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A small intergenic region drives exclusive tissue-specific expression of the adjacent genes in Arabidopsis thaliana
© Bondino and Valle; licensee BioMed Central Ltd. 2009
- Received: 8 June 2009
- Accepted: 16 October 2009
- Published: 16 October 2009
Transcription initiation by RNA polymerase II is unidirectional from most genes. In plants, divergent genes, defined as non-overlapping genes organized head-to-head, are highly represented in the Arabidopsis genome. Nevertheless, there is scarce evidence on functional analyses of these intergenic regions. The At5g06290 and At5g06280 loci are head-to-head oriented and encode a chloroplast-located 2-Cys peroxiredoxin B (2CPB) and a protein of unknown function (PUF), respectively. The 2-Cys peroxiredoxins are proteins involved in redox processes, they are part of the plant antioxidant defence and also act as chaperons. In this study, the transcriptional activity of a small intergenic region (351 bp) shared by At5g06290 and At5g06280 in Arabidopsis thaliana was characterized.
Activity of the intergenic region in both orientations was analyzed by driving the β-glucuronidase (GUS) reporter gene during the development and growth of Arabidopsis plants under physiological and stressful conditions. Results have shown that this region drives expression either of 2cpb or puf in photosynthetic or vascular tissues, respectively. GUS expression driven by the promoter in 2cpb orientation was enhanced by heat stress. On the other hand, the promoter in both orientations has shown similar down-regulation of GUS expression under low temperatures and other stress conditions such as mannitol, oxidative stress, or fungal elicitor.
The results from this study account for the first evidence of an intergenic region that, in opposite orientation, directs GUS expression in different spatially-localized Arabidopsis tissues in a mutually exclusive manner. Additionally, this is the first demonstration of a small intergenic region that drives expression of a gene whose product is involved in the chloroplast antioxidant defence such as 2cpb. Furthermore, these results contribute to show that 2cpb is related to the heat stress defensive system in leaves and roots of Arabidopsis thaliana.
- Intergenic Region
- Gene Pair
- Vascular Bundle
- Methyl Viologen
- Bidirectional Promoter
A promoter region of an eukaryotic protein-encoding gene usually consists of a core promoter region of around 50 bp nucleotides adjacent to the transcription initiation site, and multiple distal DNA regulatory elements to control transcription efficiency. There are several key genetic elements within a core promoter: the TATA box, an initiator element, the downstream promoter element usually found in TATA-less promoters, and the TFIIB-recognition element [1, 2]. The TATA boxes are usually located about 25 to 30 bp upstream of the transcription start site (TSS), while the less conserved initiator elements span the TSS. These sequences contribute to an accurate transcription initiation and to the TATA-containing promoters strength. In Arabidopsis core promoters, the TATA box is located between -50 and -20 relative to the TSS and, instead of the initiator element around the TSS, the YR rule (Y: C or T; R: A or G) applies to most of them. Another element is the pyrimidine patch (Y Patch), although its role is still unknown. These three elements are orientation-sensitive . Other promoter elements found in Arabidopsis and rice are regulatory element groups (REGs), which appear upstream of the TATA box (-20 to -400), and exist in an orientation-insensitive manner .
Divergent genes, defined as non-overlapping genes organized head-to-head in opposite orientation, represent a 36.5% of the total gene pairs when separated by less than 1 kb in the Arabidopsis genome . Nevertheless, there is scarce evidence on functional analyses of the intergenic regions between those gene pairs. Previous findings of head-to-head oriented genes sharing an intergenic region with putative bidirectional promoters were reported in Brassica napus , Capsicum annuum , and by computational analysis in rice, Arabidopsis, and black cottonwood . Large-scale studies of expression data in Arabidopsis revealed that neighbouring genes in the genome are co-expressed , and that the lengths of the intergenic sequences have opposite effects on the ability of a gene to be epigenetically regulated for differential expression . Two recent papers have shown activity of larger intergenic regions in rice (1.8 kbp) and Arabidopsis (2.1 kbp), functioning as bidirectional promoters of chymotrypsin protease inhibitor  and chlorophyll a/b-binding protein  genes, respectively. These systems were assessed in a heterologous background using onion epidermal cells , and also in stable transgenic plants, the latter intended to be used for genetic engineering-based crop improvement .
All divergent gene pairs are potential sources of bidirectional promoters. To define the function of the corresponding intergenic regions and their transcriptional regulation is of great interest for plant molecular biologists.
In this study, a divergent promoter of a protein-encoding gene pair (At5g06290 and At5g06280) with an intergenic region of 351 bp was analyzed. The At5g06290 and At5g06280 loci encode a 2-Cys peroxiredoxin B (2CPB), which are a chloroplast-located protein , and a protein of unknown function (PUF), respectively http://www.arabidopsis.org. The 2-Cys peroxiredoxins are proteins involved in redox processes, and their functions are related to the antioxidant defence of the plant , photosynthesis, abiotic stress response, and possibly chloroplast-to-cytosol signalling . In yeast, peroxiredoxins could act as molecular chaperons, increasing resistance to heat stress . The expression pattern of the At5g06290 and At5g06280 was tested by fusing the intergenic region in opposite orientation to β-glucuronidase (GUS) reporter gene during the development and growth of Arabidopsis plants as well as during stress situations.
Functional analysis of the intergenic region between At5g06280 and At5g06290 in Arabidopsis plants during their development and growth
As 2CPB is a chloroplastic protein , we analyzed the putative intracellular location of PUF using ChloroP 1.1 Server  and the deduced amino acid sequence of At5g06280. The prediction results have shown that PUF (156 residues) is likely to be a plastidic protein, because it has an amino-terminal extension indicative of chloroplast transit peptide (score 0.506). For comparison, 2CPB score was 0.598 using this web tool.
Response of Prom280:GUS and Prom290:GUS plants to various stresses
To confirm the effect of heat treatment on the induction of 2CPB, 10-day old wild-type Arabidopsis plants were submitted for 2 days at 37°C, and the total protein of leaves and roots were extracted and analyzed by SDS-PAGE and immunoblotting. Results are presented in Additional file 1. The total protein pattern has shown slight differences between control and treated plants in the leaf or root tissues, especially in higher molecular masses larger than 66 kDa. Immunoblot analysis of these tissues has shown induction of 2CPB in both leaves and roots after heat treatment (Additional file 1, bottom panel). These data indicate that heat treatment was able to increase not only 2CPB protein level in root and leaf of wild-type plants (Additional file 1), but also GUS activity in the same tissues as observed in Prom290:GUS plants (Figures 2D and 2J).
These results suggest that puf and 2cpb are stress-responsive genes, although they are not always affected in the same way by the same stress conditions.
In search of cis-elements in the promoter of puf and 2cpb
Distribution of distances between genes and their nearest neighbours in Arabidopsis genome
With the availability of complete genome sequences for a number of organisms, functionality of intergenic regions has attracted more attention. Computational analysis has shown that divergent gene pairs with intergenic regions less than 1 kb are quite abundant in the sequenced eukaryotic genomes of both plants and animals [5, 8]. The interest in studying intergenic region functionality is increasing not only to better understand divergent transcription, but also to use them as a new toolkit to manipulate genomes . In plants, particularly, very few reports about this matter are available. An example of such investigations in plants in which data from computational assistance and bidirectionalization were integrated to construct a synthetic transcriptional unit for high-level reporter-gene expression in response to specific elicitors was reported, thus yielding exciting results . In this study, it has been found that the region shared by two divergent genes in the chromosome 5 of Arabidopsis thaliana (At5g06280 and At5g06290) functions as a promoter in both orientations. In addition, this study was able to demonstrate that tissue and developmental expression patterns differed between puf and 2cpb. Head-to-head genes from other organisms such as human, mouse, and rat genomes statistically tend to perform similar functions, and gene pairs associated with the significant co-functions seem to have stronger expression correlations . In this case, the gene products of At5g06280 and At5g06290 are both presumably located in the chloroplasts, although it is unknown if their functions are related. Thus, it is known that 2CPB is located in the chloroplasts and prevents oxidative damage of chloroplast proteins . The transcript increase of 2cpb was correlated with chlorophyll distribution and also accumulated in plants with decreased catalase activity and upon heat stress . Down-regulation of 2cpb was observed upon pathogen infection, ozone and cold [40, 41]. Instead, the role of PUF remains unknown until today, and presumably it would be a chloroplast-located protein as predicted by ChloroP analysis .
When searching for At5g06280 and At5g06290 potential orthologues, it has been found that this head-to-head gene organization was not conserved among other genomes (data not shown); pointing out that most probably their gene products are not functionally related. In humans, analysis of genome-wide expression data demonstrated that a minority of bidirectional gene pairs are expressed through a mutually exclusive mechanism . In this study, the tissue-specific expression of both genes directed by the divergent promoter has shown unidirectional activity for puf in petiole and vascular bundles and unidirectional activity in the opposite direction in different tissues for 2cpb. The higher expression of 2cpb in the leaf mesophyll, but not in vascular bundles, is coincident with its function in the redox processes of chloroplasts . Taken together, these results suggest that the directionality of the promoter activity may be regulated to some degree in a tissue-specific manner. In fact, a cis-motif associated to vascular bundle expression (AACA)  was found several times in the puf direction of transcription.
Furthermore, it has been demonstrated that the divergent promoter shared by puf and 2cpb responded to temperature stress. In relation to this, the higher 2CPB levels in the leaf and root caused by heat treatment of Arabidopsis seedlings would indicate a role of this protein in temperature stress. In yeast, peroxiredoxins could alternatively function as peroxidases and molecular chaperons, increasing resistance to heat stress . It is well known that exposure of plants to high temperature leads to the production of Hsps. The yeast heat shock factor 1 binding sequence nTTCn (or nGAAn)  was found highly represented in the intergenic region of this study. Therefore, it is tempting to speculate that high temperature could stimulate 2cpb similarly to Hsp genes. Remarkably, the puf expression was repressed similarly to 2cpb by several stress conditions.
In silico analysis of this promoter using ppdb revealed that it is a TATA-less promoter in both orientations. In plant genomes putative bidirectional promoters have TATA boxes underrepresented . A recent study  suggested that TATA box-containing genes have longer intergenic upstream regions and increased variation across species because their upstream regulatory potential is greater and, therefore, more amenable to change and modulation. The TATA box appears to be responsible for promoter unidirectionality in most cases, whereas having no TATA boxes appears to be a novel mechanism of regulation by bidirectional promoters compared to unidirectional promoters. This analysis also revealed that in a short region of this promoter (28 bp) (Figure 5B), four different cis-elements are overlapped. They are: one heat shock element (CCAAT box), a Y Patch found in the majority of Arabidopsis promoters but with unknown function , and three binding sites of homeodomains-leucine zipper transcription factors, some of them being able to bind in both directions [27, 28]. These cis-elements would be leading the transcription of 2cpb, specially ATHB1, which is involved in differentiation of the palisade mesophyll cells, and ATHB5, which in turn is involved in the control of leaf morphology development . Upstream of this region there are three AACA elements in the +/- 25 bp region of puf TSS (Figure 5A). This is a negative regulatory element in vascular promoters, which represses activity in other cell types  suggesting that, in the intergenic region under analysis, this cis-element would be preventing puf transcription in mesophilic cells. The expression of puf in vascular bundle of midribs could be activated by ATHB2, which has a homeodomain too, and by the Y Patch that is located in the 28 bp region above mentioned. The 2cpb and puf putative promoter regions mentioned have an element of response to heat near them, which could explain the heat stress experiments. It was not possible to find any abiotic stress element overrepresented in the 530 bp region analyzed, suggesting that the expression pattern observed in Figure 4 could be the result of the complex interaction of the transcription factors that bind the 28 bp region. Overall, results obtained from this study indicate that the multiple stress responsiveness of the intergenic region would reside within the 351 bp.
When length is considered, the short promoter shared by 2cpb and puf belongs to a minority group of putative bidirectional promoters present in the Arabidopsis genomes. In fact, Arabidopsis genome has a bimodal distribution of distances between the 5'ends of genes on opposite strands, peaking the smaller group of gene pairs at 323 bp. This is the first intergenic region functionally studied of this small group of Arabidopsis promoters. Plants are sessile organisms and, during their growth, they occasionally are affected by adverse environmental conditions; therefore, they may rely more strongly on elaborate transcriptional response programs to survive. Then, it is highly possible that other intergenic regions of similar lengths and regulatory features could be found in plants.
In this report, it has been shown that a 351 bp intergenic region between head-to-head oriented At5g06290 and At5g06280 directs genes expression in different Arabidopsis tissues in a mutually exclusive manner. Gene products of these loci are a chloroplast-located 2-Cys peroxiredoxin B involved in the antioxidant defence, and a protein of unknown function. This is the first report of an intergenic region that drives expression of a gene involved in the chloroplast antioxidant defence. These results also show that 2CPB is induced by heat stress in the leaves and roots, suggesting a function for this protein in the heat stress defensive system of Arabidopsis thaliana.
Plant material and growth conditions
Arabidopsis thaliana ecotype Columbia (Col-7) was synchronously germinated at 4°C for 48 h and grown in soil-vermiculite mixture (2:1 v/v) in growth chambers at 20-22°C, under long day conditions (16 h light/8 h darkness). The light intensity was set at 130 μmol m-2 s-1.
When assaying stress treatments, Arabidopsis plants grown photoautotrophically on agar medium containing 0.5 X Murashige and Skoog (MS) salts (Sigma-Aldrich).
Arabidopsis plants were cultivated on agar supplemented with the stress agent: osmotic stress (100 mM mannitol), salt stress (50 mM NaCl), oxidative stress (0.1 μM methyl viologen) or fungal elicitor (1.3 mg/mL autoclaved cellulase, Onozuka R-10, Yakult Honsha, Tokio, Japan). For cold (4°C) and high (37°C) temperature stresses, the plants were grown for 10 days on MS agar without supplements under control conditions and then the temperature treatment was applied for 2 days. For higher light intensity (800 μmol m-2 s-1), the plants were grown for 10 days and the treatment was applied for 6 h.
The intergenic region with the 5'UTR regions of the genes At5g06280 and At5g06290 was isolated by PCR from an A. thaliana DNA CTAB preparation  using the primers 5'-CGCGGATCC AGTCTTTCTTCTTCTTTTTTTTTG-3' and 5'-CGCGGATCC TGACTCTGTTCTCTCTCTCTATC-3' (added Bam HI restriction site in bold). The PCR product was subcloned into pGEM-T Easy Vector (Promega, Madison, USA). DNA sequencing was used to confirm that no spurious mutations were introduced during amplification. The fragment was excised with Bam HI, and the 530 bp fragments were cloned into the Bam HI site of pBI101.1 to create the plasmids pBI280 and pBI290. The orientation of the fragment was analyzed by PCR with primers that hybridize in the pBI101.1 plasmid (5'-ACAGTTTTCGCGATCCAGAC-3' and 5'-TTATGCTTCCGGCTCGTATG-3') and the primers previously described. Escherichia coli strain DH5α was used for plasmid construction. Agrobacterium tumefaciens strain GV3101 pMP90 was transformed with plasmids by electroporation, and Arabidopsis (Col-7) plants were transformed by floral dip infiltration  with the plasmids pBI101.1, pBI280, or pBI290.
Histochemical localization of GUS activity
GUS activity was localized by staining the tissues with 0.5 mg of 5-bromo-4-chloro-3-indolyl-b-D-glucuronic acid (X-Gluc; Gold Biotechnology, St Louis, MO, USA) per mL in X-Gluc buffer containing 50 mM sodium phosphate (pH 7.2), 10 mM EDTA, 0.33 mg/mL potassium ferricyanide and 0.001% Tween 20. The tissues were vacuum-infiltrated for three rounds of one min each, and staining reactions proceeded overnight at 37°C. Chlorophyll was removed by soaking in ethanol. The photographs were taken with a binocular microscope Leika MZ16F.
Analysis of GUS activity
Quantitative analysis of GUS activity was performed on whole aerial part using the GUS activity assay , the experiment was made twice, each treatment had three biological replicates and each replicate was a pool of 10 Arabidopsis plants, except the high light treatment which had four biological replicates.
Production of 4-methylumbelliferone [MU] was measured using a DTX 880 Multimode Detector (Beckman Coulter, Fullerton, CA). Protein concentrations of the samples were determined using Bradford reagent  and BSA as a standard. The amount of MU was determined from a standard curve, and GUS activity was expressed as nmol MU/min/mg protein. The empty vector transformed plants shown a basal activity of 0.22 ± 0.08 nmoles MU/min/mg protein.
To measure the protein levels of 2CPB, 100 mg of tissue were ground to a fine powder in liquid N2 and then homogenized with 0.2 mL of buffer (25 mM Hepes (pH 7.5), 0.6 M mannitol, 0.462 mg/mL dithiothreitol, 2 mM EDTA, 0.175 mg/mL phenylmethylsulphonyl fluoride and 1% (w/v) polyvinylpolypyrrolidone). The homogenates were centrifuged at 15,000 g for 20 min, and the supernatant protein concentration was determined utilizing BSA as a standard protein as described by . The supernatant was mixed with sample buffer 10× (250 mM Tris-HCl (pH 6.8), 10% SDS, 0.5% bromophenol blue and 20% glycerol), boiled for 5 min, and separated in a 12% SDS-PAGE as described earlier . The gels were stained with Coomassie Brilliant Blue R-250. For immunoblotting, the proteins were transferred to nitrocellulose membranes using a Mini Trans-Blot cell (Bio-Rad, CA, USA) at 100 mA for 100 min. The membranes were treated with polyclonal antibody raised against rapeseed 2-Cys peroxiredoxin . Signals on the membranes were visualized with alkaline phosphatase-conjugated goat anti-rabbit IgG (SIGMA, St Louis, MO, USA).
The signal intensities were quantified from the immunoblot using the Gel-Pro Analyzer software (Media Cybernetics Inc, Silver Spring, MD) and normalized to the intensities observed in control conditions. A representative example from three independent experiments is shown.
Promoter sequence analysis
The promoter sequence was analyzed using publicly available databases, PlantCARE http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ and PLACE http://www.dna.affrc.go.jp/PLACE/signalscan.html, which are databases of plant cis-acting regulatory elements; AthaMap http://www.athamap.de/index.php, which provides a genome-wide map of potential transcription factor binding sites in Arabidopsis thaliana; and Plant Promoter Database (ppdb) http://www.ppdb.gene.nagoya-u.ac.jp, which is based on species-specific sets of promoter elements, rather than on general motifs for multiple species.
Arabidopsis promoters length analysis
Annotation data for the Arabidopsis thaliana genes was downloaded from The Arabidopsis Information Resource (TAIR) FTP server ftp://ftp.arabidopsis.org/Maps/seqviewer_data/sv_gene.data. The analysis was performed on 27,141 genes after filtering out pseudogenes and transposon-related genes ftp://ftp.arabidopsis.org/Maps/gbrowse_data/TAIR8/TAIR8_GFF3_genes_transposons.gff from 31,762 annotated genes. Start and stop positions of the transcription units along with information on the strand that encodes an mRNA were extracted. Microsoft Office Excel was used to calculate the distances between the 3' ends of the nearest neighbour genes and the distances between 5' ends of the neighbour genes. The overlapping genes were analyzed only in the graph corresponding to the 3'ends of the nearest neighbour genes and the resulting distances among them were less than zero (shown in Figure 6C, inset).
We thank Dr. Ricardo Wolosiuk, Instituto Leloir, Argentina for the generous gift of 2-CPB antisera.
The work described in this article was performed with the financial support of the Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) and the Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET) from Argentina.
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