JXB Advance Access originally published online on August 7, 2006
Journal of Experimental Botany 2006 57(12):3145-3155; doi:10.1093/jxb/erl076
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RESEARCH PAPER |
The presence of jasmonate-inducible lectin genes in some but not all Nicotiana species explains a marked intragenus difference in plant responses to hormone treatment
Laboratory of Biochemistry and Glycobiology, Department of Molecular Biotechnology, Ghent University, Coupure Links 653, 9000 Gent, Belgium
*To whom correspondence should be addressed. E-mail: ElsJM.VanDamme{at}UGent.be
Received 21 February 2006; Accepted 8 June 2006
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Abstract |
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Tobacco (Nicotiana tabacum L. cv Samsun NN) leaves accumulate a cytoplasmic/nuclear lectin, called Nictaba, in response to methyl jasmonate. To check whether, and if so to what extent, the specific induction of this lectin applies to related species, a collection of 19 Nicotiana species—covering 12 Nicotiana sections and eight Nicotiana tabacum cultivars—was screened for their capability to synthesize the jasmonate-inducible lectin. Protein analyses by agglutination assays and western blot confirmed that only nine out of the 19 species examined synthesize lectin after jasmonate treatment. Remarkably, all allotetraploid cultivars of the N. tabacum L. species tested express the lectin after jasmonate treatment. PCR analyses demonstrated that all responsive species possess one or more lectin genes, whereas no lectin gene(s) could be traced in the non-responding species. The number of introns present in the lectin genes varies between zero and two. Four tobacco species/cultivars contain both intronless Nictaba genes as well as lectin genes with introns. These findings provide the first firm evidence for a striking intragenus difference with respect to the activation of a well-defined jasmonate-inducible gene that can be correlated with the presence/absence of orthologous genes in the genomes of closely related species from a single plant genus. In addition, the differential response of closely related tobacco species illustrates that in the field of plant hormone research, care must be taken when extrapolating results obtained with a particular model system to other—even taxonomically closely related—species.
Key words: Gene loss, inducible protein, jasmonate, lectin gene, Nicotiana, Nictaba, tobacco
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Introduction |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionSupplementary dataReferences |
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Jasmonic acid (JA) and its derivatives, commonly designated as jasmonates, are an important group of plant signalling molecules that play a pivotal role in some reproductive processes as well as in the regulation of plant metabolism and defence against pathogens and insects/herbivores (for reviews, see Creelman and Mullet, 1997; Turner et al., 2002; Wasternack and Hause, 2002; Devoto and Turner, 2005). Over the last few years, it has become clear that the role of JA in plant development is complex and greatly depends on interactions with other growth regulators.
Although the effects of jasmonates on developmental and defence-related processes are well understood, the exact mechanism(s) of the signalling cascades that relate the synthesis of jasmonates and the activation of target genes still remain to be fully elucidated. The identification and characterization of Arabidopsis mutants that are defective in jasmonate biosynthesis or signalling yielded important clues to identify the mode of action of jasmonates (Devoto and Turner, 2005). For example, the isolation of methyl jasmonate-insensitive Arabidopsis coi (coronatine-insensitive) mutants, which are defined by a single locus COI1 (Feys et al., 1994), and the subsequent identification of the coi1 gene revealed that the affected locus encodes a protein with an F-box motif and a series of leucine-rich repeats, suggesting that the protein is involved in targeting of proteins for polyubiquitination and degradation. An analogous mutant was isolated from tomato (called the jasmonate-insensitive-1 or jai1 mutant) (Li et al., 2001), indicating that the mode of action of jasmonates might be the same in all plants. However, this does not imply that jasmonates provoke the same or a similar set of effects in all plants. Some changes in gene expression such as the downregulation of proteins involved in the photosynthetic apparatus and the upregulation of some defence-related proteins are commonly observed, but often the eventual outcome of the jasmonate-induced altered gene expression strongly depends on the species and tissue under investigation. The heterogeneity in jasmonate-induced responses applies not only to the synthesis of species-specific secondary metabolites but also to the accumulation of proteins. Numerous JA-responsive genes have been identified that are clearly upregulated and lead to the synthesis of jasmonate-inducible proteins (JIPs). JIPs were first described in barley leaves (Weidhase et al., 1987), but since then have been reported for most plants analysed. Comparative analyses revealed that different plant species express different JIPs. Some types of JIPs have been identified in a single species. For example, some of the JIPs from barley seedlings have not been identified yet in any other plant. Jasmonate-induced myrosinase-binding proteins seem to be confined to Arabidopsis and Brassica species (Geshi and Brandt, 1998), whereas a wide range of grass species accumulate a jacalin-related lectin in response to JA (Van Damme et al., 2004). These and other examples indicate that plants developed, in addition to a set of common JA-responsive genes, a whole variety of specialized JA-responsive genes that according to the available data have a limited taxonomic distribution. At present, there is no simple genetic explanation for the origin of these specialized JA-responsive genes. Moreover, no detailed data have been reported with respect to the distribution of such genes within a given taxonomic group.
The recent identification in tobacco (Nicotiana tabacum cv. Samsun NN) leaves of a jasmonate-induced cytoplasmic/nuclear lectin (Chen et al., 2002), that has not been found yet in any other plant species, offered a unique opportunity to fill this gap in the current knowledge of specialized JIPs because the Nicotiana tabacum agglutinin or Nictaba is, by virtue of its sugar-binding and erythroagglutinating activity, a versatile model system for large-scale screening experiments. Here the results of an extensive survey for induction of the tobacco lectin in leaves of a large collection of wild Nicotiana species and N. tabacum cultivars are reported. It was observed that plants of only some Nicotiana species respond to jasmonic acid methyl ester (JAME) treatment by the synthesis of a Nictaba orthologue. The failure of the non-responsive Nicotiana species to express the lectin is due to the absence of the Nictaba coding sequence (CDS) from their respective genomes. These observations provide the first firm evidence that even closely related species respond differently upon treatment with JAME because of the presence/absence of some target gene(s), and indicate that extreme care should be taken before generalizing the results obtained with a particular model plant to other species.
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Materials and methods |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionSupplementary dataReferences |
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Plant material and growth conditions
Seeds of different N. tabacum L. cultivars were purchased from Lehle Seeds (Round Rock, TX, USA) except those of cv. Bright Yellow-2 (BY-2) which were obtained from Dr Nagata Toshiyuki (University of Tokyo, Tokyo, Japan). A sample of N. otophora Grisebach seeds was provided by the Botanical and Experimental Garden of the Radboud University Nijmegen (The Netherlands). Seeds from all other Nicotiana species were kindly supplied by Dr Verne A Sisson (Oxford Tobacco Research Station, Oxford, NC, USA).
Seeds were surface-sterilized by consecutive treatments with a solution of 70% ethanol (2 min) and 7% (v/v) NaOCl (10 min), and extensively rinsed in sterile water. For an in vitro-culture, seeds were placed on top of solid Murashige and Skoog (MS) medium containing 4.3 g l–1 MS micro- and macronutrients containing vitamins (Duchefa, Haarlem, The Netherlands), 30 g l–1 sucrose, pH 5.7 (adjusted with 0.5 M NaOH) and 8 g l–1 plant agar (Duchefa). After germination, the plants were transferred to MS medium containing 2.15 g l–1 micro- and macronutrients, and vitamins. Plants were kept in a growth chamber at 25 °C, 70% relative humidity, and a 16 h photoperiod. To maintain a sterile culture, in vitro plantlets were vegetatively propagated through cuttings (every 6–7 weeks). For an in vivo culture, seeds were germinated in a Petri dish filled with pot soil. After the appearance of the cotyledons, plantlets were transferred into 0.5 l pots (in pot soil) and kept in a greenhouse until flowering.
Treatment of excised leaves with JAME
Tobacco leaves were treated with JAME by flotation on a solution of 50 µM JAME. Leaves were cut from 4-week-old greenhouse-grown tobacco plants or 5- to 7-week-old in vitro-grown plants and immediately transferred to Petri dishes (90 or 140 mm diameter) filled with a solution of JAME (in water). After transfer, leaves were incubated for the desired time periods under constant light. Leaves floated on water were used as a negative control. At the end of the incubation period, leaves were washed with tap water, blotted dry, and either used immediately or frozen at –80 °C until use.
Preparation of crude extracts
Leaves were homogenized with a mortar and pestle in 5 ml g–1 fresh weight 20 mM unbuffered 1,3-diaminopropane. Extracts were transferred into microcentrifuge tubes and centrifuged for 5 min at 13 000 g. The supernatants were collected and used for further analyses.
Agglutination assay
The presence of lectin activity in crude extracts was checked by simple agglutination assays. Extracts were made from pooled leaves (n=3 or 4). A 10 µl aliquot of extract was mixed with 10 µl of 1 M ammonium sulphate and added to 30 µl of a 2% suspension of trypsin-treated rabbit erythrocytes [made up in phosphate-buffered saline (PBS): 137 mM NaCl, 8 mM Na2HPO4.2H2O, 3 mM KCl, 1.5 mM KH2PO4] in a small glass tube. Agglutination was controlled visually. Samples that yielded no visible agglutination activity after incubation for 1 h were regarded as lectin negative.
The lectin concentration in the crude extracts was estimated by a semi-quantitative agglutination assay. Aliquots of 10 µl of serially 2-fold diluted (in 1 M ammonium sulphate) extracts were mixed with 40 µl of a 2% suspension of trypsin-treated rabbit erythrocytes in the wells of polystyrene 96 U-welled microtitre plates. Agglutination was assessed visually after incubation for 1 h at room temperature. The absolute lectin content in the extracts was calculated from the titre of a solution of purified Nictaba as determined in a parallel experiment in the same microtitre plate with the same batch of erythrocytes. This semi-quantitative method allowed detection of lectin concentrations as low as 0.6 µg ml–1 with an error range <12.5%.
Analytical techniques
Crude extracts were analysed by SDS–PAGE in 15% acrylamide gels as described by Laemmli (1970). Proteins were visualized by staining with Coomassie brilliant blue R250 or blotted onto polyvinylidene fluoride (PVDF, 0.45 µm) transfer membranes (BiotraceTM PVDF, PALL, Gelman Laboratory, USA). Western blot was performed using a monospecific primary antibody against Nictaba (purified by affinity chromatography on immobilized Nictaba as described in Chen et al., 2002) and a horseradish peroxidase-coupled swine anti-rabbit IgG (DAKO A/S, Denmark) as the secondary antibody. Immunodetection was assessed by a colorimetric assay using 3-amino-9-ethylcarbazole (Sigma-Aldrich, St Louis, MO, USA) as a substrate.
Isolation of nucleic acids from tobacco leaves
Tobacco leaves were frozen and ground in liquid nitrogen using a mortar and pestle. The finely powdered leaf material was transferred to 50 ml falcon tubes containing 5 ml g–1 tissue extraction buffer. For genomic DNA extraction, Tris–HCl buffer (100 mM, pH 8) containing 20 mM EDTA pH 8, 1.4 M NaCl, 2% cetyl trimethyl ammonium bromide, and 2% polyvinylpyrrolidone 40 was used. The leaf powder was thoroughly mixed with the extraction buffer, incubated for 1 h at 60 °C, and the DNA isolated using a phenol/chloroform extraction protocol (Stewart and Via, 1993). RNA was extracted using the Trizol method according to the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA).
PCR
PCR was performed according to Sambrook et al. (1989) using different sets of primers. A first set of primers was complementary to the 5' (L35: 5'-ATGCAAGGCCAGTGGATAGCCGC-3') and 3' (L36: 5'-TTAGTTTGGACGAATGTCGAAGCCC-3') ends, respectively, of the CDS of the cDNA isolated from N. tabacum cv. Samsun NN (plasmid T1.01, GenBank accession no. AF389848). The second set of primers was complementary to the 5' end (evd 42: 5'-GATAGCATCATATCATATAT-3') and 3' end (evd 43: 5'-AGAAAATCATAAAGACAAAC-3') of the 5' and 3' untranslated region (UTR), respectively, of the cDNA isolated from N. tabacum cv. Samsun NN. PCR was performed in an AmplitronIIR Thermolyne apparatus (Dubuque, IA, USA) using Taq DNA polymerase (Invitrogen) according to the manufacturer's instructions. The following programme was used: 2 min at 94 °C followed by 25 cycles of 15 s at 94 °C, 30 s at 45–48 °C and 90 s at 72 °C, and a final incubation for 5 min at 72 °C.
RT–PCR
cDNA was synthesized from the isolated RNA using the First Strand cDNA Synthesis RT–PCR kit (Invitrogen). The Platinum Supermix PCR kit (Invitrogen) was used to perform PCR. Primer combination L35–L36 was used to amplify the sequence corresponding to the open reading frame (ORF) of the cDNA encoding Nictaba. Actin was used as an internal control of the RT–PCR (forward primer 5'-GTTGCACCACCTGAAAGGAAG-3', reverse primer 5'-CAATGGGACTAAAACGCAAAA-3' derived from GenBank accession no. AY064043).
Isolation of genomic sequences of Nictaba orthologues and in silico analysis
To isolate the genomic sequences corresponding to the previously isolated cDNA encoding Nictaba (Chen et al., 2002), PCRs were performed on genomic DNA from different Nicotiana species and N. tabacum cultivars using the primer combinations L35–L36 and evd 42–evd 43. The PCR products were separated in TAE agarose gels. Single bands were recovered from the gel using the Agarose gel DNA extraction kit (Roche Diagnostics GmbH, Mannheim, Germany), ligated into the pCR2.1-TOPO vector using the TOPO TA cloning kit (Invitrogen), and transformed into Escherichia coli via heat shock transformation. A number of colonies growing on LB agar plates containing ampicillin (100 µg ml–1) were selected and checked for the presence of the insert via EcoRI digest. Plasmid DNA from positive colonies was purified using the QIAprep Spin MiniPrep kit (Qiagen, Venlo, The Netherlands) and sequenced (by the VIB Genetic Service Facility, Antwerp, Belgium). New sequence data have been deposited in GenBank under accession nos DQ155394–DQ155409 and DQ407817–DQ407819. Sequences were analysed using Chromas 1.45 (http://www.technelysium.com.au/chromas14x.html). ORFs were determined using the 6 Frame Translation tool at the BCM server (http://searchlauncher.bcm.tmc.edu/seq-util/seq-util.html). Multiple sequence alignments were generated using ClustalW 1.83 (http://www.ebi.ac.uk/clustalw/) (Higgins et al., 1994) and BioEdit 7.0.4.1
[EC]
(www.mbio.ncsu.edu/BioEdit/bioedit.html) (Hall, 1999). The output was visualized using BoxShade 3.21 (http://www.ch.embnet.org/software/BOX_form.html).
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Results and discussion |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionSupplementary dataReferences |
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JAME treatment induces lectin activity in some but not all Nicotiana species
Nictaba was originally described as a protein that is absent from untreated N. tabacum cv. Samsun NN plants, but rapidly accumulates upon administration of JAME either in the liquid or in the gas phase (Chen et al., 2002). Additional experiments with a set of tobacco cultivars confirmed that this jasmonate-induced lectin synthesis is a common phenomenon in N. tabacum. Leaves of in vitro-grown plants of the cultivars Samsun NN, Samsun nn, Havana 38, Petite Havana SR1, Xanthi NN, Xanthi Smith, and Wisconsin 38 were devoid of detectable agglutination activity but accumulated high levels of lectin (200–500 µg lectin g–1 fresh leaf) upon floating on a solution of 50 µM JAME for 72 h. A similar response was observed in leaves from greenhouse-grown plants of the same cultivars and of cv. BY-2. To confirm that the observed agglutination activity is caused by Nictaba, the crude extracts were also analysed by SDS–PAGE and western blot analysis. As shown in Fig. 1A, JAME clearly induced the synthesis of a 19 kDa protein that is apparently absent from untreated leaves. The specific recognition of this 19 kDa protein by a monospecific polyclonal anti-Nictaba antibody in western blot experiments (Fig. 1B) unambiguously demonstrated that this 19 kDa protein corresponds to Nictaba. It can be concluded, therefore, that JAME induces the synthesis of Nictaba in leaves of all tested N. tabacum cultivars.
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To check whether Nicotiana species other than N. tabacum exhibit a similar response to JAME, the induction experiments were extended to a number of wild Nicotiana species. As shown in Table 1, only nine out of the 19 Nicotiana species included in the test accumulated lectin activity after treatment with JAME. SDS–PAGE combined with western blot analysis confirmed that the observed lectin activity relies on the presence of a 19 kDa protein specifically recognized by anti-Nictaba antibodies. Similar analyses of the non-responsive Nicotiana species indicated that the lack of lectin activity is apparently accompanied by the absence of the 19 kDa lectin polypeptide (or another immunoreactive protein) in the crude extracts from the leaves. This implies that JAME induces lectin in some but certainly not in all Nicotiana species scattered across the Nicotiana sections (Knapp et al., 2004).
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Inter- and intraspecies differences in the level of JAME-induced Nictaba
The agglutination assays with crude extracts from JAME-treated leaves not only allowed responsive and non-responsive Nicotiana species to be distinguished but also indicated marked differences in lectin activity within the group of responsive plants. Determination of the lectin content in JAME-treated leaves from plants that were grown in vitro revealed that all N. tabacum cultivars accumulated relatively high concentrations of Nictaba (Fig. 2A). Although the agglutination assay is only semi-quantitative with an inherent experimental error of
12.5%, the observed differences in lectin concentrations, which ranged between 50 and 250 µg of Nictaba g–1 fresh weight, are indicative for intraspecies differences within N. tabacum for levels of JAME-induced Nictaba. The three wild tobacco species included in the same experiment (N. africana, N. glutinosa, and N. kawakamii) accumulated only 
10 µg Nictaba g–1 fresh weight, about one order of magnitude lower than the Nictaba level induced in N. tabacum cultivars.
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Since seeds of some wild tobacco species did not germinate on MS medium, greenhouse-grown plants were used for the induction experiments with some Nicotiana species. In these experiments, marked interspecies differences were also observed (Fig. 2B). Nicotiana tabacum cv. Samsun NN, which was included as a positive control, accumulated up to 15 mg of Nictaba g–1 fresh weight. Compared with N. tabacum cv. Samsun NN, the lectin concentrations in leaves of N. plumbaginifolia, N. tomentosa, and N. tomentosiformis (50, 100, and 37.5 µg lectin g–1 fresh weight, respectively) were only moderate. In N. suaveolens, the lectin concentration remained below 1 µg g–1 fresh weight, which is three orders of magnitude lower than in N. tabacum cv. Samsun NN).
Leaves from greenhouse-grown N. tabacum cv. Samsun NN plants accumulate much higher levels of Nictaba upon JAME treatment than their counterparts from in vitro-grown plants. Similar observations were made for all other N. tabacum cultivars mentioned in Table 1 (results not shown). To check whether the same applies to other Nicotiana species, a similar experiment was done with N. glutinosa. As shown in Fig. 2, for this species also the jasmonate-induced lectin content is much higher in leaves from greenhouse-grown plants than in leaves from in vitro-grown plants, indicating that normally grown plants are more responsive than plants grown under in vitro conditions at least for what concerns this specific physiological response.
Nictaba expression is transcription dependent
To check whether the jasmonate-induced synthesis of Nictaba is transcription dependent, the accumulation of corresponding mRNAs as a function of induction was followed by RT–PCR analysis on total RNA extracted from N. tabacum cv. Samsun NN. RNA from control (untreated) leaves yielded no signal, whereas RNA purified from leaves treated for 24 h with 50 µM JAME yielded a strong signal of 500 bp, indicating that the jasmonate-induced expression of Nictaba is transcription dependent (results not shown).
Failure to accumulate lectin in response to JAME treatment correlates with the absence of the corresponding genes in the non-responsive Nicotiana species
The inability of most wild tobacco species to accumulate lectin in response to JAME treatment raised the question of why these species react so differently from the lectin-expressing species. In principle, the observed lack of response can be due to either a strongly (or even completely) reduced sensitivity to JAME or the absence of lectin gene(s) in their genomes. Since it is difficult to imagine that so many tobacco species are insensitive to jasmonates, a defect at the genomic level seemed more likely. Therefore, the presence/absence of the lectin gene(s) in the genomes of the different Nicotiana species was studied using a PCR-based method. Agarose gel electrophoresis revealed that all N. tabacum cultivars and all Nicotiana species that accumulate lectin upon induction with JAME yielded one or two DNA fragments ranging in length between 500 and 800 bp (Fig. 3A), whereas no PCR-amplified fragment could be detected in the reaction mixtures with the DNA from the non-responsive species. Since one cannot a priori rule out the possibility that the apparent absence of PCR-amplified fragments was due to extensive mismatches between the sequence of the primers and the putative lectin genes, DNA from tomato (Solanum lycopersicum), which, according to transcriptome analyses expresses closely related orthologues of Nictaba (e.g. tomato seed cDNA clone cLEE1O14; GenBank accession no. AW616959), was subjected to PCR under the same conditions and with the same primers. The tomato DNA yielded PCR amplification products of the same length as the responsive Nicotiana species (500 and 800 bp, results not shown). Taking into consideration that the nucleotide sequences of Nictaba and its orthologues from tomato differ at several positions (see below), amplification of PCR fragments from tomato DNA with the Nictaba-specific primers (evd 42–evd 43) strongly suggests that the negative reaction with the DNA from the non-responsive Nicotiana species is not due to mismatches but reflects the absence of sequences corresponding to that portion of the Nictaba gene from the genome. The combined results of the lectin/western blot analyses and PCR experiments leave no doubt that the observed failure to accumulate lectin in response to JAME correlates with the absence of the corresponding CDS in the non-responsive Nicotiana species. Accordingly, it seems likely that the Nictaba gene or at least the CDS has been lost from the genome of some Nicotiana species.
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Intra- and interspecies differences in the number and exon/intron structure of genes encoding Nictaba orthologues
The results of the PCR experiments designed to check the presence of lectin gene(s) in the different tobacco cultivars/species revealed a marked heterogeneity in both the number and length of the amplified DNA fragments. Using primers complementary to the 5' and 3' ends of the 5' and 3' UTRs, respectively, fragments ranging in size between 500 and 800 bp were obtained (Fig. 3A). Depending on the species/cultivar, one or two fragments of a distinct size were amplified. Since these results were indicative of (i) the occurrence of at least two different lectin genes in some species and (ii) differences in the exon/intron structure of the respective lectin genes, the PCR-amplified fragments were sequenced. All genomic fragments can be classified in a group of (i) sequences comprising the complete continuous ORF of Nictaba and a second group of (ii) sequences in which the same ORF is interrupted by two introns (Fig. 3B). This implies that within the genus Nicotiana, the same or similar lectins are encoded by two distinct types of genes with a different exon/intron structure. A closer examination of the structure of the lectin genes from the different species and cultivars revealed several other aspects of the Nicotiana lectin genes. First, all diploid species contain either intronless or intron-containing lectin genes. Secondly, the diploid N. tomentosiformis and allotetraploid N. africana possess multiple lectin genes. Thirdly, the lectin genes of all species classified in section Tomentosae contain introns. Fourthly, N. tabacum exhibits a marked intraspecies heterogeneity for number and structure of lectin genes. Some varieties have only intronless (e.g. cv. Xanthi NN) or intron-containing (e.g. cv. BY-2) lectin gene(s), whereas others (e.g. cv. Samsun NN) have both (Table 1; see supplementary Fig. S1 at JXB online).
Alignment of the sequences of the intron-containing genes revealed that the position of the introns is strictly conserved. The first intron is located immediately behind the start codon, whereas the second is inserted between the second and third nucleotide of the triplet of amino acid residue 30 (being an aspartate or a serine). All intron boundaries exhibit the typical GT and AG dinucleotide signature at their 5' and 3' ends, respectively. The length of the first intron varies between 77 and 117 nucleotides, whereas the second intron contains between 83 and 87 nucleotides (Fig. 3B). Furthermore, intron sequences are highly conserved (see supplementary Fig. S2 at JXB online).
Proteins encoded by lectin genes from different Nicotiana species and cultivars exhibit high sequence identity
To verify whether the PCR-amplified genomic fragments encode Nictaba or a closely related orthologue, the amino acid sequence of the putative lectin polypeptides was deduced from the nucleotide sequences. For intron-containing fragments, the CDS was determined using the nucleotide sequence of the cDNA encoding Nictaba as a template. As shown in Fig. 4, all amplified fragments encode a 165 amino acid residue polypeptide either identical or similar to the Nictaba sequence from N. tabacum cv. Samsun NN (Chen et al., 2002). All sequences are identical at 141 out of the 165 positions. Within sections Tomentosae and Nicotiana, the lectin sequences from the wild Nicotiana species share 149 identical residues. In N. tomentosiformis, the three lectins differ at two positions, indicating that in this diploid the intraspecies divergence is minimal. A more pronounced sequence divergence exists between the lectins encoded by the sequences amplified from different cultivars of the allotetraploid N. tabacum, raising questions of the origin of the tobacco lectin genes.
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Some but not all lectin genes of allotetraploid N. tabacum are identical to an orthologous gene from a presumed diploid ancestor
Recent genetic studies provided clear evidence that the S and T genomes of natural allotetraploid N. tabacum are derived from the ancestors of the modern diploids N. sylvestris and N. tomentosiformis, respectively (Ren and Timko, 2001; Chase et al., 2003; Knapp et al., 2004). Taking into consideration that the maternal parent N. sylvestris does not accumulate lectin in response to JAME and no lectin gene could be amplified from its DNA, the lectin genes found in N. tabacum should be derived from the genome of the paternal parent, N. tomentosiformis. Accordingly, one would expect that the genome of N. tabacum should contain one or more lectin genes that are identical or at least similar to the lectin genes found in N. tomentosiformis. An overview of the genes and corresponding deduced sequences of the N. tabacum lectins indicates that this is clearly not the case (Table 1, Fig. 4 and Supplementary Fig. S1). First, all three N. tomentosiformis genes contain two introns, whereas lectin genes with and without introns occur in the genome of N. tabacum. Secondly, the identification of eight different lectin genes within a relatively small collection of N. tabacum cultivars implies that the set of tobacco lectin genes is not just a copy of that found in N. tomentosiformis. Thirdly, the deduced sequences of the eight N. tabacum lectin genes differ from each other at eight positions, whereas the three paralogues from N. tomentosiformis differ by only two amino acid residues. Evidently, these observations are difficult to reconcile with the concept of a simple vertical inheritance of a set of closely related lectin genes from N. tomentosiformis into modern cultivated tobacco. Therefore, the issue of a possible different origin of the N. tabacum lectin genes was corroborated in more detail by a comparative analysis of the nucleotide sequences of the lectin genes from different Nicotiana species.
To check whether the three intron-containing lectin genes found in modern N. tomentosiformis have been transmitted to N. tabacum, their nucleotide sequences were compared with those of the intron-containing N. tabacum genes. The sequence of Tomf2 is—also in the introns—nearly identical to that of BY-2 and Hav2, indicating that the Tomf2 gene might be the direct ancestor of BY-2 and Hav2. Sam2 is also nearly identical to Tomf2 but contains a three nucleotide deletion in the first intron (see supplementary Figs S1 and S2 at JXB online). None of the N. tabacum genes can be aligned perfectly with either Tomf1 (especially in the region spanning the first half of the first intron) or Tomf3, which has a 32 nucleotide insertion at the 3' end of the first intron. This indicates that at least within the tobacco cultivars included in the present experiments, no direct descendant of Tomf1 and Tomf3 could be identified.
In contrast to BY-2 and Hav2, the sequence of Wis exhibits little sequence identity with Tomf genes within the first intron, suggesting that neither of the Tomf genes is the direct ancestor of this tobacco lectin gene. Wis shares high sequence identity with the lectin gene from N. kawakamii within the first intron but differs in the second intron by a four nucleotide insertion. Since Wis also differs from the N. otophora and N. tomentosa lectin genes in the second intron, none of the wild tobacco species included in this study is a likely donor of the lectin gene identified in the tobacco cv. Wisconsin 38.
The origin of the intronless tobacco lectin genes is even more puzzling because in none of the formerly presumed donors of the T genome (i.e. N. tomentosiformis, N. tomentosa, and N. otophora) was an intronless lectin gene identified. In principle, two other explanations can be put forward. First, the intronless genes originated from an intron-containing N. tomentosiformis lectin gene through a deletion of the introns. Secondly, the intronless tobacco lectin genes were introduced into the tobacco genome through introgression from an unidentified wild Nicotiana species possessing an intronless lectin gene. If the intronless genes result from an intron deletion process, one can reasonably expect that the coding sequences of both genes should be identical. Alignment of intron-containing and intronless genes from tobacco cultivar Samsun NN indicated that the intronless gene Sam1 differs by one and four nucleotides from the CDS of the intron-containing genes Sam2 and Sam3, respectively, whereas Sam2 and Sam3 differ from each other by three nucleotides in the CDS and by two nucleotides in the intron sequences. It is possible, therefore, that at least the intronless lectin gene found in the tobacco cultivar Samsun NN originated from a corresponding intron-containing gene through the deletion of both introns. Even though intron deletion can, in principle, have led to the origin of intronless genes in tobacco, the possibility cannot be excluded that tobacco acquired an intronless gene from a wild relative (e.g. through introgression).
In conclusion, the available sequence data provide no unambiguous explanation for the origin of the N. tabacum lectin genes. Direct inheritance of a set of lectin genes from the most likely donor of the T genome (in this case N. tomentosiformis) is difficult to reconcile with both the sequence and gene structure data. A possible explanation might be that the T genome of N. tabacum is derived from a particular lineage of N. tomentosiformis. Based on the observation that repetitive sequences of geminiviral origin present in the genome of N. tabacum cultivars were found in only one out of four N. tomentosiformis varieties, it has been suggested, indeed, that the allotetraploid N. tabacum originated after divergence within N. tomentosiformis and that the spectrum of potential donors of the paternal genome can be narrowed to a genotype of N. tomentosiformis characterized by the presence of GRD53 and GRD3 repeats (Murad et al., 2002). Alternatively, other as yet unidentified events played an important role in the evolution of the N. tabacum lectin genes.
Evidence for jasmonate-independent expression of Nictaba orthologues in other Solanaceae species
To check whether proteins similar to Nictaba are expressed in Solanaceae other than Nicotiana species, the publicly accessible expressed sequence tag (EST) databases were searched for the presence of Nictaba orthologues. Expressed proteins similar to Nictaba could be identified in pepper (Capsicum annuum) (Capann), tomato (S. lycopersicum, Sollyc; Solanum hirsutum, Solhir) and potato (Solanum tuberosum) (Soltub1 and Soltub2) (Fig. 5). Whereas the diploids pepper and tomato apparently express only a single molecular form, two closely related but definitely distinct isoforms occur in allotetraploid potato, suggesting that each genome contains distinct genes encoding a Nictaba orthologue. Though the sequences of the pepper, tomato, and potato proteins share high sequence identity with Nictaba, there are two obvious differences. First, a single residue deletion is introduced in Nictaba at the boundary of the two exons, and second, the nuclear localization signal present in Nictaba is not conserved in the pepper, tomato, and potato proteins.
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The identification of ESTs encoding Nictaba orthologues in pepper, tomato, and potato indicates that the material from which the cDNA libraries were constructed synthesized lectin. Since most of these materials were derived from plants that were not treated with JAME, the expression in pepper, tomato, and potato is apparently not JAME dependent. Treatment of potato and tomato with JAME in a concentration range between 0 and 1000 µM did not result in the synthesis of detectable amounts of a Nictaba orthologue. Similar experiments with leaves from Petunia hybrid and Datura stramonium also yielded no positive results. Therefore, one can reasonably conclude that the jasmonate-induced accumulation of Nictaba orthologues is not a common phenomenon in Solanaceae species.
Biological relevance of the presence/absence of genes encoding Nictaba orthologues
Since the biological role of Nictaba is not fully understood yet, the impact of the presence/absence of the JA-inducible lectin genes in different tobacco species is difficult to assess. However, several lines of evidence suggest that Nictaba might be a defence-related protein. Preliminary experiments indicated that insect herbivory also induces the expression of Nictaba in tobacco leaves (N Lannoo, unpublished results). Furthermore, addition of purified Nictaba lectin to an artificial diet interferes with growth and development of at least some insects. Though still preliminary, these observations suggest that the evolutionary mechanism underlying the presence or absence of Nictaba genes in diverse Nicotiana species should preferentially be discussed in the broader context of plant defence gene evolution. Hitherto, little conclusive evidence has been reported for the loss of a (putative) defence-related protein within a single genus. A well-documented example is the creeping specific-1 (Crs-1) gene from creeping bentgrass (Agrostis stolonifera) that encodes a protein with an N-terminal dirigent protein domain and a C-terminal domain equivalent to the mannose-binding jacalin-related lectins. Crs-1 is apparently in the process of being lost from heterozygous populations of creeping bentgrass (Li et al., 2005). Moreover, the fact that Crs-1 could not be detected in several other Agrostis species was interpreted as an important argument to conclude that the gene is being lost from the genus. Though the molecular and genetic data leave no doubt that the Crs-1 gene is being lost, it still remains to be demonstrated that the presence/absence of the gene is correlated with the expression/absence of expression of the putative Crs-1 protein. Another intraspecific gene loss was reported for a gene encoding an insecticidal lectin in Glechoma hederacea (ground ivy) (Wang et al., 2003). Analysis of a wild population revealed that five out of 41 clones accumulated no lectin in their leaves and indicated that the failure to express the lectin was correlated with the failure to amplify the CDS from genomic DNA. It should be emphasized here that in contrast to the apparent intraspecific gene loss of the Crs-1 gene in A. stolonifera and the lectin gene in G. hederacea, there are no indications for an intraspecific loss of the Nictaba gene within N. tabacum.
Though both the Crs-1 gene product and G. hederacea lectins are considered defence-related proteins, the occurrence of mixed populations of genotypes with and without the respective genes indicates that these presumed defence genes are not essential for survival of individual plants in their natural habitat. Since in neither case was the question answered of why these genes are partly retained (or partly lost), the data described for Crs-1 and the G. hederacea lectin provide little useful information with respect to the advantage/disadvantage of the presence/absence of genes encoding Nictaba orthologues in Nicotiana species.
Even though Nictaba genes are apparently not essential for survival under normal conditions, the lectins found in the different Nicotiana species share relatively little sequence divergence. This is rather surprising because plant defence genes often evolve rather rapidly, resulting in important intra- and interspecific sequence divergence. This holds true for both generalist defences (e.g. class I chitinases in the genus Arabis and wound-induced serine protease inhibitor from Zea and related genera) (Bishop et al., 2000; Tiffin and Gaut, 2001; Tiffin et al., 2004) and specialist defences (e.g. hm1 and hm2 from Zea) (Zhang et al., 2002, Tiffin et al., 2004).
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Conclusion |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionSupplementary dataReferences |
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The present survey of the response of a collection of wild Nicotiana species and N. tabacum cultivars provides the first firm evidence for a marked intragenus difference with respect to the activation of a well-defined jasmonate-inducible gene. Moreover, it could be unambiguously demonstrated that the non-responsive Nicotiana species fail to accumulate the lectin because the CDS of the corresponding genes is absent from their genome. These findings are of paramount importance in view of the study of the jasmonate signalling pathway because they illustrate in a fairly dramatic way that the results obtained with a particular model system cannot simply be extrapolated to other plant species, especially not when applied to specialized responses.
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Supplementary data |
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TopAbstractIntroductionMaterials and methodsResults and discussionConclusionSupplementary dataReferences |
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Supplementary data (Figs S1 and S2) for this paper can be found at JXB online.
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Acknowledgements |
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This research was supported by the Fund for Scientific Research-Flanders and the Research Council of Ghent University. We thank Dr Verne A Sisson and Dr Nagata Toshiyuki for providing seeds.
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Abbreviations |
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BY-2, Bright Yellow-2; CDS, coding sequence; JA, jasmonic acid; JAME, jasmonic acid methyl ester; JIP, jasmonate-inducible protein; MS, Murashige and Skoog; Nictaba, Nicotiana tabacum agglutinin; ORF, open reading frame; UTR, untranslated region.
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