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American Society of Plant Biologists A Chloroplast Phosphate Transporter, PHT2;1, Influences Allocation of Phosphate within the Plant and Phosphate-Starvation ResponsesThe Samuel Roberts Noble Foundation, 2510 Sam Noble Parkway, Ardmore, Oklahoma, 73401 1 To whom correspondence should be addressed. E-mail mjharrison{at}noble.org; fax 580-221-7380
The uptake and distribution of Pi in plants requires multiple Pi transport systems that must function in concert to maintain homeostasis throughout growth and development. The Pi transporter PHT2;1 of Arabidopsis shares similarity with members of the Pi transporter family, which includes Na+/Pi symporters of fungal and animal origin and H+/Pi symporters of bacterial origin. Sequence comparisons between proteins of this family revealed that plant members possess extended N termini, which share features with chloroplast transit peptides. Localization of a PHT2;1–green fluorescent protein fusion protein indicates that it is present in the chloroplast envelope. A Pi transport function for PHT2;1 was confirmed in yeast using a truncated version of the protein lacking its transit peptide, which allowed targeting to the plasma membrane. To assess the in vivo role of PHT2;1 in phosphorus metabolism, we identified a null mutant, pht2;1-1. Analysis of the mutant reveals that PHT2;1 activity affects Pi allocation within the plant and modulates Pi-starvation responses, including the expression of Pi-starvation response genes and the translocation of Pi within leaves.
Phosphorus is essential for all living organisms as a structural component of nucleic acids and phospholipids, a constituent of energy transfer reactions, and a regulator in signal transduction cascades. In plants, phosphorus also is of fundamental importance to photosynthesis. Within the chloroplast, phosphate is an essential substrate for photophosphorylation and also plays a central role in the partitioning of triose phosphates, the end products of photosynthesis, between the starch and Suc biosynthetic pathways.
Plants acquire phosphorus as Pi from the soil solution. Despite its widespread occurrence in the environment, Pi often is limiting for plant growth mainly because it exists in the soil in soluble organic and inorganic forms that cannot be used directly by the plant (Marschner, 1995
The transport of Pi across membranes is a pivotal step in the regulation of Pi use. Plants require multiple Pi transport systems to mediate acquisition from diverse environments and to enable its subsequent transport to all of the cells and subcellular compartments of the plant (Bieleski, 1973
Little is known of the transport mechanisms that operate in the vascular tissue or in the shoots. Within each cell, Pi is transported between intracellular compartments, including chloroplasts/plastids and mitochondria, where it is assimilated via photooxidative and oxidative phosphorylation. Pi also is transferred into and out of the vacuole, a process that plays an essential role in the homeostatic control of cytoplasmic Pi concentration (Mimura, 1999
The triose phosphate/phosphate translocator (TPT) was the first phosphate transporter to be cloned from plants (Flügge et al., 1989
In addition to the TPT proteins, translocators that mediate phosphoenolpyruvate/phosphate exchange and Glc-6-phos-phate/phosphate exchange also are present in plastid inner envelope membranes (Fischer et al., 1997
Phosphate is mobilized across the inner mitochondrial membrane by the mitochondrial phosphate transporter. Mitochondrial phosphate transporters belong to the mitochondrial carrier family, which is characterized by a six-transmembrane-domain structure that is composed of three repeated segments of two transmembrane
In addition to these organellar Pi transporters, two other phylogenetically distinct classes of Pi transporters have been identified in plants. The first class contains transporters sharing similarity with the high-affinity H+/Pi symporter PHO84 of Saccharomyces cerevisiae (Bun-ya et al., 1991
Uptake experiments using heterologous expression systems indicate that the encoded transporters mediate high-affinity Pi transport and that H+/Pi symport is the most likely transport mechanism (Muchhal et al., 1996
Transporters of the second class share similarity with the Na+/Pi symporter PHO-4 of Neurospora crassa (Mann et al., 1989
Daram et al. (1999)
As part of a search for Na+/Pi transporters in plants, we also cloned PHT2;1. In contrast to Daram et al. (1999)
Expression and Localization Analyses in Arabidopsis Several plant Pi transporter genes have been identified based on the similarity of their deduced amino acid sequences to those of fungal H+/Pi symporters. Therefore, we used a similar strategy to identify plant orthologs of the phylogenetically distinct class of fungal Na+/Pi symporters. A BLASTP database search (Altschul et al., 1990  ) for sequences similar to that of the N. crassa PHO-4 Na+/Pi symporter (Versaw and Metzenberg, 1995) yielded two closely related plant sequences, one from Arabidopsis and another from Mesembryanthemum crystallinum, as well as sequences of cross-kingdom origin, including those of the previously confirmed mammalian and fungal Na+/Pi symporters (Kavanaugh et al., 1994; Olah et al., 1994; Martinez and Persson, 1998).
The Arabidopsis nucleotide sequence corresponds to an open reading frame (ORF), ORF02, within an 81-kb contig from chromosome III (Quigley et al., 1996
Our initial findings were similar to those reported. PHT2;1 is a single-copy gene that shows shoot-specific expression independent of external Pi concentrations (Figure 1)
. However, further analysis revealed that PHT2;1 expression varies during the photoperiod, with transcript levels 1.8-fold greater in shoot tissues harvested at the midpoint of the light phase than in tissues harvested at the midpoint of the dark phase (Figure 1). To determine whether PHT2;1 transcript levels are influenced by light, we monitored transcript levels in dark-treated plants and after exposure to light. After 6 days in darkness, PHT2;1 transcripts were barely detected, but after reexposure to light, induction occurred within 1 h (Figure 2)
. A similar pattern was detected for TPT, which was shown previously to be expressed in a light-dependent manner (Schulz et al., 1993
PHT2;1 possesses a long N-terminal extension that is not present in the fungal Na+/Pi symporters, which prompted us to examine the PHT2;1 amino acid sequence for the presence of potential targeting sequences. Several protein-sorting prediction programs (Predotar version 0.5, TargetP version 1.0, PSORT, and ChloroP 1.1) were used, and the searches yielded complex results with positive scores for mitochondrial, chloroplast, and plasma membrane targeting. To distinguish these possibilities and confirm the subcellular localization of PHT2;1, its coding region was fused to that of the GFP (Chiu et al., 1996 ) and introduced into 4-week-old Arabidopsis leaves by particle bombardment. After 24 h of incubation, green fluorescence was observed in the chloroplasts of transformed cells (Figure 3) . Confocal microscopy revealed that the GFP signal is strongest at the chloroplast periphery, whereas the chlorophyll autofluorescence is uniform. This pattern, coupled with the fact that PHT2;1 is an integral membrane protein, is consistent with localization in the chloroplast envelope. Because the envelope inner membrane is the permeability barrier between the chloroplast stroma and the cytosol and is the location of other transport proteins, PHT2;1 most likely is located within the inner membrane.
Analysis of the TPT proteins, which are located in the chloroplast envelope inner membrane, resulted in subcellular targeting predictions similar to those obtained for PHT2;1. However, a program designed specifically to identify chloroplast proteins (ChloroP 1.1) predicted correctly that the TPTs are located in the chloroplast envelope and predicts transit peptide cleavage sites that match those derived experimentally (Fischer et al., 1994 ). The same analysis applied to PHT2;1 predicted cleavage resulting in a transit peptide of 71 amino acids.
Localization and Functional Analyses in Yeast
A PHT2;1-GFP fusion clone was constructed and introduced into the yeast mutant PAM2, which is defective in the high-affinity uptake of Pi (Martinez and Persson, 1998
The Pi transport activities of PHT2;1 and PHT2;1(  1-71) then were compared in the yeast PAM2 strain. Both PHT2;1 and PHT2;1(1-71) mediate saturable Pi transport in yeast with the same pH optimum of 4.0 and with similar apparent Km values of 870 ± 10 µM and 810 ± 40 µM, respectively (Figures 5A and 5B) . This difference in apparent Km is not significant. Cells carrying the empty expression vector also exhibited saturable Pi uptake via the endogenous low-affinity transport system, with a Km value of 1010 ± 20 µM, which is significantly greater (P = 0.001) than that attributed to either PHT2;1 or PHT2;1(1-71). In addition, the transport activity of cells expressing PHT2;1(1-71) was approximately eightfold greater than that of cells expressing PHT2;1 under all assay conditions. This increased capacity for Pi transport also is reflected in cell growth.
When cultured in low-Pi medium (220 µM), the growth rate of cells expressing PHT2;1( 1-71) was greater than that of cells expressing PHT2;1, with doubling times of  5.5 and 7.5 h, respectively (Figure 5D). The transport activity of PHT2;1(1-71) was highly specific for Pi, because a 40-fold excess of anions such as sulfate, nitrate, and chloride did not compete for Pi uptake (Table 1).
Although we identified PHT2;1 based on its relatedness to a Na+/Pi symporter, we found that the Pi transport activity mediated by PHT2;1 and PHT2;1( 1-71) was unaffected regardless of whether Na+ was limited to that present solely as a contaminant in other media components or was present at the high concentration optimal for the related fungal Na+/Pi symporters (Figure 5C) (Versaw and Metzenberg, 1995; Martinez and Persson, 1998). Daram et al. (1999) also reported that PHT2;1 activity in yeast was independent of Na+. However, the maximum concentration of Na+ included in their transport assays was 250-fold less than the optimal concentration for the orthologous transporters, and Na+ dependence or enhancement of Pi transport activity may not have been detected under such conditions.
Isolation of a PHT2;1 Null Mutant
Sequencing of these junction fragments confirmed the presence of a T-DNA inserted within the PHT2;1 coding region, with left border and right border insertion sites 209 and 250 bp downstream, respectively, relative to the A of the ATG start codon (Figure 6A). Continued screening reduced the complexity of the population to a single seed pool representing 10 individual T-DNA lines. We screened individual plants from this seed pool and identified several positive for the PHT2;1 insertion. Plants hemizygous or homozygous for the PHT2;1::T-DNA locus were distinguished by PCR (Figure 6B, lanes 5 to 7) and verified by DNA gel blot analysis (Figure 6C). To determine whether the homozygous insertion line contains any additional T-DNA insertions elsewhere in the genome, we probed the same blot with the 5.2-kb T-DNA sequence.
Only one band was observed in the homozygous line (Figure 6C, lane 4), and its size is consistent with insertion of a single T-DNA. In addition, the F2 progeny of a cross between the wild type and the homozygous mutant line showed 3:1 segregation for resistance to kanamycin, consistent with the expected pattern for a single T-DNA insertion (236 kanamycin resistant:78 kanamycin susceptible;
Growth and Pi Content of pht2;1-1 Plants
To confirm that the pht2;1-1mutation rather than another closely linked mutation is responsible for the observed phenotype, we tested the ability of the wild-type PHT2;1 allele to complement the mutation. pht2;1-1 plants were transformed with either the binary vector pSKI089 as a control or PHT2;1/pSKI089, which carries a 4.2-kb segment of the genomic PHT2;1 locus spanning from 1.7 kb upstream to 0.3 kb downstream of the ORF. The leaf Pi content of pht2;1-1 plants transformed with pSKI089 was 78% that of the wild type, whereas the leaf Pi content of pht2;1-1 plants transformed with PHT2;1/pSKI089 did not differ significantly from that of wild-type plants (Table 3). A subset of these plants was examined by reverse transcriptase–mediated PCR to confirm the presence/ absence of PHT2;1 transcripts, and as expected, pht2;1-1 plants transformed with pSKI089 lacked PHT2;1 transcripts. These results indicate that PHT2;1 is sufficient for complementation of the phenotype associated with the pht2;1-1 mutation. All noted differences are significant at a confidence level of >95% (Student's t test).
Effect of Pi Supply on Plant Growth and Pi Accumulation To assess the effect of Pi supply on pht2;1-1 plants and to allow analysis of roots, we grew plants under defined nutrient conditions on agar medium and fertilized sand. As shown in Figure 7 , plants grown on agar medium and sand had similar responses to Pi supply. Under high-Pi conditions, pht2;1-1 rosette fresh weight and Pi content were 80 and 70% those of the wild type, respectively, a phenotype similar to that observed in plants grown on Metro Mix 350. Under low-Pi conditions, we found no significant differences in the fresh weight of either roots or rosettes.
The total phosphate content of whole plants also showed no significant difference (Table 4). However, pht2;1-1 root Pi content was 52 to 70% that of the wild type, and leaf Pi content was 129 to 156% that of the wild type, indicating that under low-Pi growth conditions, the pht2;1-1 mutation affects Pi allocation at the whole-plant level. The total phosphate content of wild-type and pht2;1-1 tissues showed differences equivalent to those observed for Pi, indicating that cellular Pi but not organic phosphate content is altered in the mutant (Table 4). In addition, when plants were grown under the same nutritional regimes but with exposure to continuous light, the Pi content of all tissues was diminished, yet the relative differences between the wild type and pht2;1-1 were maintained (data not shown). All noted differences between the wild type and pht2;1-1 are significant at a confidence level of >95% (Student's t test).
Increased Expression of Pi-Regulated Genes in the pht2;1-1 Background We examined the effect of the pht2;1-1 mutation on the expression of several genes known to be upregulated in response to Pi deprivation. These included AtPT1 and AtPT2, which encode high-affinity Pi transporters that are expressed predominantly in the root (Muchhal et al., 1996 ), and PAP1, which encodes a purple acid phosphatase (Patel et al., 1997). Plants were grown on washed sand, fertilized with 1 mM Pi for 3 weeks, and then fertilized weekly with 0 or 1 mM Pi for an additional 3 weeks before harvest. RNA was isolated from pooled rosettes and roots, respectively, of eight plants grown under each condition. RNA gel blot analysis showed that expression of each of the Pi-regulated genes was greater under Pi-deprived conditions in both wild-type and pht2;1-1 plants, indicating that the mechanisms responsible for this Pi deprivation response are functional in the mutant (Figure 8A) . However, when the relative levels of expression of these genes in pht2;1-1 tissues were compared with those of the wild type, it was clear that expression was greater in the mutant under Pi-deprived conditions but not under Pi-sufficient conditions.
Although the increased level of expression for any individual gene was slight, collectively, the trend for increased expression under Pi-deprived conditions was significant (P = 0.02). Similar results were observed from an additional independent experiment as well as from plants grown for 4 weeks on agar medium containing 0.2 and 4 mM Pi (data not shown). By contrast, RNA gel blot analysis using the same RNA samples described above but hybridized with a TPT cDNA probe showed no significant difference in expression between the genetic backgrounds (Figure 8B).
Retranslocation of Pi in Leaves during Pi Deprivation As shown in Figure 9 , wild-type plants displayed the expected redistribution of Pi, that is, the ratio of young-to-old leaf Pi content increased with time of Pi deprivation. The increasing ratio represents both a reduction in the Pi content of old leaves and a modest increase in young leaves. By contrast, pht2;1-1 plants did not undergo a similar redistribution over this time course; the Pi content of both old and young leaves remained nearly constant.
Although some members of the major facilitator superfamily are known to play a role in transport into organelles, until now, transporters of the PiT and PHS families have not been found in organellar membranes. The initial clue to the location of PHT2;1 came from its long N-terminal extension, which is a feature unique to the plant members of the PiT family. In other transport families, extended N-terminal regions have been associated with regulation (Harper et al., 1998 ; Pittman and Hirschi, 2001), and Daram et al. (1999) proposed this function for PHT2;1 as well. However, computer predictions indicated that the N-terminal region probably was a transit peptide, and localization of a PHT2;1-GFP fusion protein confirmed that PHT2;1 resides in the chloroplast. The ChloroP 1.1 program predicted correctly a chloroplast localization for PHT2;1 and suggested a cleavage site between amino acids 71 and 72, which results in a transit peptide similar in length to those of the TPTs (Fischer et al., 1994).
Some chloroplast transit peptides target their attached preproteins to mitochondria when expressed in fungal cells (Hurt et al., 1986
At least two possible explanations exist to account for the activity attributed to its presence. First, the uptake activity may reflect only a small, undetected portion of the expressed protein that has reached the plasma membrane via a nonspecific mechanism. Alternatively, insertion of PHT2;1 into mitochondrial membranes, where it may be functional, could have pleiotropic effects on the control of Pi homeostasis. Thus, Pi transport across the plasma membrane may be affected indirectly, which presumably would involve the endogenous low-affinity Pi transporters (Tamai et al., 1985
To investigate PHT2;1 Pi transport activity, we compared transport mediated by PHT2;1 with that mediated by PHT2;1(
Based on the in planta localization of the full-length transporter, features of the transit peptide, and functional analyses in yeast, we conclude that PHT2;1 is a low-affinity Pi transporter located in the chloroplast inner envelope membrane. The pH dependence of Pi transport and the inhibitory effect of protonophores (Daram et al., 1999
The low affinity for Pi displayed by PHT2;1 is consistent with the relatively high Pi concentration expected within the cytoplasm. Although measurement of the Pi content of subcellular compartments is difficult and may not accurately represent the metabolically active concentration, estimates of 10 to 15 mM for the cytoplasm (Mimura, 1999
Most transgenic plants with diminished levels of TPT lack a visible phenotype (Riesmeier et al., 1993
Our investigation of the null mutant pht2;1-1 reveals that PHT2;1 function affects the accumulation of Pi in leaves and the allocation of Pi throughout the plant. Under high-Pi growth conditions, the reduction in leaf Pi content and the overall reduction in growth are consistent with Pi limitation of photosynthesis (Walker and Sivak, 1986 It is possible that TPT is solely responsible for the remaining import activity. However, other uncharacterized transporters also may share this function. Considering this possibility, it is interesting that at least five sequences exist within the Arabidopsis genome that encode proteins with similarity to mammalian type I Na+/Pi symporters, three of which are predicted to be targeted to the chloroplast. It might be expected that under low-Pi growth conditions, the pht2;1-1 phenotypes would be similar to or even more extreme than those observed under high-Pi conditions. However, although the Pi content of pht2;1-1 roots was diminished during growth under low-Pi conditions, the root and rosette mass were comparable to those of the wild type, whereas the Pi content of the leaves was significantly greater than that of the wild type. These data indicate that under low-Pi conditions, the allocation of Pi within pht2;1-1 is altered, and apparently, the increased levels of Pi in leaves is sufficient to enable growth comparable to that of the wild type.
It is known that Pi deficiency is accompanied by increases in the translocation and retranslocation of Pi between shoots and roots (Bieleski, 1973
By contrast, in microorganisms, Pi-starvation response systems and the mechanisms of their regulation have been well characterized (Wanner, 1993
The phosphate status of the shoot appears to control Pi uptake and other Pi-starvation responses in the roots, and although the mechanism is unknown, it is thought to involve retranslocation of Pi from the shoots to the root (Drew and Saker, 1984 Similarly, one possible explanation for the phenotypes observed in pht2;1-1 is that under low-Pi growth conditions, retranslocation of Pi between the shoot and the root is impaired, resulting in higher Pi concentrations in the leaves and enhanced expression of Pi-starvation response genes in the roots. It is not known whether pht2;1-1 is impaired in its ability to retranslocate Pi to the roots; however, it is clear that pht2;1-1 fails to retranslocate Pi efficiently from old leaves to young leaves, a phenotype that is consistent with this hypothesis. The mechanisms by which plants coordinate Pi acquisition and allocation to meet the varying demands of both photosynthetic and nonphotosynthetic tissues remain largely unknown. The pht2;1-1 mutant reveals that PHT2;1 is an important component of this process and demonstrates that this novel chloroplast transporter influences the allocation of Pi throughout the plant and affects the expression of Pi-starvation responses. The mechanisms by which PHT2;1 affects these processes will be the subject of future investigations.
It is possible that the involvement is indirect and that the distribution of Pi within the plant is controlled in response to the relative levels of Pi in the various subcellular compartments, including the chloroplast. Alternatively, PHT2;1 might be involved directly, as in yeast and E. coli, in which protein complexes that include Pi transporters are involved in the sensing and signaling of phosphate status and affect downstream responses through interaction with regulatory proteins (Webb and Cox, 1994
Plasmids PHT2;1 cDNA was amplified from Arabidopsis thaliana ecotype Columbia RNA by reverse transcriptase–mediated (RT)–PCR using an oligonucleotide-(dT)18 primer in the reverse transcriptase reaction, followed by PCR with gene-specific primers based on the ATORF02 sequence. PCR primers were designed to introduce unique BamHI and XhoI sites at the 5' and 3' ends of the gene, respectively (primer B, 5'-CGTGGATCCATGACTCTTCCTTATCG-3'; primer E, 5'-GTCCTCGAGCATGTTCTTCGTATAAC-3'). The amplified 1.8-kb cDNA was cloned into the yeast expression vector pWV3, creating PHT2;1/pWV3. The vector pWV3 is a derivative of pGAD GH (Clontech, Palo Alto, CA), in which the region encoding the GAL4 activation domain has been deleted. Both strands of the PHT2;1 insert were sequenced to ensure the integrity of the amplified sequence.
For localization experiments in Arabidopsis, a PCR-generated SalI fragment containing the complete open reading frame (ORF) of PHT2;1 was cloned in frame to the 5' end of green fluorescent protein (GFP) in the plasmid CaMV35S-sGFP(S65T)-nos (Chiu et al., 1996
For complementation of pht2;1-1, a 4.2-kb segment of the PHT2;1 locus, spanning from 1.7 kb upstream to 0.3 kb downstream of the ORF, was amplified by PCR from Arabidopsis ecotype Columbia genomic DNA. The 4.2-kb segment was digested with SpeI and cloned into the binary vector pSKI089 to create PHT2;1/pSKI089. The vector pSKI089 is a derivative of pSKI006 (Weigel et al., 2000
Localization of GFP Fusion Proteins
For localization experiments in yeast, the chimeric constructs PHT2;1-GFP/pWV3 and PHT2;1( Arabidopsis and yeast cells were imaged with a Bio-Rad 1024 ES confocal laser scanning microscope equipped with a x63 water-immersion objective (numerical aperture of 1.2). Optical sections were scanned three times and the signals were averaged according to the Kalman equation (LaserSharp MRC-1024 software, Bio-Rad, Hercules, CA). Fluorescence from GFP was imaged using the 488-nm line of the krypton-argon laser, and emission was detected at 522 nm, whereas chlorophyll autofluorescence was excited at 488 nm, and emission was detected at 680 nm. Fluorescence from MitoTracker Red was excited by light at 568 nm, and emission was detected at 590 nm. Concurrent transmitted light images were recorded with differential interference contrast optics and the transmitted light detector.
Yeast Pi Transport and Growth Assays The standard growth/uptake medium contained an equivalent concentration of Na+ ions at the same pH value, as determined by titration with NaOH. Competition assays were conducted in 25 mM Na-citrate buffer, pH 4.0, containing 2% Glc. For growth comparisons, transformed cells were grown to the midlogarithmic phase in liquid low-Pi medium (SD-Leu medium containing 220 µM KH2PO4 and 25 mM Na-citrate, pH 4.0), harvested by centrifugation, and suspended in fresh medium. Cultures were incubated at 30°C with agitation (150 rpm), and OD600 was monitored over time as a measurement of growth.
Plant Growth Conditions
For growth on agar-solidified medium, surface-sterilized seeds were grown for 4 weeks on half-strength Murashige and Skoog (1962) For growth on sand, plants were transferred to 36-cell packs (one plant per cell) containing acid-washed sand for an additional 3 weeks and fertilized weekly with half-strength Hoagland solution containing either 0.01 or 1.0 mM Pi. For Pi-starvation experiments, plants were grown on washed sand and fertilized weekly for 3 weeks with half-strength Hoagland solution containing 1 mM Pi, then fertilized weekly with half-strength Hoagland solution containing 0 or 1 mM Pi for an additional 3 weeks before tissues were harvested. A single lot of agar, which contributed 7.6 µM Pi to the medium when used at 0.7% (w/v), was used throughout this study. Pi was added to medium and fertilizer as KH2PO4. When the Pi concentration was varied, K2SO4 was added to maintain a constant K+ concentration.
PHT2;1 Mutant Screen Both strands of the amplified junction fragments were sequenced to confirm the presence of PHT2;1 sequence and to determine the T-DNA insertion site. Seeds from the positive pool of T-DNA lines were grown, and individual plants were screened by PCR as described above. Plants hemizygous or homozygous for the PHT2;1::T-DNA locus were distinguished by PCR using a combination of two PHT2;1-specific primers (primer C, 5'-CTTCCTTATCGTTTCTCTTCCG-3'; primer D, 5'-ATATACAAGCCCAAACCCAACC-3') and the right border primer (primer G). An individual line homozygous for the T-DNA insertion was identified and verified by DNA gel blot analysis. We named this line and the corresponding mutant allele pht2;1-1.
Complementation of pht2;1-1 A subset of the transformed plants were analyzed further by RT-PCR to confirm the presence of PHT2;1 transcripts. RT-PCR was conducted on 1 µg of total RNA isolated from leaves of individual plants. A 582-bp fragment of the PHT2;1 gene was amplified using 5'-CTCCTCTCTTCCTTAGCTGCAGCTG-3' and 5'-GCGAATGAC-ATGAAGCAAGCGGAGAG-3'. As a control, a 492-bp EIF-4A2 gene fragment was amplified using 5'-GCAAGAGAATCTTCTTAGGGGTATCTATGC-3' and 5'-GGTGGGAGAAGCTGGAATATGTCATAG-3'. The primers chosen for the amplification of PHT2;1 and EIF-4A2 gene fragments were from separate exons, and all primers were included in each PCR procedure.
Pi Content Determination
Plant Nucleic Acid Isolation and Blotting Upon request, all novel materials described in this article will be made available in a timely manner for noncommercial research purposes. No restrictions or conditions will be placed on the use of any materials described in this article that would limit their use for noncommercial research purposes.
Accession Numbers
We thank G. May and W. Schneider for critical review of the manuscript and the members of the Plant Biology Division for helpful discussions. This work was supported by The Samuel Roberts Noble Foundation.
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.002220. Received February 8, 2002; accepted April 24, 2002.
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Abstract
INTRODUCTION
-helices separated by a hydrophilic loop (Laloi, 1999
2 = 0.004, P = 0.94). RNA gel blot analysis of leaf RNA confirmed that the homozygous mutant lacks PHT2;1 transcripts (
