JXB Advance Access originally published online on May 24, 2007
Journal of Experimental Botany 2007 58(8):2203-2216; doi:10.1093/jxb/erm078
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RESEARCH PAPER |
Identification and expression profiling of low oxygen regulated genes from Citrus flavedo tissues using RT-PCR differential display
Group of Biotechnology of Pharmaceutical Plants, Laboratory of Pharmacognosy, Department of Pharmaceutical Sciences, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece
To whom correspondence should be addressed. E-mail: kanellis{at}pharm.auth.gr
Received 12 February 2007; Revised 16 March 2007 Accepted 19 March 2007
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Abstract |
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The molecular basis for the adaptation of fruit tissues to low oxygen treatments remains largely unknown. RT-PCR differential display (DD) was employed to isolate anoxic and/or hypoxic genes whose expression responded to short, low-oxygen regimes. This approach led to the isolation, cloning, successful sequencing, and bioinformatic analysis of 98 transcripts from Citrus flavedo tissues that were differentially expressed in DD gels in response to 0, 0.5, 3, and 21% O2 for 24 h. RNA blot analysis of 25 DD clones revealed that 11 genes were induced under hypoxia and/or anoxia, 11 exhibited constitutive expression and three transcripts were suppressed by low oxygen levels. Almost half of the DD cDNAs were either of unknown function or shared no apparent homology to any expressed sequences in the GenBank/EMBL databases. Six DD genes were similar to molecules of the following functions: C-compound and carbohydrate utilization, plant development, amino acid metabolism, and biosynthesis of brasinosteroids. Time-course and stress-related experiments of low O2-regulated genes indicated that these genes responded differently in terms of their earliness, band intensity, and their specificity to stresses, showing that some of them can be termed hypoxia- or anoxia-induced genes.
Key words: Anoxia, Citrus, Citrus sinensis L., flavedo, gene expression, hypoxia, mRNA differential display, orange fruit, stress
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Ripening of climacteric fruit is the result of co-ordinated metabolic events, which through dramatic changes in anatomy, physiology, biochemistry, and gene expression lead to the transformation of an unattractive fruit into an appealing one (Giovannoni, 2001). These alterations affect many physical parameters, such as colour, flavour, and texture of the fruit (Seymour et al., 1993). The plant hormone ethylene initiates and sustains a number of these changes in climacteric fruit (Alexander and Grierson, 2002). Although ethylene seems to play only a minor role in the ripening of non-climacteric fruit such as Citrus, both types of fruit share common regulatory cascades (Adams-Phillips et al., 2004). Overall, ripening is suggested to be under developmental control (Giovannoni, 2004; Vrebalov et al., 2002).
The quality of fresh fruit and vegetables during storage is greatly influenced by temperature, relative humidity, and atmospheric composition (oxygen, carbon dioxide, and ethylene) of their environment. Application of low-oxygen atmospheres prevents and/or retards the rate of ripening of fruit, resulting in the prolongation of their commercial life (Kader, 1986). In addition, ultra-low oxygen or anaerobic conditions have recently been applied as new post-harvest quarantine treatments to restrict insect infestations (Shellie et al., 1997; Shellie, 2002). Furthermore, there are a number of standard commercial practices such as wax coatings, packing in plastic liners, etc in which the supply of oxygen to fruit in general may be limited. Despite these commercial applications of low-oxygen regimes, the precise mode of action of low oxygen in fruit tissues and ripening is not well understood. In general, plants have developed strategies to cope with low oxygen concentrations. For example, a rapid decrease in respiration, a drop in the adenylate energy charge, and a co-ordinated down-regulation of the Krebs cycle and glycolysis are the first metabolic responses of tissues to low oxygen concentrations (Geigenberger, 2003; Solomos, 1982). However, the entire process, beginning with the sensing of oxygen levels and including the adaptation to oxygen deficiency, irrespective of plant tissue (root or plant organs) subjected to stress, includes a battery of physiological, biochemical, and molecular mechanisms, which have not been unequivocally defined (Drew, 1997; Fukao and Bailey-Serres, 2004; Geigenberger, 2003; Kanellis, 1994; Solomos and Kanellis, 1997).
Until recently, the general perception of fruit metabolism under low oxygen atmospheres was often limited to the suppression of respiration and to the induction of alcoholic fermentation in combination with the inhibitory effects of low oxygen on ethylene biosynthesis and action (Kanellis, 1994; Solomos and Kanellis, 1997). It must be noted, however, that research on hypoxia/anoxia is primarily focused on elucidating the regulation of expression of hypoxic/anaerobic genes and the molecular basis for the adaptation to the low oxygen stress in root or vegetative tissues (Chang et al., 2000; Geigenberger, 2003; Subbaiah and Sachs, 2003; Fukao and Bailey-Serres, 2004; Bailey-Serres and Chang, 2005). Yet little has been done to clarify these aspects in fruit or other detached plant organs subjected to controlled or modified atmosphere treatments. Within this context, gene expression in roots or cell cultures was modified under low oxygen regimes, and trans-elements and cis-factors have been identified (Olive et al., 1991; Dolferus et al., 1994; Hoeren et al., 1998; Klok et al., 2002; Paul et al., 2004). In addition to known anaerobic polypeptides (ANPs), other hypoxia- or anoxia-induced genes identified consist of transcription factors (de Vetten and Ferl, 1995; Hoeren et al., 1998), signal transduction elements (Baxter-Burrell et al., 2002; Dordas et al., 2003), non-symbiotic haemoglobin (Dordas et al., 2004), ethylene biosynthetic genes (Olson et al., 1995; Vriezen et al., 1999), nitrogen metabolism (Mattana et al., 1994), and cell wall loosening (Saab and Sachs, 1996).
In the past, it has been shown that transferring ripening-initiated avocado fruit to 2.5% oxygen for 6 d suppressed cellulase activity (Kanellis et al., 1989a), as well as the abundance of immunoreactive protein and its mRNA (Kanellis et al., 1989b, c). This treatment also produced an alteration in the profile of avocado total proteins, which involved suppression, enhancement, and induction of new polypeptides and genes (Kanellis et al., 1989a, 1991, 1993). In addition, it has been shown that the range of oxygen levels (2.5–5.5%) which suppressed the induction of ripening enzymes, at the protein and mRNA levels, was similar to those oxygen levels that induced the synthesis of new isoenzymes of alcohol dehydrogenase (Kanellis et al., 1991). Analysis of mRNA populations in preclimacteric avocado fruit revealed that low oxygen levels induced new mRNA species possibly implicated in the adaptive mechanism under low oxygen, suppressed those synthesized de novo, or kept the house-keeping and/or pre-existing mRNAs unchanged, indicating that the low oxygen response is complex and involves more than a simple adaptation in energy metabolism (Loulakakis et al., 2006).
Comprehensive expression analysis is an important tool for isolating differentially expressed and functionally important stress-regulated genes, as well as for revealing the genetic networks in which they take part. This can be achieved by applying different techniques, such as RT-PCR differential display, serial analysis of gene expression (SAGE), subtractive hybridization, and cDNA microarray. RT-PCR differential display (Liang and Pardee, 1992, 1998) has been widely used to isolate genes whose expression profiles have been altered under different abiotic or biotic cues because of its technical simplicity and lack of requirement for previous genomic information of the species of interest (Kuno et al., 2000; Carginale et al., 2004; Basse, 2005; Lang et al., 2005).
We are interested in understanding the molecular basis for the adaptation of fruit tissues to low oxygen treatments. RT-PCR differential display was used to identify low oxygen-responsive transcripts from orange fruit tissues following treatments with low oxygen levels. Citrus fruit flavedo (the outer coloured part of the fruit peel) tissues were chosen to avoid gas exchange or oxygen gradient problems. In addition, orange is a non-climacteric fruit, and thus a suitable fruit tissue for isolating hypoxia/anoxia responsive genes that lack the ethylene involvement. This approach led to the identification of novel genes that are up- or down-regulated in response to low oxygen regimes.
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Materials and methods |
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Fruit material and gas treatments
Ripe Citrus sinensis L. Osbeck (cv. Navel) fruit were harvested from an orchard in the southern part of Greece and were transported to the laboratory where they were sorted by colour and size. Fruits were placed in air-tight 50 l containers and were subjected to various oxygen concentrations (0, 0.5, 3, and 21% O2) with a constant gas flow rate of 600 ml min–1 for 24 h at 22 °C. A different set of fruits was used for the time-course experiment, under the same conditions. Samples were collected after 1, 3, 6, 12, 24, and 72 h. For heat and cold stress, fruit were incubated for 0, 6, 12, or 24 h, at 40 °C or 4 °C, respectively. For wounding treatment, the fruit's flavedo was cut into uniform stripes using a sharp knife and sampled after 0, 6, 12, or 24 h, while for ethylene-treated samples, fruits were kept for 0, 6, 12, or 24 h in ethrel (600 ppm) at 20 °C.
All experiments were performed in triplicate, and 15 oranges were used for each sample. The flavedo was collected from each fruit and frozen directly in liquid N2. Fruit tissues were stored in –80 °C.
RT-PCR differential display (DD)
A modified protocol established by Sokolov and Prockop (1994) was used to perform RT-PCR differential display techniques. In addition, the DD PROFILETM kit (Display Systems Biotech Inc, Vista, CA, USA) was used. The DD method was applied on cDNAs synthesized from Citrus flavedo total RNAs. Ten µg of total RNA, treated with DNase, were used for the cDNA synthesis. Reverse transcription was performed using AMV-Reverse transcriptase (Promega GmbH, Mannheim, Germany), or M-MuLV (New England Biolabs, Hertfordshire, UK) or Omniscript (Qiagen, Hilden, Germany) and random hexamers (Amersham Pharmacia Biotech AB, Buckinghamshire, UK) as primers. The reaction volume was 25 µl and 2 µl of the synthesized cDNA was used as template for the subsequent PCR reactions. For the PCR reactions, 2 µM dNTPs, 200 nM primers, 1.5 mM Mg, and 1 unit of Taq DNA polymerase (Display Systems Biotech Inc, Vista, CA, USA) were mixed with 0.5 µl of 35S-dATP (Amersham Pharmacia Biotech AB, Buckinghamshire, UK). 5 µl from each reaction were analysed in a 6% acrylamide gel. The gel was dried and exposed on X-AR autoradiographic (Kodak) film at room temperature. After autoradiography the appropriate bands were isolated from the gel and reamplified by PCR, under the same conditions, but without radiolabelled dATP. The primers used for PCR amplification (BS57, GGAAGCAGCT; BS58, CAGTGAGCGT; BS52, CAAGCGAGGT; BS54, AACGCGCAAC; H4, GTCAGGCCAC) were decameres (GC content higher than 60%), without stop codons in their sequences.
The amplified bands were fragmented on 2% agarose gels, purified using the Qiaquick gel extraction kit (Qiagen, Hilden, Germany) and cloned in pGEM-T Easy vector (Promega GmbH, Mannheim, Germany). These cloned bands, upon sequencing, were also used as probes in the RNA blot analysis. Probes were synthesized with the ‘RadPrime DNA labeling system’ (Invitrogen, Life Technologies Carlsbad, CA).
Reverse northern
In order to screen multiple clones isolated from differential display gels, reverse northern analysis was essentially performed according to Zegzouti et al. (1997). The clones showing differential expression were blotted on membranes and hybridized either to total cDNA probes made from orange flavedo fruit held in 0% O2 for 24 h or to total cDNA probes from air-treated fruit. Equal counts from each labelled cDNA preparation were used for hybridization. After autoradiography the intensity of each band was measured using the Kodak 1D image analysis software and expressed as Net Intensity (NI) and Relative Intensity (RI). NI refers to the net values given by the Kodak 1D image analysis software, whereas RI is the ratio of the values in different oxygen levels to the values in air.
RNA extraction and blot analysis
Frozen fruit flavedo tissue, 10 g, was ground in liquid nitrogen to a fine powder and total RNA was extracted as described by Griffiths et al. (1999). For blot hybridizations, 10–20 µg total RNAs were separated electrophoretically on denaturing formaldehyde agarose gels and blotted onto Nylon membranes according to the manufacturer's instructions (Schleicher and Schuell, GmbH, Dassel, Germany). [
32P]dCTP-labelled probes were prepared using the ‘RadPrime DNA labeling system’ (Invitrogen, Life Technologies Carlsbad, CA). The membranes were hybridized as described by Church and Gilbert (1984). After hybridization and autoradiography, membranes were stripped using boiling 0.1% SDS and used for further hybridizations.
Sequencing
Plasmid DNA was sequenced using the SequiTherm EXCELTM II DNA sequencing kit (Epicentre, Madison, Wisconsin) and fluorescence-labelled primers (MWG, Germany). Sequencing reactions were analysed in a DNA Sequencer Long ReadIR 4200 IR2 (Li-Cor, USA).
Full-length isolation (5' and 3' RACE)
To obtain the full-length sequence of four selected DD fragments, 5' and 3' RACE experiments were performed, using the 5' RACE System for Rapid Amplification of cDNA Ends, Version 2.0 (Invitrogen, Life Technologies Carlsbad, CA). Gene specific primers were designed based on the sequence information obtained from the DD fragments, for each of the four transcripts (Table 1). These primers were used for first strand cDNA synthesis from RNA samples isolated from Citrus fruit subjected to 0% O2 for 24 h. The resulting PCR fragments were cloned in pGEM-T Easy vector (Promega GmbH, Mannheim, Germany) and individual clones were sequenced.
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For the 3' RACE experiments, first strand cDNA from 0% O2 24 h RNA samples was synthesized using an oligodT primer (5'-GGCCACGCGTCGACTAGTA(T)17-3') and M-MuLV Reverse Transcriptase (New England Biolabs, Hertfordshire, UK). Subsequent PCR amplification was performed using the AUAP primer (5'-GGCCACGCGTCGACTAGTAC-3') and gene-specific primers. The resulting PCR fragments were cloned as above and individual clones were sequenced. The primers used for the RACE experiments are summarized in Table 1.
Anaerobic response elements
DD clones that revealed increased levels of transcript during hypoxic and/or anoxic conditions were searched with BLASTX algorithm against the A. thaliana proteome (http://mips.gsf.de/proj/plant/jsf/athal/searchjsp/searchSequence.jsp). The promoters of the genes that exhibited significant similarity to the DD clones sequence were extracted from the Atcis database (http://arabidopsis.med.ohio-state.edu/AtcisDB/). It is clear that the promoter data for Arabidopsis Atcis database are predicted. Frequencies of 6-mer ‘words’ in our query set of sequences (on both strands) were compared with the frequencies of the words in the current genomic sequence set of 1000 bp long upstream regions using the TAIR motif finder program (http://www.arabidopsis.org/tools/bulk/motiffinder/index.jsp). The presence of already known anaerobic response elements (GT-motif, G-box like, pARE-1, pARE-2, pARE-3, and SURE-a-like motif, a sugar-responsive element that has been found to be over represented in hypoxia-induced plant promoters) (Liu et al., 2005; Mohanty et al., 2005) was manually examined in the above motifs.
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Results |
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Isolation of low oxygen regulated genes
The use of the differential display method (DD) has led to the isolation of a number of genes from orange flavedo fruit tissues exhibiting over-expression, suppression, or constitutive expression during hypoxic or anoxic treatments. Two DD approaches were performed to isolate low oxygen regulated genes: (i) cDNA was synthesized from Citrus fruit total RNAs using random hexamers. DNA fragments were amplified by PCR reactions using decamers as primers and the cDNA described above as template in order to isolate putative differentially expressed genes, and (ii) the DD PROFILETM kit (Display Systems Biotech Inc, Vista, CA, USA). The application of both approaches resulted in the isolation of a total of 34 bands from various DD gels: 26 and eight bands seemed to be induced or suppressed by low oxygen, respectively. Following successful re-amplification by PCR using the corresponding primer sets and separation in agarose gels, the above 34 PCR bands were cloned. Sequence analysis of five individual clones from each DD band revealed that a number of the isolated 34 bands resulted in more than one cloned DNA fragment, due to co-migration of these DNA fragments during electrophoresis in the DD gel. Within this context, 98 clones were produced originating from the 34 PCR bands. To confirm that this pattern of DD expression represented true differences in gene expression and not artefacts due to the PCR amplification process, these 98 clones were used for reverse northern (RN) analysis using as probes cDNAs produced from total RNA isolated from air and hypoxic flavedo tissues (data not shown). Reverse northern results gave the first estimation of the expression levels of the spotted genes in hypoxia- or anoxia-treated fruits. To verify the results of reverse northern analysis, a total of 25 individual clones, out of the 98 produced, were used as probes in the RNA blot analysis of Citrus fruit subjected to hypoxia/anoxia. The 25 clones were selected on the basis of the following criteria: (i) the intensity of the spots of the individual clones in the reverse northern; (ii) the correlation between the intensity of the clones in the reverse northern analysis and in the original DD gel; (iii) the correlation between the size of the cloned fragments and the estimated size of the original differential display bands in the DD gels; and (iv) the relative abundance of individual clones after cloning the DD band. The nucleotide sequences of selected clones have been deposited in GenBank, and their accession numbers along with the results of the BLASTX search are shown in Table 2.
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Verification of the DD cDNAs expression by RNA blot analysis
Hypoxia or anoxia induced a variety of genes
Firstly, the 25 selected clones were assessed for low oxygen regulation by RNA blots using total RNA isolated from flavedo tissues of orange fruit subjected to 0, 0.5, 3, and 21% O2 for 24 h. Based on the low oxygen response (Fig. 1), these clones were clustered in three groups according to their expression profiles during a 24 h treatment (Table 2). Group I, consisting of 11 genes, showed an increase in their mRNA accumulation; group II, with 11 genes (only three genes are depicted in Fig. 1), exhibited constitutive expression; and group III, with three genes, displayed decreased gene expression (Table 2; Fig. 1). Based on this expression pattern, 11 out of the 25 clones can be characterized as hypoxic and/or anoxic genes and three out of 11 can be referred to as only anoxic ones (Fig. 1).
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20 µg) was fractionated in 1% agarose-formaldehyde gel, blotted to Hybond N-membranes and probed with 32P-labelled low oxygen regulated genes. Equal loading, integrity, and transfer were assessed by methylene blue staining of ribosomal RNA (25s rRNA band is shown).All 11 low-oxygen induced genes verified by RNA blot analysis showed similarities with a variety of metabolic enzymes. Some of these clones were further analysed by isolating the full-length cDNAs of 15d, 26b, and 32a using 5' and 3' RACE techniques (Tables 1, 2) in order to elicit more information on nucleotide sequence. Clone 15d showed 86% identities to pyruvate decarboxylase. The isolation of this known hypoxic/anoxic gene provides evidence that our experimental conditions provoked the anaerobic and hypoxic response to Citrus flavedo tissues. The rest of the low-oxygen induced genes were found to show similarity to varying degrees with different genes; for example, clone 3c showed low similarity (47% identities) with an auxin-induced protein-like from Oryza sativa (japonica cultivar-group); clone 3e exhibited similarities (86% identities) with a Phaseolus vulgaris putative phosphatase; clone 6c similarity (60% identities) with A. thaliana putative serine esterase; clone 11c similarity (98% identities) with limonoid UDP-glucosyltransferase; clone 18d 81% identities with Medicago truncatula, hypoxia-responsive family protein; clone 25e 91% identities with Medicago truncatula xylose isomerase family protein; clone 26b exhibited similarity (88% identities) with glutamate decarboxylase; clone 27a was similar to A. thaliana SPX (SYG1/Pho81/XPR1) domain-containing protein; clone 28c exhibited no significant similarities with known sequences deposited in the gene banks; and 32a displayed 82% identity with A. thaliana cytochrome P450 (Table 2). It is interesting to note that a number of genes not previously reported to be inducible by hypoxia/anoxia were identified in the present study.
DD clones constitutively-expressed in hypoxia or anoxia
For simplicity, Fig. 1 depicts three out of 11 clones that can be characterized as constitutive, since neither hypoxia nor anoxia influenced their RNA accumulation pattern during a 24 h holding period in low O2 environment. These clones showed similarities with the following (Table 2): a protein of unknown function expressed in Arabidopsis thaliana (clone 1c); unknown sequence (clone 10a); 26S ribosomal RNA (clone 12b); Vigna radiata calmodulin (clone 12d); Nicotiana tabacum hsp70 protein (clone 15b); Citrus sinensis elongation factor 1 (clone 20d); putative Arabidopsis thaliana DegP protease, (clone 21d); Citrus sinensis similar to fimbriata-associated protein (clone 22a); Nicotiana tomentosiformis mitochondrial ATPase (clone 22e), a protein detected under disease stress; Veronica incana F1-ATPase alpha subunit (atp1) (clone 24b); and Citrus sinensis ClpP protease (ClpP) proteolytic subunit (clone 28b) (Table 2).
DD clones suppressed by low O2
Three clones were suppressed by a 24 h exposure to low O2 levels (Fig. 1). These clones showed similarities with Medicago truncatula, mono-oxygenase family protein (clone 11b); Citrusxparadisi, dehydrin (ERD10) (clone 13d); and with Phaseolus vulgaris, pvgbss1b for granule-bound starch synthase (clone 33i).
Other DD clones
Eleven clones showed very low or no hybridization signal even after an over-exposure (data not shown). These clones were the following: 3d (similar to Arabidopsis CYP94C1, haem binding/iron ion binding/mono-oxygenase/oxygen binding), 23b (similar to auxin:hydrogen symporter, Arabidopsis thaliana), 31d (similar to 10-formyltetrahydrofolate synthase), and clones 4a, 7a, 12a, 22c, 25a, 27b, 29c, 32c that exhibited no significant similarity with deposited sequences.
Time-course of mRNA accumulation of low O2-responsive genes
Time-course experiments of mRNA accumulation of low O2-responsive genes were performed in Citrus fruit that were held in 0, 0.5, 3, and 21% O2 for 0, 1, 3, 6, 12, 24, and 72 h. Total RNA was isolated from Citrus flavedo tissues and subjected to RNA blot analysis (Figs 2, 3, 4).
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Time-course of mRNA accumulation of low O2-induced DD clones
Figure 2 demonstrates that low O2-induced genes responded differently in terms of the timing of their induction and their hybridization signal intensity during the 72 h holding period in low O2 regimes. For example, transcript accumulation of the clones showing homology with auxin-induced protein-like from Oryza sativa (clone 3c), pyruvate decarboxylase (clone 15d), limonoid UDP-glucosyltransferase (clone 11c), and hypoxia-responsive family protein (clone 18d) could be detected as early as 1 h after imposition of low O2 stress and especially in 3% or 0.5% O2. These four messages seem to be induced mainly by hypoxia, thus they can be termed hypoxic genes. Clones showing homology with a Phaseolus vulgaris putative phosphatase (clone 3e), with xylose isomerase (clone 25e), with glutamate decarboxylase (clone 26b), similarity to A. thaliana SPX (SYG1/Pho81/XPR1) domain-containing protein (clone 27a), no similarities with known sequences (clone 28c), and similarity with cytochrome P450 (clone 32a), were mainly expressed after 12 h in 0.5% or 0% O2 and thus can be characterized as anoxic genes.
Time-course of transcript levels of constitutively expressed DD clones under hypoxia and/or anoxia
A number of DD clones were not affected by low O2 levels after a 24 h holding period. However, a question arose whether a longer period in low O2 was required for the induction of these genes. Low O2 (0, 0.5, 3%) for 72 h failed to affect the gene expression of the 11 constitutively expressed DD clones (data not shown). In Fig. 3 only three clones, i.e. 10a, 24b, and 28b are presented.
Time-course of mRNA levels of low O2-suppressed DD clones
Next, the time-course of gene suppression of DD clones exhibiting a decrease in RNA accumulation in fruit held in low O2 for 24 h were studied (Fig. 4). Clones showing homologies with a Medicago truncatula aromatic ring hydroxylase (clone 11b), dehydrin (ERD10-clone 13d) (Porat et al., 2004) and with Phaseolus vulgaris mRNA for granule-bound starch synthase (clone 33i) were suppressed by hypoxia and/or anoxia; however, the effectiveness of this suppression was evident after 12 h in low O2 (Fig. 4; Porat et al., 2004, for dehydrin).
Stress regulation of low O2-responsive genes
In order to investigate whether the low O2-responsive DD clones were also regulated by other stresses or hormones, gene expression of selected clones was studied in Citrus fruit held in 4 °C, 40 °C, ethrel (ethylene) and wounded for 0, 6, 12, and 24 h.
Stress regulation of low O2-induced genes
Figure 5 demonstrates that among low O2-induced genes, i.e. 3e, 15d, 18d, 26b, 27a, 28c, and 32a, the clones showing similarities with glutamate decarboxylase (26b), no significant similarity with known sequences (28c), and similarity with cytochrome P450 (32a), seem to be mainly regulated by low O2. The present data suggest that the above clones may participate in the adaptation mechanism of Citrus fruit specifically to low O2 stress. Putative phosphatase-clone 3e seems to be induced by low temperature (4 °C), wounding, and high temperature (40 °C) after 6 h and transiently by ethrel after 6 h. Pyruvate decarboxylase (15d) was induced by 40 °C after 24 h, wounding after 6 h, and transiently by ethrel after 12 h. Hypoxia-responsive family protein (18d) seems to be induced by all stresses and ethylene. SPX (SYG1/Pho81/XPR1) domain-containing protein (27a) is induced by low temperature, wounding, and ethylene. This suggests that these four low O2-induced genes are not specific to low O2 stress, but rather they may participate in the adaptation process of Citrus tissues to a number of stresses.
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In silico promoter analysis for common regulatory motifs
Promoter regions of genes that respond similarly to specific stresses may share common regulatory elements. 5' up-stream region analysis may provide indirect evidence for the identification of unknown genes or of those that help others to be clustered in groups of common function. Because of the limited information on the Citrus genome, an in silico promoter analysis was undertaken of the corresponding homologous Arabidopsis hypoxic/anaerobic genes identified in this study and common DNA regulatory motifs located in their 5' regions were sought. The search was conducted at the Arabidopsis thaliana proteome (see Materials and methods) and the corresponding Arabidopsis gene promoters were studied for the presence of already known anaerobic response elements (GT-motif, G-box like, pARE-1, pARE-2, pARE-3) and SURE-a-like motif, a sugar-responsive element that has been found to be over-represented in hypoxia-induced plant promoters (Liu et al., 2005; Mohanty et al., 2005) (Table 3). Up to 1000 bp immediately upstream of the ATG of each Arabidopsis homologue of the differentially expressed genes was retrieved and analysed for over-represented 6-mer motifs in both strands (Table 3). The 5' region of the known anaerobic gene, PDC, shares both the GT-motif and the G-box-like motif, whereas others, like the Arabidopsis gene homologous to 3c, contain all four known anaerobic motifs (Table 3). GT-motif (AAACC) is present in the 5' up-stream regions in eight out of 11 genes showing anaerobic induction, suggesting a common regulatory function. However, surprisingly, none of the known anaerobic response elements analysed in this study is present in 6c, 11c, and 28c homologues, suggesting that other, not yet identified anaerobic motifs may be present in these promoters or that the specific genes in Arabidopsis are not affected by low oxygen conditions.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Data on low oxygen-responsive genes isolated from fruit tissues is limited. Our previous studies revealed that the action of low oxygen on fruit ripening is intricate, involving suppression of the de novo synthesized ripening proteins and mRNA transcripts, induction of new proteins and mRNA species, and constitutive expression of the house-keeping and/or pre-existing proteins or mRNA species (Kanellis et al., 1989a, b, 1991, 1993; Loulakakis et al., 2006). In the present study, RT-PCR differential display (Liang and Pardee, 1992, 1998) in Citrus fruit was used to elucidate the molecular mechanisms governing the response of fruit tissues to short-term low oxygen stress. This approach led to the isolation, successful cloning, sequencing, and bioinformatic analysis of 98 transcripts that were differentially expressed in DD gels in response to hypoxia and anoxia in Citrus flavedo tissues. RNA blot analysis on 25 DD clones revealed that 11 genes were induced under hypoxia and/or anoxia, 11 exhibited constitutive expression and three transcripts were suppressed by low oxygen regimes (Figs 1, 2, 3, 4
). Only two genes, PDC (15d) and GAD (26b), participating in fermentation and GABA biosynthesis, respectively, overlapped with known hypoxic/anoxic responsive genes shown to be induced in root or vegetative tissues of Arabidopsis or other plant species (Klok et al., 2002; Agarwal and Grover, 2005; Branco-Price et al., 2005; Fenglong et al., 2005; Liu et al., 2005; Loreti et al., 2005), suggesting common hypoxia/anoxia regulatory mechanisms well conserved among different plant tissues and species, at least for these two genes. On the other hand, it is known that shoots and roots even of the same plant respond differently to hypoxia/anoxia (Ellis et al., 1999). On this evidence, Citrus flavedo tissues (this study) may be expected to react to low oxygen stress by expressing a different set of genes, implying distinctive mechanisms of adaptation to this stress. As encapsulated in Table 2, half of the isolated low oxygen-induced cDNAs are either of unknown function in terms of their involvement in hypoxia/anoxia or they share no apparent homology to any expressed sequences in the GenBank/EMBL databases (3c, 6c, 18d, 27a, and 28C). The other six DD genes are similar to molecules of the following functions: C-compound and carbohydrate utilization (11c, 15d, and 25e), plant development (3e), amino acid metabolism (26b), and biosynthesis of brasinosteroids (32a). Of the novel genes identified, 3c (an auxin-induced protein-like), 3e (a putative phosphatase), 6c (a putative serine esterase), 18d (a hypoxia-responsive family protein), 25e (a xylose isomerase family protein), 27a [a SPX (SYG1/Pho81/XPR1) domain-containing protein], 28c (no significant similarity with known sequences), and 32a (a P450, member of the CYP90A family participating in the brassinolide biosynthesis) were shown for the first time to be induced by hypoxia/anoxia. It is not known whether the induction of these genes is specific to Citrus flavedo tissues, or common to other plant tissues, since bio-informatic analysis revealed no similarity with the recently identified low oxygen regulated genes in roots or vegetative tissues (Klok et al., 2002; Agarwal and Grover, 2005; Branco-Price et al., 2005; Fenglong et al., 2005; Liu et al., 2005; Loreti et al., 2005). The differences observed in transcript profiles between the genes identified in the present study and the above reports might be ascribed to the difference in tissues used (Citrus flavedo versus roots and vegetative tissues) and in techniques applied (DD versus DNA microarrays).
Among these genes, 28c and 32a seem to be induced only by anoxia, as shown by the absence of any observable alteration in their gene expression profile in response to low and high temperature, wounding, and ethylene (Fig. 5). The 28c clone displayed no significant similarity with deposited sequences in the gene banks. It showed 100% identity with a cDNA clone isolated from Star Ruby grapefruit temperature-conditioned flavedo (http://www.ncbi.nlm.nih.gov). It is not known how these two proteins participate in the adaptation of Citrus fruit to hypoxia/anoxia. 32a showed a high degree of similarities with a member of the CP90A family, a cytochrome P450 mono-oxygenase which converts 6-deoxocathasterone to 6-deoxoteasterone in the late C6 oxidation pathway and cathasterone to teasterone in the early C6 oxidation pathway of brassinolide biosynthesis. Mutants display de-etiolation and derepression of light-induced genes in the dark, dwarfism, male sterility, and activation of stress-regulated genes in the light. This is the first demonstration of this P450 gene response in anoxia. It is interesting to note that the Arabidopsis plants grown at 2.5 kPa O2 look like det2 (Li et al., 1996) and cpd (Szekeres et al., 1996) mutants, which are deficient in the brassinosteroid biosynthetic pathway (Ramonell et al., 2001). The det2 mutant showed phenotypic characteristics that look like a wild-type plant grown in hypoxia: it exhibited thicker leaves than the wild type, was insensitive to the dwarfing effects of low oxygen and displayed male sterility. These authors observed that by adding brassinolide to the wild-type plants in hypoxia they were able to alleviate the hypoxic symptoms, resulting in leaf thickness close to normal. The authors have pinpointed brassinolides as possible mediators of distinct aspects of the developmental responses to hypoxia/anoxia, an inference sustained by the fact that brassinolides require molecular oxygen at several steps in their biosynthesis.
Clone 3c codes for an auxin responsive protein. This family consists of the protein products of the ARG7 auxin responsive genes, none of which have any identified functional role. In a recent study, the induction of a number of auxin-responsive genes (At3g23030 [IAA2], At5g19140, and At1g19840) was recorded, while a gene encoding an auxin carrier protein (At2g17500) was repressed by low-oxygen stress (Liu et al., 2005). Contrary to the present study and to that of Liu et al. (2005), Loretti et al. (2005) found that a number of auxin responsive genes were suppressed by hypoxia with the addition of sucrose to alleviate the negative effect of hypoxia. In the hormonal regulation of hypoxia/anoxia, only the involvement of ethylene as a signalling molecule has been documented (Drew, 1997; He et al., 1996). However the available information is inadequate to propose the participation of auxin in hypoxic/anoxic tolerance (Liu et al., 2005).
Clone 3e, encoding a putative phosphatase, exhibited a high degree of similarities to nodulin PvNOD33, whose expression is induced during Phaseolus vulgaris nodule development (Roussis et al., 2003). In silico analysis of EST databases indicated that sequences highly homologous to the PvNOD33 putative polypeptide are expressed in different legume (soybean and Medicago truncatula) and non-legume (tomato, A. thaliana, V. vinifera, and Zea mays) plant species. V. vinifera GRIP31 and Z. mays ZSS3 are the only plant-characterized PvNOD33 homologues. Their pattern of expression showed that the GRIP31 increased in vine ripening berries (Davies and Robinson, 2000), while the ZSS3 augmented during glucose starvation (Chevalier et al., 1995). However, the function of these proteins is far from clear. The observation that the expression of PvNOD33 was highly correlated with the consumption of large amounts of sucrose in nodules (Roussis et al., 2003) in order to supply carbon skeletons for ammonia assimilation, cellulose and starch biosynthesis, and for the energy demands of both the plant cell and the bacteroids, suggests a possible role of 3e in carbon metabolism during hypoxia/anoxia in Citrus flavedo tissue.
6c shows similarity with a putative serine esterase with unknown function. These proteins contain a serine esterase catalytic motif (GHSMGG) and exhibit a variety of catalytic functions. It is not known how this type of enzyme participates in the anoxia/hypoxia stress.
The nucleotide sequence of the 11c clone corresponded to a limonoid UDP-glucosyltransferase gene recently isolated from Citrus fruit (Kita et al., 2000). This protein regulates the conversion of limonoid aglycones to glucosides in Citrus fruit, thus controlling the removal of bitterness from orange fruit. However, it is not known how this gene may participate in the adaptation to low oxygen.
Clone 18d exhibited homology with a family of proteins known to be involved in the response to hypoxia. Members of this family mostly come from diverse eukaryotic organisms; however, eubacterial members have been isolated as well. This region was found at the N-terminus of the member proteins which are predicted to be transmembrane (Gracey et al., 2001).
25e showed similarities with xylose isomerase, an enzyme which is found in micro-organisms and plants (Kristo et al., 1996), and which catalyses the interconversion of D-xylose to D-xylulose. It can also isomerize D-ribose to D-ribulose and D-glucose to D-fructose. It may be inferred from this that xylose isomerase may contribute to the mobilization and channelling of sugars to glycolysis to sustain fermentation for the production of ATP.
The 26b clone codes for glutamate decarboxylase (GAD), participating in the biosynthesis of 
-aminobutyric acid (GABA). Until recently, it was thought that GABA levels were controlled by the activity of GAD which increased in response to various stimuli, such as low oxygen, heat shock, wounding, phytohormones (Bown and Shelp, 1997; Kinnersley and Turano, 2000; Bouche and Fromm, 2004) and low temperature (Fig. 5). This is accomplished by increases in cytosolic H+ or calcium/calmodulin (CaM) concentrations that directly affect the activity of the enzyme. However, this study and that by Klock et al. (2002) showed that gene induction may also take part in the increases in GAD activity in response to anoxia, implying that fruit tissues have developed mechanisms of adaptation to low oxygen stress similar to those of other plant tissues. In addition, Citrus GAD gene expression (this study) seems to be induced only by anoxia and not by other stresses (Fig. 5).
Clone 27a exhibited similarities with a ‘SPX domain-containing protein’, which is named after SYG1/Pho81/XPR1 proteins. This 180 residue length domain was found at the amino terminus of a variety of proteins. In the yeast protein SYG1, the N-terminus directly binds to the G-protein ß-subunit and inhibits transduction of the mating pheromone signal, suggesting that all the members of this family are involved in G-protein associated signal transduction (Spain et al., 1995; Battini et al., 1999). The N-termini of several proteins involved in the regulation of phosphate transport, including the putative phosphate level sensors PHO81 from Saccharomyces cerevisiae and NUC-2 from Neurospora crassa, are also members of this family (Lenburg and O'Shea, 1996). Several members are annotated as XPR1 proteins. The similarity between SYG1, phosphate regulators, and XPR1 sequences has been previously noted, as has the additional similarity to several predicted proteins of unknown function from Drosophila melanogaster, Arabidopsis thaliana, Caenorhabditis elegans, Schizosaccharomyces pombe, and Saccharomyces cerevisiae (Battini et al., 1999; Tailor et al., 1999). In addition, given the similarities between XPR1 and SYG1 and phosphate regulatory proteins, it has been proposed that XPR1 might be involved in G-protein associated signal transduction, and may itself function as a phosphate sensor (Battini et al., 1999).
In the course of this study, a number of genes were shown to exhibit suppressed expression in low oxygen regimes (Table 2; Figs 1, 4). Among them, clone 11b showed homology with a Medicago truncatula peptidase having a cysteine peptidase active site with aromatic-ring hydroxylase. This mono-oxygenase belongs to a family which includes diverse enzymes that utilize FAD. Clone 13d codes for a Citrus sinensis dehydrin (ERD10) (Porat et al., 2004), which might protect the fruit from chilling temperatures. Dehydrins, in general, may participate in the stabilization of membrane structures under stress conditions (Koag et al., 2003). Clone 33i shows similarities with proteins that transfer UDP, ADP, GDP, or CMP linked sugars. However, the physiological significance of these suppressed genes remains to be examined.
In silico analysis revealed that established regulatory elements of known promoters (Liu et al., 2005; Mohanty et al., 2005) are present in the 5' motifs of most of the corresponding homologues of the Arabidopsis hypoxic/anaerobic genes identified in this study, indicating control by the same set of transcription factors. However, these elements should be isolated and functionally characterized from the Citrus anaerobic genes in order to pinpoint common anaerobic regulatory elements within plant species.
Most of the genes isolated with mRNA DD in Citrus flavedo differ from the genes identified using other techniques in various species (Klok et al., 2002; Agarwal and Grover, 2005; Branco-Price et al., 2005; Fenglong et al., 2005; Liu et al., 2005; Loreti et al., 2005) indicating that the different techniques can supplement each other. Further, the present work provides new insight into functions and processes that were not previously connected with anoxia/hypoxia. More detailed characterization needs to be performed for some genes showing promising, novel, but unidentified functions in hypoxia/anoxia. For example, functional analysis of the cytochrome P450, auxin-induced protein-like, putative phosphatase, serine esterase, xylose isomerase, and SPX (SYG1/Pho81/XPR1) domain-containing protein may shed light on the understanding of the molecular basis of a plant organ's response to low oxygen.
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Acknowledgements |
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We are grateful to Professor Theo Solomos for critically reading the manuscript. This work was supported by grants from EU (FAIR-CT98-4096) and from General Secretariat for Research and Technology of Greece (GSRT) to AKK.
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Footnotes |
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* These authors contributed equally to this work.
Present address: Institute of Agrobiotechnology, CERTH, 6th km Charilaou-Thermi Road, Thermi GR-570 01, Greece.
On sabbatical leave; present address: Laboratory of Food Processing and Engineering, Division of Food Science and Technology, Department of Agricultural Sciences, Aristotle University of Thessaloniki, 541 24 Thessaloniki, Greece.
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