JXB Advance Access published online on June 11, 2007
Journal of Experimental Botany, doi:10.1093/jxb/erm123
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© 2007 The Author(s).
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.This paper is available online free of all access charges (see http://jxb.oxfordjournals.org/open_access.html for further details)
RESEARCH PAPER |
Isolation and comparative analysis of the wheat TaPT2 promoter: identification in silico of new putative regulatory motifs conserved between monocots and dicots
1Millennium Nucleus in Plant Cell Biology and Center of Plant Biotechnology, Andres Bello University, Av. República 217, 837-0146, Santiago, Chile
2Laboratorio de Genética Molecular Vegetal, Facultad de Ciencias, Universidad de Chile, Las Palmeras 3425, Ñuñoa, Santiago, Chile
3Instituto de Investigaciones Agropecuarias, Unidad de Biotecnología, INIA-Carillanca, Casilla 58-D, Temuco, Chile
4Departamento de Botánica, Universidad de Concepción, Casilla 160-C, Concepción, Chile
To whom correspondence should be addressed. E-mail: hsilva{at}unab.cl
Received 5 February 2007; Revised 30 April 2007 Accepted 1 May 2007
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Abstract |
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Phosphorus deficiency is one of the major nutrient stresses affecting plant growth. Plants respond to phosphate (Pi) deficiency through multiple strategies, including the synthesis of high-affinity Pi transporters. In this study, the expression pattern of one putative wheat high-affinity phosphate transporter, TaPT2, was examined in roots and leaves under Pi-deficient conditions. TaPT2 transcript levels increased in roots of Pi-starved plants. A 579 bp fragment of the TaPT2 promoter is sufficient to drive the expression of the GUS reporter gene specifically in roots of Pi-deprived wheat. This TaPT2 promoter fragment was also able to drive expression of the GUS reporter gene in transgenic Arabidopsis thaliana, under similar growth conditions. Conserved regions and candidate regulatory motifs were detected by comparing this promoter with Pi transporter promoters from barley, rice, and Arabidopsis. Altogether, these results indicate that there are conserved cis-acting elements and trans-acting factors that enable the TaPT2 promoter to be regulated in a tissue-specific and Pi-dependent fashion in both monocots and dicots.
Key words: Arabidopsis thaliana, high affinity phosphate transporter, promoter, Triticum aestivum
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Introduction |
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Phosphorus is involved in several key biological processes such as metabolism, respiration, photosynthesis, enzyme regulation, and signalling. Phosphorus is also a structural component of many macromolecules (Eastmond and Graham, 2003; Kruger and von Schaewen, 2003; Meijer and Munnik, 2003; Fernie et al., 2004; Chen et al., 2005; Peñaloza et al., 2005). The bioavailability of phosphate (Pi) in the soil is a worldwide problem for plant growth and increased productivity. Pi is often found in nature associated with metals, which decreases its bioavailability (Ticconi and Abel, 2004). A common agricultural solution to the problem of Pi bioavailability is to enrich the soil with bioavailable Pi fertilizers. However, only about 1% of the Pi applied to the soil is used by plants. Additionally, most of the Pi fertilizers come from phosphoric rocks, a raw material that is expected to be depleted by the end of the century at the present rate of use (Raghothama, 1999).
Plants must maintain intracellular Pi levels at a millimolar range, even when soil Pi concentrations are at a micromolar level (Reisenauer, 1966). To cope with low soil Pi, plants have developed strategies for overcoming Pi starvation. Such strategies involve changes in root architecture (Lopez-Bucio et al., 2003), association with mycorrhiza (Karandashov et al., 2004; Karandashov and Bucher, 2005), expression and secretion of acid phosphatases (Haran et al., 2000), secretion of organic acids (Diatloff et al., 2004), induction of microRNAs (Fujii et al., 2005), and synthesis of high-affinity phosphate transporters (Karthikeyan et al., 2002).
The molecular mechanisms that coordinate this global Pi deficiency response are not well understood. However, it is known to be transcriptionally regulated (Raghothama, 1999). Furthermore, Affymetrix experiments suggest that specific sets of transcription factors are involved in regulating the early and late responses to Pi deficiency in Arabidopsis (Misson et al., 2005).
One of the best-conserved adaptations of plants to low Pi supply is the induced expression of high-affinity Pi transporter genes. These are known as Pht1 transporters to distinguish them from the low-affinity Pi transporters which are in the Pht2 family. Structurally, the Pht1 transporters are integral membrane proteins with two groups of six membrane-spanning segments linked by a hydrophilic region (Persson et al., 2003). In Arabidopsis thaliana, Pht1 transporters are divided into nine groups, several of which are expressed exclusively in roots during phosphate starvation (Mudge et al., 2002).
There is a high level of conservation among the high-affinity Pi transporters of different plants, suggesting that their function has been maintained throughout evolution. However, not all the high-affinity Pi transporters are transcriptionally regulated in the same way. Some are expressed in the presence or absence of Pi, in different tissues, in different cell types, or preferentially expressed in mycorrhizal associations, possibly suggesting an evolutionary divergence of their physiological function. Part of this divergence could be associated with the regulatory non-coding regions of these transporters.
Although the function and regulation of these high-affinity Pi transporters have been well studied in model dicot plants such as Arabidopsis (Daram et al., 1998; Mukatira et al., 2001; Misson et al., 2004), they are not well characterized in cereals and other monocots.
Based on its contribution to the food industry, wheat is one of the major worldwide crops and is the main annual crop in Chile (www.odepa.gob.cl). In Chile, however, wheat is usually grown in acidic soils with low Pi bioavailability. This situation is a major problem because young wheat plants need Pi for the development of tillers and spikelets (Romer and Schilling, 1986). To date, several entries for Pi transporters in wheat can be found in public databases, but their function and mechanisms of regulation have not been studied in detail. Davies et al. (2002) isolated eight different high-affinity Pi transporters from wheat root cDNA libraries and they analysed the expression levels of some of them in different wheat varieties. They observed that different transporters have different expression patterns within the same variety and also that the same transporter could be expressed differentially in different varieties.
In the present work, the expression pattern of one of these putative high-affinity Pi transporters, TaPT2 (GeneBank accession number AJ344241), in wheat (Dalcahue variety) is examined under phosphate-deficient conditions. A 579 bp fragment was isolated upstream of the first ATG codon of this gene, and it was demonstrated that this sequence can specifically drive the expression of the ß-glucuronidase (GUS) reporter gene in wheat roots under Pi-deficient conditions. Interestingly, the isolated promoter can carry out the same regulation in Arabidopsis thaliana, suggesting that it contains conserved trans-acting factors and cis-acting elements that enable this promoter to be regulated in a tissue-specific and Pi-dependent fashion in both dicots and monocots. Bioinformatically, four highly conserved motifs were identified in both the wheat TaPT2 promoter and the Pi transporter promoters from monocots and Arabidopsis that could explain their conserved heterologous regulation.
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Materials and methods |
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Plant growth, transformation, and Pi treatments
Wild-type and transgenic Arabidopsis thaliana (ecotype Columbia) were grown in a mixture of soil and vermiculite (3:1) in a growth chamber with a 16 h light cycle (140 µE m–2 s–1) at 22 °C. Alternatively, seeds were surface sterilized as described in Gonzalez et al. (2006) and grown under the same conditions as soil-grown plants in plates with Murashige–Skoog medium containing 0.8% agar, 0.1% sucrose, and 50 mg l–1 kanamycin for transgenic lines.
Transgenic Arabidopsis were obtained using the Agrobacterium tumefaciens-mediated floral dip method (Clough and Bent, 1998). Triticum aestivum, variety Bobwhite, was transformed with the TaPT2 promoter–uidA fusion using biobalistics (Pelligrinineschi et al., 2000) at INIA-Carillanca.
Treatments in the presence of phosphate or under phosphate-deficient conditions were performed to determine the effects on the wheat TaPT2 transcript levels and the activity of the TaPT2 promoter–uidA fusion. Treatments were performed as described by Peñaloza et al. (2002), with the following modifications: Arabidopsis and wheat seeds were germinated in 10 ml flasks placed on a shaker and maintained for 5 d in a nutritive solution containing bioavailable Pi [1 mM NH4NO3, 3 mM KNO3, 2 mM Ca(NO3), 1 mM MgSO4, 0.07 µM (NH4)6Mo7O24, 22 µM H3BO3, 1.6 µM ZnSO4, 0.4 µM CuSO4, 2 µM MnSO4, 20 µM Fe EDTA, 0.1 mM KH2PO4]. After 5 d, the plants were washed with sterile distilled water and grown in the presence and absence of bioavailable Pi (in 10 ml flasks placed on a shaker). These solutions are identical to the germination solution except that the solution without Pi (Pi-deficient solution) does not contain KH2PO4. The pH was adjusted daily to 5.8. Solutions were maintained sterile and changed every 4 d.
Total RNA extraction
Between 50 mg and 100 mg of tissue was ground to a fine powder in liquid nitrogen. Total RNA was isolated using Trizol reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The final pellet was dissolved in RNase-free water and stored at –80 °C. RNA quality and quantity were determined as described by Meisel et al. (2005).
Northern blot and reverse transcriptase (RT)-PCR
Northern blot experiments were carried out as described by Silva et al. (1999). Total RNA (20 µg), extracted from whole roots or leaves, was fractionated by electrophoresis in an agarose gel containing 22% formaldehyde and capillary transferred to a nylon membrane (Hybond N+; Amersham Biosciences) using 10x SSC (1x SSC: 0.15 M NaCl, 0.015 M sodium citrate). The RNA was UV-cross-linked to the nylon membrane using a Spectroline TS-312A transilluminator. The membrane was prehybridized for 2 h in prehybridization solution [5x SSC, 50 mM sodium phosphate, pH 6.8, 5x Denhart solution (1x Denhart solution: 0.02% Ficoll; 0.02% PVP, 0.02% BSA), 1 mM EDTA, pH 8.0, 1% SDS, 10 g ml–1 salmon sperm DNA]. The membrane was hybridized with the 32P-labelled probe in hybridization solution (identical to prehybridization solution except that it contains 0.1% SDS) at 65 °C for 16 h. The membrane was subsequently washed with 5x SSC, 0.1% SDS for 15 min at 65 °C and for 30 min with 2x SSC, 0.1% SDS at 65 °C. Radioactivity was detected by exposing the membrane to an autoradiography film for 24 h.
All DNA 32P-radiolabelled probes were obtained by PCR using the Fermentas Hexalabel kit. The primers used to prepare TaPT2 and Actin probes are shown in Table 1. An 18S rRNA EST of Arabidopsis cloned in a pBluescript vector (H8B10T7), kindly donated by L. Holuigue, was used as a template to prepare a probe against 18S rRNA using the SK and KS primers. The probes were purified using the CONCERT Matrix Gel Extraction System (GIBCO BRL).
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RT-PCR experiments were carried out using total RNA extracted from whole roots or leaves. First strand cDNA was synthesized from 2.5 µg of the DNase-treated RNA in a final volume of 20 µl. The reaction mix was prepared using the RevertAid H Minus M-MuLV reverse transcriptase kit (Fermentas, Vilnius, Lithuania) using anchored oligo(dT) of 18-mers according to the manufacturer's instructions. The primers used to amplify the internal regions of TaPT2 (12F–PT2 and 553R–PT2) are shown in Table 1. The conditions for the PCR are as follows: cDNAs were diluted 100 times and 1 µl of each dilution was used as a template. The reactions were performed in a final volume of 20 µl, containing 0.5 µM of primers, 0.2 mM dNTPs, 2.0 mM MgCl2, 5 U of Taq polymerase, and buffer. The conditions of the PCR were: 94 °C for 3 min; 40 cycles of 94 °C for 30 s, 60 °C for 3 min, 72 °C for 2 min; 72 °C for 5 min.
Cloning of the TaPT2 promoter
The wheat genomic DNA (Triticum aestivum variety Dalcahue) was extracted as described by Das et al. (1990) except that 100–300 mg of 3-week-old wheat leaves were used. The Universal Genome WalkerTM Kit (Clontech Laboratories, Inc., Palo Alto, CA, USA) was used to isolate a 579 bp fragment of the TaPT2 promoter. Ten 6 bp-recognizing and blunt end-forming restriction enzymes (DraI, EcoRV, PvuII, StuI, SspI, SmaI, MscI, ScaI, Eco105I, and HpaI) were used to digest the isolated genomic DNA. DNA fragments containing the adaptors at both ends were used as templates for amplifying the TaPT2 promoter region. PT2–GSP1 and PT2–GSP2 specific primers were designed to isolate the promoter (Table 1 and Fig. 2). The first PCR, using AP1 and PT2–GSP1 primers, was performed using Synergy DNA polymerase (Genecraft, Münster, Germany). The second PCR, using AP2 and PT2–GSP2 primers, was performed using the Advantage Genomic Polymerase Mix (Clontech). The amplified fragment, corresponding to the StuI library, was cloned in the pGEM-T vector (Promega, Madison, WI, USA). The TaPT2 promoter sequence was amplifying using the AP2 and PT2–BamHI primers (Table 1), cloned into the pGEM-T vector, and re-sequenced. The fragment was then released from the pGEM-T with a BamHI–SalI sequential digestion, isolated, and transcriptionally fused to the uidA reporter gene in the promoterless binary vector pBI101.1 (Jefferson et al., 1987). The binary vector was introduced into Agrobacterium tumefaciens (GV3101) for Arabidopsis transformation as described earlier.
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Bioinformatics analyses
A phylogenetic tree analysis was performed among TaPT2 and other plant and fungal Pi transporters (see Supplementary data available at JXB online). To identify predicted conserved motifs, approximately 600 bp upstream of the predicted start, codons were analysed using the YMF 3.0 program (Blanchette and Sinha, 2001; http://wingless.cs.washington.edu/YMF/YMFWeb/YMFInput.pl). Only statistically significant motifs (Z score value >6.5) were selected (Sinha and Tompa, 2002). The motifs identified by this method were also found using the programs Motif Sampler (Thijs et al., 2002) and MEME (Bailey and Elkan, 1995). Nucleotide entry codes (GenBank accession number or MIPS code for Arabidopsis) and references for each sequence analysed are: HvPT1, HvPT2, and HvPT4 to HvPT8 (AF543197 [GenBank] , AY187019 [GenBank] , AY187024 [GenBank] , AY187021 [GenBank] , AF543198 [GenBank] , AY187022 [GenBank] and AY187023, respectively) (Schünmann et al., 2004a; Glassop et al., 2005); AtPT1 to AtPT9 (At5g43350, At5g43370, At5g43360, At2g38940, At2g32830, At5g43340, At3g54700, At1g20860 and At1g76430, respectively) (Mudge et al., 2002); OsPT1 to OsPT13 (AF536961, AF536962, AF536963, AF536964, AF536965, AF536966, AF536967, AF536968, AF536969, AF536970, AF536971 [GenBank] , AF536972 and AF536973, respectively) (Paszkowski et al., 2002). The 600 bp 5' proximal to the ATG codon of the OsPT1 to OsPT13 were obtained from the publicly available BAC sequences.
Analysis of putative transcription factor-binding sites was carried out using the PLACE database (http://www.dna.affrc.go.jp/htdocs/PLACE/) (Higo et al., 1999).
Note: the plant phosphate transporter genes used in this work have been referred to using simplified nomenclature. According to the Commission on Plant Gene Nomenclature (http://mbclserver.rutgers.edu/CPGN/Guide.html), the official name for TaPT2 is TRIae;Pht1;2; for TaPHT1MYC, TRIae;Pht1;myc; for HvPT1 to HvPT8, HORvu;Pht1;1 to HORvu;Pht1;8; for OsPT1 to OsPT13, ORYsa;Pht1;1 to ORYsa;Pht1;13; for AtPT1 to AtPT9, ARAth;Pht1;1 to ARAth;Pht1;9; for AtPht2, ARAth;Pht2;1; for LePT1, LePT2, and LePT4, LYCes;Pht1;1, LYCes;Pht1;2, and LYCes;Pht1;4; for MtPT1 to MtPT4, MEDtr;Pht1;1 to MEDtr;Pht1;4; for StPT3 and StPT4, SOLtu;Pht1;3 and SOLtu;Pht1;4; for Zm PT6, ZEAma;Pht1;6.
GUS activity measurement
Histochemical staining for GUS activity was performed as described by Meisel and Lam (1996) and Jefferson et al. (1987), with the following modifications. Transgenic wheat and Arabidopsis seedlings with different Pi treatment times were used directly for vacuum infiltration in 50 mM NaH2PO4, pH 7.0, 0.1 mM X-Gluc, 10 mM EDTA, 0.1% Triton X-100. Samples were incubated at 37 °C until sufficient staining had developed (24–72 h). Plant material was then fixed in 4% formaldehyde, 4% acetic acid, 28.5% ethanol for 30 min. Subsequent incubation in 70% ethanol for 1 h, 100% ethanol for 1 h, 70% ethanol for 1 h, followed by incubation in distilled water, was performed to remove chlorophyll from the plant material.
Fluorometric GUS assays were performed as described by Jefferson et al. (1987). The wheat and Arabidopsis root and leaf tissues were ground in the presence of liquid nitrogen in a mortar, and transferred to a microtube. One millilitre of the extraction buffer [50 mM NaH2PO4, pH 7.0, 1 mM EDTA, 0.1% Triton X-100, 0.1% (w/v) sodium laurylsarcosine, and 5 mM dithiothreitol] was added and mixed. After centrifuging for 10 min at 12 000 g at 4 °C, the supernatant was transferred to a microtube. The fluorogenic reaction was carried out in 2 ml volume containing 1 mM 4-methyl umbelliferyl glucuronide in an extraction buffer supplemented with a 50 µl aliquot of the protein extract supernatants. The protein quantity of the sample extracts was determined using BSA (bovine serum albumin) as a standard, according to Bradford (1976).
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Analysis of the TaPT2 protein sequence
The predicted TaPT2 protein sequence (GeneBank accession number CAC69856) comprises 525 amino acids. As was predicted by the TMPred program (Hofmann and Stoffel, 1993) and like other Pht1 family members, TaPT2 contains 12 conserved potential membrane-spanning domains arranged in a 6 + 6 configuration around a large low-conserved central cytoplasmic loop (data not shown). Furthermore, the TaPT2 protein sequence contains high-affinity transporter-conserved sequence motifs such as phosphorylation sites for protein kinase C and casein kinase II, as well as a potential site for N-glycosylation (data not shown; Smith et al., 2000).
An unrooted phylogenetic tree shows that TaPT2 is clustered with the high-affinity Pi monocot transporters (rice and barley) not associated with mycorrhizas that are expressed in roots during Pi starvation (HvPT1, HvPT2, and OsPT2) (Paszkowski et al., 2002; Rae et al., 2003; Schünmann et al., 2004a) (see Supplementary Fig. S1 at JXB online). The TaPT2 amino acid sequence is highly conserved with the Hordeum vulgare orthologue (98% identity with HvPT1 and 96% identity with HvPT2; data not shown).
TaPT2 expression in Dalcahue wheat variety in the presence or absence of bioavailable Pi
The expression of TaPT2 in wheat (Dalcahue variety) was first evaluated by northern blot analyses. Total RNA was extracted from leaves and roots of plants grown for 7 d in the presence of Pi or under Pi-starvation conditions. In the presence of 0.1 mM Pi, no TaPT2 transcripts were observed in leaf extracts whereas a weak band was observed in roots (Fig. 1A). In the absence of Pi, TaPT2 transcripts were detected at high levels in roots but not in leaves. Since there is a high level of sequence conservation between TaPT2 and other Pi transporters (TaPT1 has a 93% sequence identity at the nucleotide level to TaPT1; data not shown) which may result in cross-hybridization, the northern blot results were confirmed by RT-PCR using specific primers for TaPT2 (Fig. 1B). As described for the wheat variety Jing411 (Davies et al., 2002), the expression of TaPT2 is induced specifically in Pi-deprived roots of the Dalcahue wheat variety.
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Identification and analysis of the TaPT2 promoter region
To gain further insights into the regulatory mechanism of TaPT2 gene expression, the promoter region of TaPT2 was isolated using the Genome Walker technique (Siebert et al., 1995). A single 579 bp fragment, upstream of the start codon was isolated and cloned (Fig. 2). Using the PLACE algorithm (Higo et al., 1999) some known cis regulatory motifs in this sequence were identified. One motif contains the sequence ATATT (rootmotiftapox1). This motif, identified previously in the promoter regions of the rolD gene (Agrobacterium rhizogenes Ri plasmid) and the TTG2 gene (Arabidopsis thaliana), appears to be associated with root gene expression (Elmayan and Tepfer, 1995; Johnson et al., 2002). One base pair variants of the motifs associated with transcriptional responses to Pi starvation, P1BS (GNATATNC; Rubio et al., 2001) and the PHO-like element [G(G/T/A) (C/T/A)GTGG; Mukatira et al., 2001], two that are different from the consensus P1BS at the 3' end, and one that is different from the PHO-like element at the last base of the 5' end (Fig. 2), were found.
To identify new conserved motifs in the TaPT2 promoter, sequences approximately 600 bp immediately upstream of the translation start site of 13 rice phosphate transporters, seven barley phosphate transporters, and nine Arabidopsis phosphate transporters were compared with the wheat TaPT2 promoter sequence. Using the program YMF3.0 (Blanchette and Sinha, 2001), four statistically significant motifs were identified in the promoter regions of these phosphate transporters: motif 1 = SGCCGGCS (Z score 16.32), motif 2 = CTATNTATA (Z score 8.57), motif 3 = ATAAGTC (Z score 7.86), and motif 4 = ATATRCA (Z score 6.69) (Fig. 3).
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In agreement with the low divergence observed between the TaPT2, HvPT1, and HvPT2 transporters (Fig. S1 available at JXB online), a highly conserved promoter region was found among these three genes. As shown in Fig. 3, the TaPT2 promoter shares a 116 bp region with 77% identity to the HvPT1 promoter (sequence alignment marked with one asterisk) and 255 bp with 78% identity to the HvPT2 promoter (sequence alignment marked with two asterisks). Three of the four different motifs identified (motifs 1, 2, and 4) are present within this highly conserved promoter region (Fig. 3). One of these motifs is found exclusively in the promoters of monocot transporter genes (motif 1: SGCCGGCS). Motifs 2, 3, and 4 were identified in the promoters of phosphate transporter genes from both monocots and dicots (Arabidopsis). Notably, all four motifs are absent in the promoters of transporters expressed specifically in shoots and mycorrhiza: HvPT6, HvPT8, and OsPT11, respectively (Paszkowski et al., 2002; Rae et al., 2003; Glassop et al., 2005). Furthermore, motif 4 (ATATRCA) is similar to the P1BS motif (GNATATNC). Only one motif 2 (CTATNTATA) site was found in the promoter of the flower-specific transporter AtPT6 (Mudge et al., 2002).
The isolated TaPT2 promoter is sufficient to drive Pi-deficient expression of GUS in the roots of transgenic wheat
To determine if the TaPT2 579 bp promoter fragment that was isolated is sufficient to drive Pi-deficiency expression in the roots of wheat, this fragment was cloned upstream of the uidA (GUS) gene in the binary plasmid pBI101.1 and transgenic wheat (Bobwhite variety) was generated. Three independent transgenic lines that weakly expressed GUS activity were obtained. The GUS activity of the best T3 homozygous line (line with the highest level of GUS expression) was analyzed fluorimetrically and histochemically in plants grown in the presence or absence of Pi (Fig. 4). As observed for TaPT2 expression in the Dalcahue variety (Fig. 1), the isolated TaPT2 promoter triggered GUS expression in roots in the absence of Pi. These results suggest that the isolated 579 bp TaPT2 promoter contains cis elements that are able to regulate root-specific expression under Pi-deficient conditions, such as P1BS or PHO-like motifs.
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Isolated TaPT2 promoter drives Pi deficiency-specific expression of GUS in roots of transgenic Arabidopsis thaliana
The conservation of two of the putative regulatory motifs in the Arabidopsis thaliana high-affinity Pi transporter promoters, identified with the different prediction programs, suggests a probable Pi-deficiency induction of the TaPT2 promoter in this dicot. To test whether the Pi-deficient induction of this promoter is conserved between monocots and dicots, Arabidopsis transgenic plants were generated using the same promoter–GUS construct used to transform wheat. GUS activity of seven homozygous T3 independent lines was analysed histochemically in the presence and absence of Pi. Results of the best transgenic line obtained (G3R, line with the highest level of GUS expression) are shown in Fig. 5. Interestingly, as was observed with the expression of TaPT2 in the wheat variety Dalcahue (Fig. 1), as well as the activity of the TaPT2 promoter fused to GUS in transgenic wheat (Fig. 4), the isolated TaPT2 promoter also triggered GUS expression specifically in the roots of Pi-deprived Arabidopsis plants. These results demonstrate that the regulatory elements present in the isolated TaPT2 promoter are sufficient to induce root-specific Pi-deficiency expression in both monocots and dicots. Additionally, this suggests that there is also conservation between monocots and dicots of the trans-acting factors that interact with these cis-acting elements.
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Discussion |
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In this work, it was demonstrated that the promoter region of the wheat high-affinity Pi transporter, TaPT2, contains regions sufficient to up-regulate gene expression in Pi-deprived roots of both monocot and dicot species.
Davies et al. (2002) have demonstrated previously that the TaPT2 gene is expressed primarily in roots of Pi-deprived plants of the Chinese wheat Jing411, a variety that has low tolerance to Pi deficiency. This change in TaPT2 gene expression in response to Pi deficiency was not, however, detected in three other varieties tolerant of the changes in external Pi conditions (Davies et al., 2002).
In the present study it was demonstrated that a wheat cultivar of Chilean origin (cv. Dalcahue) also shows alterations in the expression levels of TaPT2 under Pi-deficient conditions (Fig. 1). Dalcahue is a spring variety from the south of Chile that is tolerant of acidic soil and tolerant of low Pi levels (http://www.inia.cl/semillas/trigosrecomendacion.htm). This finding suggests that the differential expression of TaPT2 in roots under different Pi conditions may not necessarily be associated with tolerance under low Pi conditions, as pointed out by Davies et al. (2002). It is possible that the different TaPT2 expression levels in different wheat varieties could be due to differences in the regulatory sequences that are present in their promoters. A full-length TaPT2 cDNA from the wheat variety Xiaoyan 54 (GenBank accession number AJ344248) contains an 83 bp region from the 5' UTR. This region is only 70% identical to the same region isolated in this work (data not shown), and has significant differences in the P1BS-like motif. This suggests that the remaining upstream regulatory region may also be different. It will be very interesting to identify and compare the TaPT2 regulatory sequences from varieties such as Xiaoyan54 that differ from the Dalcahue variety in terms of TaPT2 transcript levels, and/or tolerance to low Pi levels. This type of analysis may provide insight into identifying the cis-elements responsible for this type of transcriptional regulation.
High-affinity Pi transporters have been identified in both dicots and monocots. Some of these transporters have been shown to be expressed exclusively in the aerial portion of the plant, others exclusively in specific mycorrhiza associations, and yet others are expressed only in roots of Pi-starved plants. The high-affinity Pi transporters whose expression is altered depending upon Pi conditions include the TaPT2 from wheat, its putative barley orthologues, HvPT1 and HvPT2 (Smith et al., 1999; Schünmann et al., 2004a), the rice orthologue OsPT2 (Paszkowski et al., 2002), and several members of the Pht1 Arabidopsis family (Mudge et al., 2002). The high sequence identity existing within the coding regions of TaPT2, HvPt1, and HvPT2 extends into the promoter regions, suggesting that the transcriptional regulation of these genes may be evolutionarily conserved. Schünmann et al. (2004b) have shown that a minimum regulatory region (the P1BS motif) within the HvPT1 promoter is necessary to regulate expression in a tissue- and Pi-specific manner. Although a complete P1BS motif has not been identified in the TaPT2 promoter, one of the four motifs that have been identified (motif 4: ATATRCA) contains five of the base pairs found in the P1BS motif. The high conservation of this candidate motif in the promoters of others Pi transporters in rice, barley, and Arabidopsis suggests that this motif is functionally conserved and may play a role in inducing expression under Pi-deficient conditions in the root. This motif was not found in the sequence of transporter promoters that are expressed in flowers (AtPT6), shoots (HvPT6), or specifically with mycorrhiza (OsPT11 and HvPT8).
Some studies have shown that there is high conservation in the regulatory mechanisms that define the phosphate-signalling pathway, among them the transcription factor PHR1 which has been studied in Arabidopsis thaliana, Oryza sativa, and the unicellular alga Chlamydomonas reinhardtii (Rubio et al., 2001; Bari et al., 2006). However, until now, there was no evidence that this pathway exists in wheat. Analyses of the TaPT2 promoter fragment in wheat and Arabidopsis provide indirect evidence that suggests that this pathway is present and functionally conserved in wheat.
There are very few examples in the literature of monocot gene promoters that are functional in a dicot plant (Liu et al., 2003; Iwamoto et al., 2004). Koyama et al. (2005) have shown that the promoter of the Arabidopsis high-affinity Pi transporter, AtPT1, is able to drive GUS expression in a root-specific and Pi-dependent manner in both Arabidopsis and rice. Karandashov et al. (2004) tested the activity of mycorrhiza-specific Pi transporter promoters of two dicot species (potato and Medicago truncatula) and a monocot (rice) in several dicot host plants. They observed that only the dicot promoters were functionally regulated in dicots, whereas the promoter from rice was not. The results from Karadashov et al. (2004), therefore suggests that not all promoters will behave the same in dicots and monocots.
In this paper, it has been demonstrated that an isolated fragment of the TaPT2 promoter is sufficient to drive GUS expression in a Pi-dependent manner in the roots of both monocots (wheat) and dicots (Arabidopsis). This demonstrates that the transcriptional regulation of these high-affinity Pi transporters is conserved between monocots and dicots. Additionally, this suggests that the Pi mycorrhiza-specific transporters are not regulated in a similar manner.
The analyses of the transgenic wheat used in the present study and Arabidopsis lines show that the TaPT2 promoter activity is low. This observation agrees with the low activity observed by Schünmann et al. (2004a) in the HvPT1 and HvPT2 promoters. This low level of activity may be due to the absence of regulatory regions further upstream of the 579 bp fragment or regulatory regions in an intron. It has been observed in monocots that an intron between the promoter and the coding region increases the expression level considerably without changing the expression pattern (Schünmann et al., 2004a).
It is important to mention that in both dicots and monocots (Arabidopsis and wheat, respectively) maximal GUS expression was detected after 7 d of Pi deprivation. This result is similar to that observed in Arabidopsis microarray analyses performed by Misson et al. (2005).
The conservation of the TaPT2 promoter activity in both wheat and Arabidopsis suggests that the model plant Arabidopsis thaliana may be used to study the transcriptional regulation of this and/or other wheat transporters, the trans-acting factors that may regulate this promoter, and the functionality of the new motifs identified in this work.
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Supplementary data |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
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A phylogenetic tree analysis was performed among TaPT2 and other plant and fungal Pi transporters.
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Acknowledgements |
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The authors wish to thank Dr. Loreto Holuigue from the Catholic University of Chile for providing us with the rRNA vector and Dr Patricio Arce from the Catholic University of Chile for providing access to the fluorometer. This research was supported by Grants from the Fundación para la Innovación Agraria (FIA, BIOT 01-A-36), Chile and ICM, Millennium Nucleus in Plant Cell Biology, PCB (P02-009-F), Chile.
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Footnotes |
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* These authors contributed equally to this work.
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References |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionSupplementary dataReferences |
|---|
Bailey TL, Elkan C. The value of prior knowledge in discovering motifs with MEME. In: Proceedings of the Third International Conference on Intelligent Systems in Molecular Biology—Rawlings C, Clark D, Altman R, Hunter L, Lengauer T, Wodak S, eds. (1995) Menlo Park, CA: The AAAI Press. 21–29.
Bari R, Pant BD, Stitt M, Scheible WR. PHO2, MicroRNA399, and PHR1 define a phosphate-signaling pathway in plants. Plant Physiology (2006) 141:988–999.
Blanchette M, Sinha S. Separating real motifs from their artifacts. Bioinformatics (2001) 17:S30–S38.[Abstract]
Bradford MM. A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry (1976) 72:248–254.[CrossRef][Web of Science][Medline]
Chen S, Hajirezaei M, Peisker M, Tschiersch H, Sonnewald U, Bornke F. Decreased sucrose-6-phosphate phosphatase level in transgenic tobacco inhibits photosynthesis, alters carbohydrate partitioning, and reduces growth. Planta (2005) 221:479–492.[CrossRef][Web of Science][Medline]
Clough SJ, Bent AF. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant Journal (1998) 16:735–743.[CrossRef][Web of Science][Medline]
Daram P, Brunner S, Persson BL, Amrhein N, Bucher M. Functional analysis and cell-specific expression of a phosphate transporter from tomato. Planta (1998) 206:225–233.[CrossRef][Web of Science][Medline]
Das OP, Alvarez C, Chaudhuri S, Messing J. Molecular methods for genetic analysis of maize. Methods in Molecular and Cellular Biology (1990) 1:213–222.
Davies TGE, Ying J, Xu Q, Li ZS, Li J, Gordon-Weeks R. Expression analysis of putative high-affinity phosphate transporters in Chinese winter wheats. Plant, Cell and Environment (2002) 25:1325–1339.[CrossRef]
Diatloff E, Roberts M, Sanders D, Roberts SK. Characterization of anion channels in the plasma membrane of Arabidopsis epidermal root cells and the identification of a citrate-permeable channel induced by phosphate starvation. Plant Physiology (2004) 136:4136–4149.
Eastmond PJ, Graham IA. Trehalose metabolism: a regulatory role for trehalose-6-phosphate? Current Opinion in Plant Biology (2003) 6:231–235.[CrossRef][Web of Science][Medline]
Elmayan T, Tepfer M. Evaluation in tobacco of the organ specificity and strength of the rolD promoter, domain A of the 35S promoter and the 35S2 promoter. Transgenic Research (1995) 4:388–396.[CrossRef][Web of Science][Medline]
Fernie AR, Carrari F, Sweetlove LJ. Respiratory metabolism: glycolysis, the TCA cycle and mitochondrial electron transport. Current Opinion in Plant Biology (2004) 7:254–261.[CrossRef][Web of Science][Medline]
Fujii H, Chiou TJ, Lin SI, Aung K, Zhu JK. A miRNA involved in phosphate-starvation response in Arabidopsis. Current Biology (2005) 15:2038–2043.[CrossRef][Web of Science][Medline]
Glassop D, Smith SE, Smith FW. Cereal phosphate transporters associated with the mycorrhizal pathway of phosphate uptake into roots. Planta (2005) 222:688–698.[CrossRef][Web of Science][Medline]
Gonzalez J, Reyes F, Salas C, Santiago M, Codriansky Y, Coliheuque N, Silva H. Arabidopsis thaliana: a model host plant to study plant–pathogen interaction using Chilean field isolates of Botrytis cinerea. Biological Research (2006) 39:221–228.[Web of Science][Medline]
Haran S, Logendra S, Seskar M, Bratanova M, Raskin I. Characterization of Arabidopsis acid phosphatase promoter and regulation of acid phosphatase expression. Plant Physiology (2000) 124:615–626.
Higo K, Ugawa Y, Iwamoto M, Korenaga T. Plant cis-acting regulatory DNA elements (PLACE) database. Nucleic Acids Research (1999) 27:297–300.
Hofmann K, Stoffel W. TMbase – a database of membrane spanning proteins segments. Biological Chemistry Hoppe-Seyler (1993) 347:166.
Iwamoto M, Higo H, Higo K. Strong expression of the rice catalase gene CatB promoter in protoplasts and roots of both monocots and dicots. Plant Physiology and Biochemistry (2004) 42:241–249.[CrossRef][Web of Science][Medline]
Jefferson RA, Kavanagh TA, Bevan MW. Gus fusions: ß-glucuronidase as a sensitive and versatile gene-fusion marker in higher plants. EMBO Journal (1987) 6:3901–3907.[Web of Science][Medline]
Johnson CS, Kolevski B, Smyth DR. TRANSPARENT TESTA GLABRA2, a trichome and seed coat development gene of Arabidopsis, encodes a WRKY transcription factor. The Plant Cell (2002) 14:1359–1375.
Karandashov V, Bucher M. Symbiotic phosphate transport in arbuscular mycorrhizas. Trends in Plant Science (2005) 10:22–29.[CrossRef][Web of Science][Medline]
Karandashov V, Nagy R, Wegmuller S, Amrhein N, Bucher M. Evolutionary conservation of a phosphate transporter in the arbuscular mycorrhizal symbiosis. Proceedings of the National Academy of Sciences, USA (2004) 101:6285–6290.
Karthikeyan AS, Varadarajan DK, Mukatira UT, D'Urzo MP, Damsz B, Raghothama KG. Regulated expression of Arabidopsis phosphate transporters. Plant Physiology (2002) 130:221–233.
Koyama T, Ono T, Shimizu M, et al. Promoter of Arabidopsis thaliana phosphate transporter gene drives root-specific expression of transgene in rice. Journal of Bioscience and Bioengineering (2005) 99:38–42.[CrossRef][Web of Science][Medline]
Kruger NJ, von Schaewen A. The oxidative pentose phosphate pathway: structure and organisation. Current Opinion in Plant Biology (2003) 6:236–246.[CrossRef][Web of Science][Medline]
Liu ZZ, Wang JL, Huang X, Xu WH, Liu ZM, Fang RX. The promoter of a rice glycine-rich protein gene, Osgrp-2, confers vascular-specific expression in transgenic plants. Planta (2003) 216:824–833.[Web of Science][Medline]
Lopez-Bucio J, Cruz-Ramirez A, Herrera-Estrella L. The role of nutrient availability in regulating root architecture. Current Opinion in Plant Biology (2003) 6:280–287.[CrossRef][Web of Science][Medline]
Meijer HJ, Munnik T. Phospholipid-based signaling in plants. Annual Review of Plant Biology (2003) 54:265–306.[CrossRef][Medline]
Meisel LA, Fonseca B, Gonzalez S, Baeza-Yates R, Cambiazo V, Campos R, Gonzalez M, Orellana A, Retamales J, Silva H. A rapid and efficient method for purifying high quality total RNA from peaches (Prunus persica) for functional genomics analyses. Biological Research (2005) 38:83–88.[Web of Science][Medline]
Meisel LA, Lam E. The conserved ELK-homeodomain of KNOTTED-1 contains two regions that signal nuclear localization. Plant Molecular Biology (1996) 30:1–14.[CrossRef][Web of Science][Medline]
Misson J, Raghothama KG, Jain A, et al. A genome-wide transcriptional analysis using Arabidopsis thaliana Affymetrix gene chips determined plant responses to phosphate deprivation. Proceedings of the National Academy of Sciences, USA (2005) 102:11934–11939.
Misson J, Thibaud MC, Bechtold N, Raghothama KG, Nussaume L. Transcriptional regulation and functional properties of Arabidopsis Pht1;4, a high affinity transporter contributing greatly to phosphate uptake in phosphate deprived plants. Plant Molecular Biology (2004) 55:727–741.[CrossRef][Web of Science][Medline]
Mudge SR, Rae AL, Diatloff E, Smith FW. Expression analysis suggests novel roles for members of the Pht1 family of phosphate transporters in Arabidopsis. The Plant Journal (2002) 31:341–353.[CrossRef][Web of Science][Medline]
Mukatira UT, Liu C, Varadarajan DK, Raghothama KG. Negative regulation of phosphate starvation-induced genes. Plant Physiology (2001) 127:1854–1862.
Paszkowski U, Kroken S, Roux C, Briggs SP. Rice phosphate transporters include an evolutionarily divergent gene specifically activated in arbuscular mycorrhizal symbiosis. Proceedings of the National Academy of Sciences, USA (2002) 99:13324–13329.
Pelligrinineschi AP, Fennell S, Mclean S, Brito RM, Velazquez L, Salgado M, Olivares JJ, Hernandez R, Hoisington D. Routine transformation system for use with CIMMYT wheat varieties. In: Koholi M, Francis M, eds. Application of biotechnologies to wheat breeding. Proceedings of a Conference at La Estanzuela, Uruguay. 19–20 November (2000) 1998:111–120.
Peñaloza E, Gutierrez A, Martínez J, Muñoz G, Bravo L, Corcuera LJ. Differential gene expression in proteoid root clusters of white lupin (Lupinus albus). Physiologia Plantarum (2002) 116:28–36.[CrossRef][Medline]
Peñaloza E, Muñoz G, Salvo-Garrido H, Silva H, Corcuera LJ. Phosphate deficiency regulates phosphoenolpyruvate carboxylase expression in proteoid root clusters of white lupin. Journal of Experimental Biology (2005) 56:145–153.
Persson BL, Lagerstedt JO, Pratt JR, Pattison-Granberg J, Lundh K, Shokrollahzadeh S, Lundh F. Regulation of phosphate acquisition in Saccharomyces cerevisiae. Current Genetics (2003) 43:225–244.[CrossRef][Web of Science][Medline]
Rae AL, Cybinski DH, Jarmey JM, Smith FW. Characterization of two phosphate transporters from barley: evidence for diverse function and kinetic properties among members of the Pht1 family. Plant Molecular Biology (2003) 53:27–36.[CrossRef][Web of Science][Medline]
Raghothama KG. Phosphate acquisition. Annual Review of Plant Physiology and Plant Molecular Biology (1999) 50:665–693.[CrossRef][Web of Science][Medline]
Reisenauer HM. Mineral nutrients in soil solution. Altman PL, Dittmer DS, eds. (1966) In: Altman PL, Dittmer DS, eds. Enviromental Biology. Bethesda, MD: Federation of American Societies for Experimental Biology, 507–508.
Romer W, Schilling G. Phosphorus requirement of the wheat plant at various stages of its life cycle. Plant and Soil (1986) 91:221–229.[CrossRef][Web of Science]
Rubio V, Linhares F, Solano R, Martín AC, Iglesias J, Leyva A, Paz-Ares JA. A conserved MYB transcription factor involved in phosphate starvation signaling both in vascular plants and in unicellular algae. Genes & Development (2001) 15:2122–2133.
Schünmann PHD, Richardson AE, Smith FW, Delhaize E. Characterization of promoter expression patterns derived from the Pht1 phosphate transporter genes of barley (Hordeum vulgare L.). Journal of Experimental Botany (2004a) 55:855–865.
Schünmann PHD, Richardson AE, Vickers CE, Delhaize E. Promoter analysis of the barley Pht1;1 phosphate transporter gene identifies regions controlling root expression and responsiveness to phosphate deprivation. Plant Physiology (2004b) 136:4205–4214.
Siebert P, Chenchik A, Kellogg D, Lukyanov K, Lukyanov S. An improved PCR method for walking in uncloned genomic DNA. Nucleic Acids Research (1995) 23:1087–1088.
Silva H, Yoshioka K, Dooner HK, Klessig DF. Characterization of a new Arabidopsis mutant exhibiting enhanced disease resistance. Molecular Plant–Microbe Interactions (1999) 12:1053–1063.[CrossRef]
Sinha S, Tompa M. Discovery of novel transcription factor binding sites by statistical overrepresentation. Nucleic Acids Research (2002) 30:5549–5560.
Smith FW, Cybinsky DH, Rae AL. Regulation of expression of genes encoding phosphate transporters in barley roots. In: Plant nutrition – molecular biology and genetics—Gissel-Nielsen G, Jensen A, eds. (1999) Dordrecht: Kluwer Academic Publishers. 145–150.
Smith FW, Rae AL, Hawkesford MJ. Molecular mechanisms of phosphate and sulphate transport in plants. Biochimica et Biophysica Acta (2000) 1465:236–245.[Medline]
Thijs G, Marchal K, Lescot M, Rombauts S, De Moor B, Rouzé P, Moreau Y. A Gibbs sampling method to detect overrepresented motifs in the upstream regions of coexpressed genes. Journal of Computational Biology (2002) 9:447–464.[CrossRef][Web of Science][Medline]
Ticconi CA, Abel S. Short on phosphate: plant surveillance and countermeasures. Trends in Plant Science (2004) 9:548–555.[CrossRef][Web of Science][Medline]
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