification kit (Clontech). Single stranded cDNA was further used for the amplification of the LTP fragment using the reverse specific primer LTP5 with the Advantage 2 PCR Enzyme System (Clontech). The following cycles were performed: 5 cycles of 94 ˚C – 30 sec, 58 ˚C – 40 sec and 68 ˚C – 30 sec; 28 cycles of 94 ˚C – 30 sec, 53 ˚C – 40 sec and 68 ˚C – 30 sec; and a final extension of 72 ˚C – 3 min. The resulting product (650 nt) was cloned into the vector pGEM-T Easy (Promega) and automatically sequenced. Total RNA for Northern blot analysis was isolated from frozen tissues using the TRIZOL Reagent (Gibco). RNA was separated by elec-
trophoresis through denaturant 1.5 % (w/v) agarose gels and transferred to Hybond-N nylon membrane (Amersham Pharmacia Biotech). For hybridisation analysis, a PCR fragment synthesised with primers LTP3 and oligo-dT (LTP301 probe) was labelled by incorporation of 32 P-dCTP by random priming. The probe was hybridised as suggested by the membrane supplier and blots were washed at high stringency with a final wash of 0.1× SSPE and 0.1% SDS for 30 min at 65 ˚C.
Results and Discussion In a previous work we characterised the N-terminal amino acid sequence of an antifungal protein purified from sunflower seeds (Ha-AP10). Based on that sequence, a degenerate primer was designed and used in RT-PCR reactions using cDNA prepared from dried seeds. The PCR fragments obtained were cloned and an antisense primer, designed from the 3′ UTR of one of these partial clones and designated LTP301, was then used for the preparation of the full-length clone. The clone obtained (Ha-LTP5, GenBank Accession Number AF529201) presents an ORF of 116 amino acids and showed a significant identity with some previously reported LTP clones. The putative protein presents a highly hydrophobic pre-region with the typical features of eukaryotic secretory signal sequences (PC-gene, Accelrys). Hence, the mature protein consists of 90 amino acids and the putative N-terminal residue of the mature protein (Ile) should coincide with that determined by chemical sequencing of Ha-AP10 (Fig. 1). In addition, the mature protein encoded by Ha-LTP5 has a molecular mass of 9284.8 Da, a calculated pI of 9.5, and it presents hydrophobic features, characteristics of which have been used for the purification of Ha-AP10 from seeds. However, the deduced N-terminal amino acid sequence differs from the N-terminal chemically determined sequence of HaAP10 at one amino acid residue (position 20), which was
Figure 1. Multiple alignment of the deduced mature amino acid sequence of Ha-LTP5 (Accession Number AF529201), amino acid sequence of Ha-AP10 protein (Accession Number P82007), and its homologous cDNA sequences (BLASTP 2.2.1). Alignments were performed with the Clustal W program. Accession numbers AAF76930 and NP_200739: LTP4 and LTP precursor-like from Arabidopsis thaliana; Q9M5x7: LTP precursor from Malus × domestica; AY059472: LTP precursor from Davidia involucrata; Q39950: Sdi-9 drought-induced LTP-like from Helianthus annuus. Amino acid residues defining the LTP consensus are indicated by asterisks.
A putative lipid transfer protein cDNA from sunflower identified as Ser by Edman degradation (Regente and de la Canal 2000), but appears as a Gly in Ha-LTP5. This could be explained as a mistake of identification in the chemical reaction taking into account that it was the last detectable amino acid. Alternatively, the clone Ha-LTP5 may be a member of a multigene family and the protein Ha-AP10 could be the product of another closely related member of this family. In support of this and in accordance with evidence obtained in other plants (Kader 1997), Southern blot analysis showed the presence of several bands hybridising to a LTP301 probe (not shown). The deduced amino acid sequence of the mature Ha-LTP5 was 51 % identical to the putative lipid transfer protein 4 from Arabidopsis thaliana (AAF76930) and also homologous to other plant LTP clones (Fig. 1). Even though the homology detected is not high (63 %), the cysteine residues and other residues that define the LTP consensus motif (Broekaert et al. 1997) are present in conserved positions. This is also the case of Sdi-9, a sunflower LTP-like protein induced by stress (Ouvrard et al. 1996), which shows limited homology revealing the diversity of LTPs at an intraspecific level. LTPs from dicotyledoneous plants have been found to be expressed in a variety of plant organs and tissues including leaves, stems, flower organs, embryos and cotyledons, and a particular pattern of expression is frequently associated with a specific member of a gene family (Kader 1997). We have analysed the presence of Ha-LTP5 transcripts by Northern blot analysis using a probe (LTP301) containing the complete 3′ non coding region because LTP genes from a given plant have been shown to share a weak identity in their 3′ UTR. Figure 2 shows that dried seeds accumulate significant amounts of Ha-LTP5 transcripts, but no hybridisation signals could be observed in roots, hypocotyls, cotyledons, leaves and flowers, even after long exposure. This result, which requires confirmation by alternative methods, is in accordance with the distribution of the antifungal protein Ha-AP10 determined by Western blot and indicates that under basal environmental conditions Ha-LTP5 would be expressed only in seeds. A
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Figure 2. RNA gel blot analysis of the expression of Ha-LTP5 in sunflower. Total RNA (15 µg) was hybridised first to a radioactive probe containing part of the coding region of Ha-LTP5 and the complete 3′ UTR (A) and then to a control probe encoding 18S RNA (B). Lanes: 1, root; 2, hypocotyl; 3, cotyledon; 4, first leaf; all from 12-day-old seedlings. Lanes 5, adult leaf, and 6, mature flowers from 3 month old plants; 7, dried seeds.
high expression of LTP genes in flowers and in aerial portions of the plant has been described for various dicot species (Kader 1996). Instead, Ha-LTP5 presents an unusual pattern of expression, the detailed analysis of which would bring relevant clues to elucidate its role in planta. Acknowledgements. The authors acknowledge funding by the University of Mar del Plata and the ANPCyT from Argentina, and the International Foundation for Science, Sweden. MR and LdlC are members of the CONICET, Argentina.
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