A novel gene, named NgAOX1a, was isolated from Nicotiana glutinosa by RT-PCR (reverse transcription-PCR). The full-length cDNA of NgAOX1a was 1448 bp, including a 1062-bp ORF (open reading frame), a 124 bp 5′ UTR (untranslated region) and a 262 bp 3′ UTR. The ORF encodes a 353-amino-acid protein which contains two conserved cysteine residues, four iron-binding motifs, five α-helix regions and six conserved histidine residues. The phylogenetic tree showed that NgAOX1a belongs to the AOX1 (alternative oxidase 1)-type group. Alignment analysis showed that NgAOX1a shares a high similarity with other known AOXs. Four exons and three introns were detected in the genomic DNA sequence, and Southern-blotting analysis suggested that NgAOX1a is a single-copy gene. A series of putative cis-acting elements were examined in the 5′-flanking region of NgAOX1a. Northern-blotting analysis showed that the transcript levels of NgAOX1a can be markedly accumulated when tobacco seedlings are treated with various abiotic stimuli, such as exogenous signalling molecules for plant defence response, salicylic acid and H2O2, and the exogenous TCA (tricarboxylic acid) cycle metabolite citrate. However, it could be suppressed by abiotic stress, such as CoCl2, an inhibitor of ethylene, which indicates that the expression of NgAOX1a may be regulated by ethylene. In addition, NgAOX1a can also be strongly induced by three viral pathogens, tobacco mosaic virus, potato virus X and potato virus Y. These results indicate that NgAOX1a may be involved in multi-signal transduction pathways and may play an important role in defence response.

INTRODUCTION

Plant mitochondria possess a bifurcated electron-transport chain. In addition to the cyanide-sensitive cyt (cytochrome) respiratory pathway found in all eukaryotes, plants have a second cyanide-insensitive alternative pathway [1,2]. Electron flow via the cyt pathway is coupled to ATP production, whereas electron transfer through the alternative pathway branches from the cyt pathway at the ubiquinone pool and reduces oxygen to water without conservation of energy in the form of ATP, which is lost as heat. This reaction consists of a single step that is solely mediated by AOX (alternative oxidase).

AOX is the terminal oxidase of the cyanide-insensitive alternative pathway in the mitochondrial inner membranes. AOX proteins are found in every examined species and in almost every plant organ [3]. Since the first cloning of the AOX gene from the thermogenic plant Sauromatum guttatum, a variety of AOX genes have subsequently been identified and isolated from both monocots and dicots, including Arabidopsis, soya bean, cucumber, wheat, tobacco, rice, cotton, cowpea etc. Several studies have shown that AOX proteins are encoded by a small multi-gene family in these plants. On the basis of the phylogenetic analysis of amino acid sequences, AOX genes can be grouped into two different types, AOX1 and AOX2. AOX1 genes are inducible by stress stimuli in many tissues of both monocot and dicot species [4,5]. For example, AOX1 can be induced by slicing or aging in potato tubers, ET (ethylene) treatment of fruits and storage tissues, cycloheximide treatment of cucumber cotyledons, light treatment of rice and cold treatment of tobacco and wheat. Moreover, AOX1 can be induced by SA (salicylic acid) in many different species. Because SA induction is related to defence-response genes in plants [6], AOX1 may be involved in defence response. However, AOX2 genes are usually constitutive or developmentally expressed only in eudicot species [4,5].

As early as 1994, the NtAOX1a (Nicotiana tabacam AOX1a) gene was cloned from tobacco (N. tabacam L. cv Bright Yellow), and a cell-suspension system of tobacco was established to investigate the expression of NtAOX1a. Afterwards, cell cultures of tobacco (N. tabacam L. cv Petit Havana) were also established to investigate signals regulating the expression of AOX1 [5]. However, cells in culture may have a metabolic state different from that of intact plant tissue [79]. Also, cells in culture may behave similarly to some, but not all, plant tissue types [10]. For example, tobacco culture cells grown at low inorganic phosphate have increased AOX expression [11], but tobacco leaves grown at low inorganic phosphate do not [12]. In order to obtain experimental data on the structural and regulatory patterns of AOX in plant tissue, Nicotiana glutinosa was chosen for research.

In the present study, we selected the leaf tissues of N. glutinosa as experimental material, isolated an inducible AOX gene described as NgAOX1a (N. glutinosa AOX1a) and investigated the expression characteristics of the NgAOX1a gene under different conditions. The study of the NgAOX1a gene will provide a useful reference for further study of the function of the AOX gene in stress defence and signal transduction in the whole plant of N. glutinosa.

MATERIALS AND METHODS

Plant materials and treatments

A mixture of seeds (N. glutinosa) and compost were sown on the surface of sterilized compost in pots covered with plastic film for germination at 26°C in a greenhouse. After germination, the film was uncovered and the germinated seedlings were grown in the greenhouse. The 3-month-old plants were subjected to different treatments. Some of them were respectively sprayed with 10 mM citrate, 1 mM SA, 10 mM H2O2 and 1 mM CoCl2, whereas others were inoculated with the viral pathogens TMV (tobacco mosaic virus), PVX (potato virus X) and PVY (potato virus Y). TMV, PVX and PVY were mechanically inoculated on to leaf discs or whole leaves of 3-month-old plants [13]. Uninoculated leaves were collected every 2 days. All collected leaves of treated seedlings were immediately frozen in liquid nitrogen and stored at −80°C until use.

RNA extraction, cDNA synthesis and DNA isolation

Total RNA for cDNA synthesis was extracted from the seedling leaves after 3 days of 1 mM SA treatment using the RNeasy plant mini kit (Qiagen) according to the manufacturer's protocol. Total RNA for Northern-blotting analysis was extracted from leaf materials which were treated using Trizol reagent (Invitrogen) in accordance with the manufacturer's instructions. All RNA samples thus obtained were treated with DNaseI (Promega) to remove the potential genomic DNA contamination and stored at −80°C prior to cDNA synthesis and expression analysis.

The first-strand cDNA was synthesized from approx. 2 μg of total RNA using the adaptor primer B26 (Supplementary Table S1 at http://www.bioscirep.org/bsr/028/bsr0280259add.htm) and MMLV (Moloney-murine-leukaemia virus) reverse transcriptase at 42°C for 1 h.

Genomic DNA was isolated from 3-month-old plants by the CTAB (cetyltrimethylammonium bromide) method [14]. The quality and concentration of RNA and DNA samples were examined using EB (ethidium bromide)-strained agarose gel electrophoresis and spectrophotometric analysis.

Primers

The primers used in the present study are listed in Supplementary Table S1.

Cloning of the internal conservative sequence of NgAOX1a

In order to obtain the internal conservative fragment of the NgAOX1a gene, two degenerate PCR primers, AP1 and AP2 (Supplementary Table S1), were designed and synthesized, corresponding to the conserved amino acids and nucleotide sequences of the AOX1a gene from Arabidopsis, cotton, soya-bean and N. tabacum from the GenBank® database. RT-PCR (reverse transcription-PCR) was performed under the following conditions: the template was first pre-denatured at 94°C for 5 min, and then subjected to 35 cycles of amplification (94°C for 50 s, 53°C for 50 s and 72°C for 30 s), followed by an extension for 5 min at 72°C. The reaction mixture was subjected to electrophoresis on a 1% agarose gel, purified with an agarose gel DNA purification kit (TaKaRa), ligated to PMD18-T vector (TaKaRa), transformed into competent Escherichia coli DH5α cells and the DNA was then sequenced.

5′ RACE (rapid amplification of cDNA ends) of NgAOX1a

The first-strand cDNA was purified with the Wizard DNA Clean-Up system (Promega) in accordance with the manufacturer's instructions, polyadenylated at its 5′ end with dGTP using terminal deoxynucleotidyl transferase (TaKaRa), and then followed by ethanol precipitation and resuspension in distilled de-ionized water. The 5′-ready cDNA was obtained and used as the template for the primary PCR. On the basis of the internal conservative sequence obtained from N. glutinosa, the 5′-specific primers, W5 and N5 (Supplementary Table S1), were designed and synthesized for 5′ RACE of NgAOX1a. The W5 primer, together with the abridged anchor primer (AAP, Supplementary Table S1), was used for primary amplification under the following PCR conditions: pre-denaturation at 94°C for 5 min, followed by 35 cycles of amplification (94°C for 50 s, 53°C for 50 s and 72°C for 1 min). After the final cycle, the amplification was extended for 5 min at 72°C. The PCR product was used as the template for the nested PCR amplification with the N5 nested primer and the abridged universal amplification primer (AUAP, Supplementary Table S1). The reaction condition of the secondary PCR was the same as the primary PCR amplification, except that the annealing temperature was 55°C. The products of the secondary PCR were subjected to electrophoresis, purified and cloned into PMD18-T vector, followed by sequencing as described above.

3′ RACE of NgAOX1a

Two specific primers, W3 and N3 (Supplementary Table S1), were also designed and synthesized for 3′ RACE of NgAOX1a on the basis of the internal sequence. The primary PCR was carried out with the primers W3 and B26 and the first-strand cDNA as the template. The primary PCR products and the N3 primer, coupled with the B25 (Supplementary Table S1) primer, were employed along with the secondary PCR. Both primary and secondary PCR amplification were conducted as follows: pre-denaturation at 94°C for 5 min; 35 cycles of amplification (94°C for 50 s, 50/55°C for 50 s and 72°C for 1 min); and a final extension step at 72°C for 5 min. The products of secondary PCR were subjected to electrophoresis, purified and cloned into the PMD18-T vector, and then sequenced.

Cloning of the full-length cDNA and genomic fragment of NgAOX1a

The sequences of 3′ and 5′ cDNA with the internal conservative region obtained were assembled to generate a putative full-length cDNA of NgAOX1a. According to the deduced cDNA, a pair of gene-specific primers, F1 and F2 (Supplementary Table S1), were designed and synthesized. The cDNA sequence and genomic fragment were amplified by PCR from the templates of the first-strand cDNA and genomic DNA respectively using gene-specific primers F1 and F2. The general PCR conditions were as follows: 94°C for 5 min; 35 cycles of amplification (94°C for 50 s, 52/54°C for 50 s, 72°C for 2 min); extended for 5 min at 72°C. The PCR products were subjected to electrophoresis, purified, cloned into the PMD18-T vector and sequenced.

Amplification of the promoter region of NgAOX1a

To obtain the fragment of the promoter region, genomic DNA (approx. 5 μg) was completely digested for 24 h with BglII at 37°C, purified with anhydrous alcohol, then ligated to the cassette and used as the template. The primers WPr used in the primary PCR and NPr used in nested PCR (Supplementary Table S1) were designed and synthesized on the basis of the 5′ end of the genomic fragment. LA-PCR (long and accurate PCR) was carried out according to the instructions of the LA-PCR in vitro Cloning Kit (TaKaRa). The PCR products were purified and cloned into pMD18-T vector, followed by sequencing.

Southern-blotting analysis

The samples of genomic DNA (30 μg/sample) from 3-month-old plants were digested for 48 h at 37°C with EcoRI, EcoRV, HindIII and XbaI (TaKaRa), which did not cut within the probe region, then subjected to electrophoresis on a 1% agarose gel and blotted on to a Hybond-N+ Nylon membrane (Amersham Pharmacia) by capillary blotting according to a method described by Sambrook et al. [15]. The probe, a 533-bp fragment of the 5′-end cDNA (see Figure 2), was amplified with the primers B5 and B3 (Supplementary Table S1) by RT-PCR, and labelled with [α-32P]dCTP according to the manufacturer's instructions (Primer-a-Gene® Labeling System; Promega). Filters were pre-hybridized overnight at 42°C in blocking reagent (6×SSC, 5×Denhardt's buffer, 0.5% SDS and 1 mg/ml single-stranded DNA), hybridized at 42°C for 36–48 h with the probe, and washed three times for 10 min at 42°C in 2× SSC/0.2% SDS and 0.2× SSC/0.2% SDS (1× SSC is 0.15 M NaCl/0.015 M). After the high-stringency wash, filters were exposed to a phosphorimager for 2–3 days, and then the hybridization signals were scanned with a phosphorimaging screen.

Northern-blotting analysis

The samples of total RNA (30 μg/sample) were denatured and separated by electrophoresis using 1% formaldehyde agarose gels and blotted as described above. Equal loading of samples was confirmed by including EB in the sample loading buffer, allowing visualization of RNA under the gel imaging system. The hybridization probe, conditions and procedures were the same as those described for Southern blotting. All Southern-and Northernblotting analysis experiments were repeated twice using independent samples and representative results are presented.

RESULTS

Cloning and sequence analysis of NgAOX1a

Alignment of AOX sequences from several species showed several highly conserved regions of amino acids. According to the conserved regions, two degenerate primers were designed, synthesized and used for PCR amplification of N. glutinosa cDNA. After the electrophoresis, a single band of the predicted size (approx. 300 bp) was examined, and showed high similarity with the NtAOX1a (accession number S71335) gene. Then the fragments of 5′ and 3′ ends were obtained using 5′ and 3′ RACE with the specific primers W5, N5, W3 and N3. They were approx. 900 bp and 600 bp in length respectively. After comparison and analysis of the overlapped regions of the three fragments, the full-length cDNA sequence of NgAOX1a was deduced, including the 5′ and 3′ UTRs (untranslated regions). A fragment, approx. 1100 bp, was obtained by RT-PCR with the specific primers F1 and F2, which was identical with the deduced cDNA of NgAOX1a.

Sequences analysis revealed that the full-length cDNA of NgAOX1a (GenBank® accession no. EF523518) was 1448 bp, including a 1062-bp ORF (open reading frame), a 124 bp 5′ UTR and a 262 bp 3′ UTR. The ORF was predicted to encode a polypeptide of 353 amino-acid residues with a calculated molecular mass of approx. 39.8 kDa and a theoretical isoelectric point of 8.92.

Characterization and phylogenetic analysis at the protein level

Sequences alignment using DNAMAN (version 5.2.2; Lynoon BioSoft Company) showed that NgAOX1a shared 67.4, 67.7, 69.7 and 93.48% similarity with the AOX from soya bean, Arabidopsis, cotton and N. tabacum respectively (Figure 1). The N-terminal end, which is the least conserved region among all the AOXs, may be a mitochondrial targeting signal [16]. Five predicted α-helical regions involved in formation of a hydro-bridged bi-nuclear iron centre and four iron-binding motifs (EXXH, FXHR and EEE-Y) [17] were detected in NgAOX1a. Two cysteine residues (C126 and C176) involved in dimerization via S–S bond formation [18], and six histidine residues (H144, H197, H224, H265, H326 and H331) involved in metal binding, were also present in NgAOX1a, which were completely conserved throughout the plant AOX sequences [17]. Mutation of C126 resulted in an alternative oxidase which was no longer activated by pyruvate [1921]. The structural conservation strongly suggests that NgAOX1a encodes functional AOX proteins.

Alignment of the deduced amino-acid sequences of AOXs

Figure 1
Alignment of the deduced amino-acid sequences of AOXs

The amino-acid sequences used for multi-alignment analysis were AtAOX1 (Arabidopsis thaliana, accession number NM_113135), GhAOX1 (Gossypium hirsutum, DQ250028), GmAOX1 (Glycine max, X68702), NtAOX1 (S71335) and NgAOX1 (EF523518). Letters on a black background correspond to identical amino acid residues. Five α-helical regions are labelled by Roman numbers (I–V). Two highly conserved cysteine residues are indicated by asterisks (*) and six completely conserved histidine residues are marked by arrowheads (▼). The positions of iron-binding motifs (EXXH, FXHR and EEE-Y) are underlined.

Figure 1
Alignment of the deduced amino-acid sequences of AOXs

The amino-acid sequences used for multi-alignment analysis were AtAOX1 (Arabidopsis thaliana, accession number NM_113135), GhAOX1 (Gossypium hirsutum, DQ250028), GmAOX1 (Glycine max, X68702), NtAOX1 (S71335) and NgAOX1 (EF523518). Letters on a black background correspond to identical amino acid residues. Five α-helical regions are labelled by Roman numbers (I–V). Two highly conserved cysteine residues are indicated by asterisks (*) and six completely conserved histidine residues are marked by arrowheads (▼). The positions of iron-binding motifs (EXXH, FXHR and EEE-Y) are underlined.

A phylogenetic tree of AOX proteins was constructed by inputting the protein sequences of different plant species into DNAMAN (Supplementary Figure S1 at http://www.bioscirep.org/bsr/028/bsr0280259add.htm). According to the phylogenetic tree, NgAOX1a had higher identity with AOX1-type plants than AOX2-type. The analysis results strongly suggest that NgAOX1a may belong to the AOX1-type gene family.

Structure analysis of the genomic sequence of NgAOX1a

In order to isolate the genomic fragment and study the exon–intron structures of NgAOX1a, PCR products were amplified using genomic DNA as template with the specific primers F1 and F2. A fragment of approx. 1640 bp was obtained. The genomic DNA sequence (GenBank® accession no. EF523519), approx. 1991 bp, has four exons and three introns (120 bp, 321 bp and 123 bp) (Figure 2). The intron insertion positions were relatively conserved and resembled most of other AOX genes, except the AOX2 gene of Arabidopsis [22] and the rice AOX1b gene [23]. Three introns had high AT contents (72.5% for intron 1, 71.4% for intron 2 and 69.9% for intron 3) and had conserved 5′-GU and AG-3′ sequences at 5′ and 3′ splice sites, which are all typical structural characteristics of plant introns [24,25].

Genomic and mRNA structures of NgAOX1a

Figure 2
Genomic and mRNA structures of NgAOX1a

The genomic DNA region of NgAOX1a with three introns (dark shading) and the mRNA with the coding region marked are shown. The probe used in hybridization is indicated.

Figure 2
Genomic and mRNA structures of NgAOX1a

The genomic DNA region of NgAOX1a with three introns (dark shading) and the mRNA with the coding region marked are shown. The probe used in hybridization is indicated.

To elucidate how many copies of the NgAOX1a gene were present in N. glutinosa genome, Southern-blotting analysis was performed under high-stringency conditions. Genomic DNA was extracted from leaf tissues and digested completely with the restriction enzymes EcoRI, EcoRV, HindIII and XbaI. To avoid cross-hybridization, the highly specific fragment, corresponding to the 5′-end partial coding sequence of the NgAOX1a gene without recognition sites for the four restriction enzymes, was used as the probe (Figure 2). The result showed that only one hybridization band was observed in four different restriction enzyme-digested DNA lanes (Supplementary Figure S2 at http://www.bioscirep.org/bsr/028/bsr0280259add.htm), indicating that NgAOX1a is a single-copy gene.

Identification of partial putative cis-acting elements in the 5′-flanking region of NgAOX1a

Agarose-gel electrophoresis analysis showed that a band of the 5′-flanking region of NgAOX1a, approx. 650 bp, was generated with two specific primers, WPr and NPr, by LA-PCR. After removing the region overlapping with the genomic sequence, the 5′-flanking region (approx. 518 bp) and the putative transcription start site were defined (Figure 3). The 5′-flanking region showed a high AT content (approx. 70%), which is commonly found in other plant promoters. Then the putative cis-acting elements in the 5′-flanking region were predicted using the PlantCARE database (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/). The TATA boxes and CAAT boxes were identified in the 5′-flanking region upstream of the putative transcription site. Several important cis-acting elements for gene regulation were also found, which include: (1) an ACE (ACGT-containing element), an ATTAAT-motif and a G-box, involved in light responsiveness; (2) an HSE (heat-shock element) involved in heat stress responsiveness and an element involved in circadian control; (3) an MBSI (MYB DNA-binding site I) involved in flavonoid biosynthetic gene regulation; and (4) three Skn-1 motifs, required for endosperm expression.

Nucleotide sequence and putative cis-acting elements of the 5′-flanking region of NgAOX1a

Figure 3
Nucleotide sequence and putative cis-acting elements of the 5′-flanking region of NgAOX1a

The transcriptional and translational start sites are marked with arrows. The putative core promoter consensus sequences are highlighted in grey, and the putative cis-acting elements are boxed.

Figure 3
Nucleotide sequence and putative cis-acting elements of the 5′-flanking region of NgAOX1a

The transcriptional and translational start sites are marked with arrows. The putative core promoter consensus sequences are highlighted in grey, and the putative cis-acting elements are boxed.

Expression profiles of NgAOX1a in response to abiotic stress

Citrate is the first organic acid in the TCA (tricarboxylic acid) cycle, and its accumulation may act as a signal at both the biochemical and gene expression levels to increase AOX respiration [26]. The available data suggests that the level of citrate is significant for the regulation of mitochondrial AOX in plants [27]. Thus, in the present study, we evaluated the expression of NgAOX1a in response to citrate supplied exogenously. As shown in Figure 4(A), exogenous citrate caused an increase in the accumulation of the NgAOX1a mRNA within 1 h after treatment, reaching a maximum at 6 h, and it then decreased. The result suggests that citrate may have an effect on the transcription of NgAOX1a in N. glutinosa. This result indicates that the expression of NgAOX1a may be regulated in response to this metabolite in the TCA cycle.

Expression patterns of NgAOX1a under abiotic stresses

Figure 4
Expression patterns of NgAOX1a under abiotic stresses

Northern-blotting analysis were performed by total RNA extracted from leaves at marked time after treated with 10 mM citrate (A), 1 mM SA (B), 10 mM H2O2 (C) and 1 mM CoCl2 (D). The EB-stained rRNA is included as the loading control. The plants which were not treated were used as controls (ck). d, day.

Figure 4
Expression patterns of NgAOX1a under abiotic stresses

Northern-blotting analysis were performed by total RNA extracted from leaves at marked time after treated with 10 mM citrate (A), 1 mM SA (B), 10 mM H2O2 (C) and 1 mM CoCl2 (D). The EB-stained rRNA is included as the loading control. The plants which were not treated were used as controls (ck). d, day.

SA is known as a signalling molecule that induces defence- or stress-related genes in plants. It is an important component of signal transduction cascades activating defence response against pathogen attack in plants [28]. ROS (reactive oxygen species) also act as signal molecules in defence responses in plants [29]. Thus, in the present study, we validated the expression of NgAOX1a under SA and H2O2 treatment. Northern-blotting analysis (Figure 4B) showed that the increase in NgAOX1a accumulation was evident within 2 days, peaking at 4 days, and then decreased to the control level. This result indicated that NgAOX1a expression was up-regulated in response to SA treatment. In response to H2O2 treatment, the transcription of NgAOX1a mRNA was increased at 1 h, peaked at 4 h, followed by a decline to control level (Figure 4C). The result suggests that H2O2, as a regulatory signal, may rapidly regulate NgAOX1a gene expression. These results indicate that NgAOX1a is a signalling-molecule-responsive gene, and its expression level is up-regulated strongly by signalling molecules SA and H2O2. These results indicate that NgAOX1a may be associated with the defence response against pathogen attack activated by SA and H2O2.

CoCl2 was also selected to induce NgAOX1a. To our surprise, the transcript of NgAOX1a was slowly decreased after CoCl2 treatment, reached its lowest level at 10 h and then increased to the level before treatment (Figure 4D). The result suggests that the accumulation of NgAOX1a transcript may be suppressed by CoCl2.

The different expression patterns of NgAOX1a in response to citrate, SA, H2O2 and CoCl2 indicate that the NgAOX1a gene may be involved in different signalling pathways and defence responses induced by multi-signal molecules.

mRNA accumulation of NgAOX1a under biotic stresses

In order to verify further whether the NgAOX1a gene is involved in pathogen attack response, viral pathogens, including TMV, PVX and PVY, were inoculated on to three lower leaves per plant. Total RNA was prepared from un-inoculated leaves of TMV-, PVX- and PVY-inoculated plants, and the transcript levels were estimated by Northern-blotting analysis. As shown in Figure 5(A), the inoculation of TMV resulted in a dramatic increase in the NgAOX1a mRNA accumulation within 1 dpi (day post-inoculation) and peaked at 4 dpi, followed by a decline to control levels at 8 dpi. The NgAOX1a gene expression pattern of plants inoculated with PVX (Figure 5B) was consistent with the expression pattern of plants inoculated with PVY (Figure 5C). The transcription accumulation of NgAOX1a reached a maximum at 8 dpi. After that, the expression level decreased gradually to the normal level at 14 dpi. These results suggest that NgAOX1a can be slowly induced, but may be regulated positively, in response to the viral pathogens TMV, PVX and PVY.

Expression patterns of NgAOX1a under biotic stresses

Figure 5
Expression patterns of NgAOX1a under biotic stresses

Northern-blotting analyses were performed with total RNA isolated from leaves at the indicated times after inoculation with TMV (A), PVX (B) and PVY (C). The EB-stained rRNA is included as the loading control. The plants which were not inoculated with viral pathogens were used as controls (ck). d, day.

Figure 5
Expression patterns of NgAOX1a under biotic stresses

Northern-blotting analyses were performed with total RNA isolated from leaves at the indicated times after inoculation with TMV (A), PVX (B) and PVY (C). The EB-stained rRNA is included as the loading control. The plants which were not inoculated with viral pathogens were used as controls (ck). d, day.

DISCUSSION

AOX, the terminal oxidase of the alternative pathway, is a single homodimeric protein encoded by a small family of nuclear genes, which can respond to many environmental stresses and exogenous signal molecules. Many members of the AOX gene family have been isolated from Arabidopsis [16], soya bean [30], wheat [31], rice [23], N. tabacam [32,33] etc. In the present study, we cloned a novel inducible AOX gene by RT-PCR from N. glutinosa, named NgAOX1a. The amino-acid sequence comparisons reveal that NgAOX1a contains five α-helical regions, four iron-binding motifs, two completely conserved cysteine residues and six completely conserved histidine residues, which are the typical structural characteristics of an AOX protein (Figure 1). We also examined the copy number of NgAOX1a. Southern-blotting analysis confirmed that NgAOX1a is a single-copy gene (Supplementary Figure S2). Furthermore, we investigated the transcriptional profiles of NgAOX1a under a variety of abiotic and biotic stresses.

Several studies have reported that SA is a key component in plant signal transduction pathway(s) involved in the defence response against bacterial, fungal and viral pathogens [34,35]. Mitochondrial ROS are also signals involved in the regulation of a subset of antiviral-resistance responses [36,37]. Therefore, we treated plants with 1 mM SA and 10 mM H2O2 respectively. Northern-blotting analysis demonstrated that NgAOX1a can be involved in the signal transduction pathway(s) activated by SA and H2O2. To gain more direct evidence, we examined its expression profiles following the inoculation of TMV, PVX and PVY respectively. The transcript levels of NgAOX1a were found to be markedly induced by three viral pathogens. These results show that NgAOX1a may be involved in the defence response, depending on SA and H2O2, and there may be an association between NgAOX1a and pathogen resistance.

Citrate and/or other TCA cycle intermediates may be important signal metabolites. Researchers have found that two separate pathways for mitochondria-to-nucleus signalling of AOX may exist, involving ROS and the other organic acids [5]. Correspondingly, we examined the effect of citrate on NgAOX1a expression. Northern blotting showed that the accumulation of NgAOX1a mRNA was induced within 6 h by citrate and decreased quickly. This result shows that an increase of citrate in the TCA cycle is accompanied by a rapid and dramatic increase in the mRNA level of the nuclear gene NgAOX1a, which is similar to the expression profile described by Vanlerberghe et al. [26]. However, the NgAOX1a gene of N. glutinosa can reach a high level at 6 h, whereas NtAOX1a of cultured cells reached this level at 8 h. This result indicates that NgAOX1a might be more easily regulated by the level of citrate in plant tissues than in suspension cells. It further demonstrates that NgAOX1a may be involved in the signal transduction pathway depending on the organic acids present in plant tissues.

The ET signal pathway may regulate the expression of some of the genes involved in plant defence [38]. Since CoCl2 is considered to be an inhibitor of ET, we evaluated the effect of CoCl2 on NgAOX1a expression. The result shows that the expression of NgAOX1a can be inhibited when treated with CoCl2, a phenomenon similar to the expression of the AOX gene in aged potato tuber slices. This result suggests that ET may be a signal molecule regulating NgAOX1a expression. It also indicates that AOX may be associated with the ET signal pathway. It is identical with the result showing the ET-dependent pathway being required for AOX expression [3].

Since expression of NgAOX1a was activated by citrate, SA, H2O2 and three viral pathogens and suppressed by CoCl2, it is reasonable to hypothesize that NgAOX1a is a gene that participates in at least three signalling pathways and plays a role in defence responses in N. glutinosa.

Since NgAOX1a can respond to SA, H2O2, citrate and CoCl2, there may be responsive elements in the 5′-flanking region. Although we found the light- and heat-stress-responsive elements in the 5′-partial-flanking region, there might be many other elements, particularly the elements responsive to SA, H2O2, citrate and CoCl2. In the future, the transgenic plants and the full-length promoter sequence will be obtained. This will aid the study of the function of the gene in multi-signal pathways, the relationship between the NgAOX1a gene and viral pathogens in N. glutinosa, and the further investigation of the function of the NgAOX1a gene in antiviral defence mechanisms.

Abbreviations

     
  • AOX

    alternative oxidase

  •  
  • cyt

    cytochrome

  •  
  • dpi

    day post-inoculation

  •  
  • EB

    ethidium bromide

  •  
  • ET

    ethylene

  •  
  • LA-PCR

    long and accurate PCR

  •  
  • Ng

    Nicotiana glutinosa

  •  
  • Nt

    Nicotiana tabacum

  •  
  • ORF

    open reading frame

  •  
  • PVX

    potato virus X

  •  
  • PVY

    potato virus Y

  •  
  • RACE

    rapid amplification of cDNA ends

  •  
  • ROS

    reactive oxygen species

  •  
  • RT-PCR

    reverse transcription-PCR

  •  
  • SA

    salicylic acid

  •  
  • TCA cycle

    tricarboxylic acid cycle

  •  
  • TMV

    tobacco mosaic virus

  •  
  • UTR

    untranslated region

We thank Dr A.-Q. Sun (College of Agriculture, Shandong Agricultural University, Tai'an, Shandong, 271018, People's Republic of China) and Assistant Professor S. Lu (Department of Entomology and Plant Pathology, Mississippi State University, MS, U.S.A.) for critical reading of the manuscript.

FUNDING

This work was supported financially in part by the National Natural Science Foundation of China (grants 30370928 and 30571215).

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Supplementary data