Of the mineral nutrients essential for plant growth, phosphorus plays the widest diversity of roles and a lack of phosphorus has profound effects on cellular metabolism. At least eight members of the Arabidopsis Pht1 phosphate (Pi) transporter family are expressed in roots and Pht1;1 and Pht1;4 show the highest transcript levels. The spatial and temporal expression patterns of these two genes show extensive overlap. To elucidate the in planta roles of Pht1;1 and Pht1;4, we identified loss-of-function mutants and also created a double mutant, lacking both Pht1;1 and Pht1;4. Consistent with their spatial expression patterns, membrane location and designation as high-affinity Pi transporters, Pht1;1 and Pht1;4 contribute to Pi transport in roots during growth under low-Pi conditions. In addition, during growth under high-Pi conditions, the double mutant shows a 75% reduction in Pi uptake capacity relative to wildtype. Thus, Pht1;1 and Pht1;4 play significant roles in Pi acquisition from both low- and high-Pi environments.
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Plant roots encounter heterogeneous, constantly fluctuating environments from which they must acquire a range of essential mineral nutrients in order to survive (Marschner, 1995; Schachtman et al., 1998). Phosphorus (P) is one of these mineral nutrients and is of particular importance because of the complex array of metabolic processes in which it is involved. As a central component of photosynthesis, carbon metabolism and energy transfer reactions, its supply, concentrations and allocation throughout the plant have to be maintained within critical limits for optimal growth and development (Schachtman et al., 1998). Although P is abundant in biological systems, the concentrations of plant-available P in soils throughout the world are low and often limit plant growth (Bieleski, 1973; Bieleski and Ferguson, 1983). In agricultural situations, P limitation impacts crop yield and while the application of P-based fertilizers can address these deficiencies, the immediate recovery of P is estimated to be less than 10%. The remainder is either immobilized in the soil or carried into ground water and rivers, causing pollution (Holford, 1997; Richardson, 1994). An understanding of the mechanisms underlying P acquisition by plants could provide a starting point from which to breed or engineer plants with superior P acquisition characteristics, or to improve P-fertilizer usage.
Plants acquire P exclusively in its ionic forms from the soil solution and have evolved a range of adaptive responses to enhance its acquisition. Phosphate (Pi) status is coupled to plant development and Pi deficiency triggers modifications in root growth and architecture and also secretion of enzymes including RNAses and phosphatases (Abel et al., 2000; Bariola et al., 1994; Duff et al., 1991; Lynch, 1995; Ma et al., 2003; Ticconi et al., 2004; Williamson et al., 2001). Together these responses enable both the exploitation of increased volumes of soil and the liberation of Pi from complex forms. A further mechanism of improving Pi acquisition is the formation of symbiotic associations with arbuscular mycorrhizal fungi. These associations are widespread throughout the plant kingdom and result in an increased Pi supply for the plant (Harrison, 1999; Smith and Read, 1997).
Pi transport from the soil into the root cells is the first and crucial step in the assimilation of P. To achieve this, the plant has to transport Pi against a significant concentration gradient, the concentration of Pi in the cell being 1000–10 000 times greater than the soil solution. In addition, the transport system(s) must overcome the negative membrane potential of the plant cell. Pi uptake systems in plant roots operate over a broad range of Pi concentrations and initial studies revealed biphasic kinetics that were interpreted to result from a combination of low- and high-affinity transport systems (Bieleski, 1973; Bieleski and Ferguson, 1983; Epstein, 1972; Marschner, 1995). Similar observations and interpretations were made for other mineral nutrients, including N, where the subsequent cloning of both high- and low-affinity nitrate transporters supported the interpretations made from the transport studies (Trueman et al., 1996; Tsay et al., 1993).
Analysis of the rice and Arabidopsis genomes revealed at least 13 and nine members of the Pht1 family in these species, respectively (Mudge et al., 2002; Okumura et al., 1998; Paszkowski et al., 2002). In rice, 10 of the 13 Pi transporters are expressed in roots and one is expressed only following development of an AM symbiosis (Paszkowski et al., 2002). The latter finding correlates with potato and M. truncatula where single mycorrhiza-specific Pi transporters have been described (Harrison et al., 2002; Rausch et al., 2001). In Arabidopsis eight of the nine members of the Pht1 family are expressed in roots and transcript levels are elevated in response to Pi deprivation. The spatial and temporal expression patterns have been catalogued recently but their individual roles and contribution to Pi transport in the plant are unknown (Karthikeyan et al., 2002; Mudge et al., 2002).
As a first step to understand the in planta roles of the individual Pi transporters, we isolated mutants defective in the expression of Pht1;1 and Pht1;4. A double mutant, pht1;1Δ4Δ, lacking both Pht1;1 and Pht1;4 was created to study the effects of the loss of both transporters. Analyses of the mutants revealed that Pht1;1 and Pht1;4 play a significant role in Pi uptake into roots during growth in a wide range of external Pi concentrations.
Phosphate transporter insertion mutants
Of the eight members of the ArabidopsisPht1 family expressed in roots, Pht1;1 and Pht1;4 are the most highly expressed and transcript levels increase substantially in response to Pi deprivation. Promoter analyses indicate a partial overlap in their spatial expression patterns with the strongest expression in the epidermis, particularly in the root hair cells and also in the tip of lateral roots (Karthikeyan et al., 2002; Muchhal et al., 1996; Mudge et al., 2002; Smith et al., 1997). As observed for M. truncatula and tomato Pht1 family Pi transporters (Chiou et al., 2001; Liu et al., 1998a), Pht1;1 is located in the plasma membrane (Figure S1).
To determine the contribution of Pht1;1 and Pht1;4 to Pi uptake in Arabidopsis roots, we used a reverse genetic approach to search for loss of function mutants. Two Pht1;1 mutants, pht1;1-1 and pht1;1-2, and two Pht1;4 mutants, pht1;4-1 and pht1;4-2, were identified (Figure 1a). The pht1;1-1 and pht1;4-1 mutants are in the Ws ecotype, while pht1;1-2 and pht1;4-2 are ecotype Columbia. In each case, the presence and location of the T-DNA or transposon insertion was verified by junction PCR and sequencing, and homozygous lines were identified by PCR. In pht1;1-1, a partial T-DNA, comprising 290 nucleotides of the left-border region of T-DNA, was present in exon 3 of the Pht1;1 gene. The T-DNA had inserted between 769 and 801 nucleotides downstream of the ATG start codon of Pht1;1 and 31 nucleotides of the Pht1;1 coding region had been deleted. The pht1;1-2 mutant contained a T-DNA insert between 596 and 614 nucleotides downstream of the ATG codon, with 19 nucleotides deleted from the Pht1;1 coding sequence. In pht1;4-1, the T-DNA had inserted between 992 and 941 nucleotides upstream of the ATG codon and 50 nucleotides of the Pht1;4 intron had been deleted. The pht1;4-2 mutant contained a single dSpm transposon insertion in exon 2 of the Pht1;4 gene between 1233 and 1255 nucleotides downstream of the ATG codon. In each line, DNA gel blot analyses were consistent with the presence of single insertions in the genomes (Figure 1b). The three hybridizing fragments visible in the pht1;1-2 blot are the result of XbaI sites within the T-DNA insert coupled with left borders at both ends of the insertion. For pht1;4-2, a line homozygous for the insertion was backcrossed to wildtype. The F2 progeny segregated 3:1 for resistance to kanamycin (92 kanamycin-resistant: 35 kanamycin-susceptible; χ2 = 0.316, P ≫ 0.05), a further indication of a single transposon insertion in the genome.
RNA gel blot and RT-PCR analyses were used to monitor the expression of Pht1;1 and Pht1;4 in wildtype and mutant lines (Figure 1c,d). Plants were grown under low-Pi and high-Pi conditions either in sand (Figure 1c), or in semi-solid, nutrient media (Figure 1d). As an indicator of the Pi status, we monitored expression of At4, a gene whose expression is strongly upregulated by Pi starvation (Burleigh and Harrison, 1999). At4 transcripts were elevated in both wildtype and mutant lines grown under low-Pi conditions, indicating that the plants were Pi-starved. Consistent with previous data, Pht1;1 and Pht1;4 transcript levels increased significantly in wildtype roots following Pi-starvation (Karthikeyan et al., 2002; Muchhal et al., 1996; Mudge et al., 2002; Smith et al., 1997). In the pht1;1-1 mutant, Pht1;1 transcripts were not detected in plants grown under high-Pi conditions but under low-Pi conditions, the Pht1;1 probe detected a transcript that was larger than the wildtype Pht1;1 transcript (Figure 1c). The transcript is larger as a result of the T-DNA insertion and as transcript levels were significantly lower than those observed in the wildtype, it appears that the insertion affects the stability of the mRNA. Sequencing revealed that this chimeric transcript contained a T-DNA insert of 180 nucleotides. Although the T-DNA insertion in the Pht1;1 gene was 290 nucleotides, 110 nucleotides from the 3′ region of the T-DNA was missing probably as a result of an RNA splicing event with a splice site arising from the T-DNA insertion. Translation of this mutant transcript would not produce a full-length Pht1;1 protein. The T-DNA disrupts the open reading frame after amino acid 206 and contains multiple, in-frame stop codons, preventing translation of a larger product. A partial protein of 206 amino acids is less than half the size of the mature protein (524 amino acids) and analyses of other 12 transmembrane domain transporters indicate that a partial protein of this size would not be functional (Caspari et al., 1994; Will et al., 1998).
RT-PCR analyses revealed that the pht1;1-2 mutant is a null allele and Pht1;1 transcripts were not present under high- or low-Pi growth conditions (Figure 1d, right panel). The transcripts detected in the pht1;1-2 mutant by Northern blot analysis (Figure 1d, left panel) arise from cross-hybridization of the Pht1;1 probe with two related genes, Pht1;2 and Pht1;3. These two genes share 89 and 97% nucleotide identity with Pht1;1. They are expressed at lower levels than Pht1;1 and transcript levels are elevated following Pi starvation (Mudge et al., 2002; Smith et al., 1997). The Pht1;2/Pht1;3 transcripts are less apparent when the plants are grown in sand (Figure 1c). Using growth condition similar to those used in Figure 1d, we were able to detect expression of Pht1;2/Pht1;3 genes in the pht1;1-1 mutant also (data not shown).
In both of the Pht1;4 mutants, the Pht1;4 transcript was not detectable under either low- or high-Pi growth conditions, even after 3 days exposure (Figure 1c). Furthermore, the Pht1;4 transcript could not be amplified by RT-PCR, indicating that the mutants are null alleles.
As previous analyses indicated a partial overlap in the expression patterns of the Pht1;1 and Pht1;4 genes, we created a double mutant to enable evaluation of loss of both Pi transporters. The double mutant, pht1;1Δ4Δ, was generated by crossing pht1;1-1 to pht1;4-1 and the double homozygote was identified from the F2 generation by PCR.
Expression of members of the Pht1 phosphate transporter gene family in pht1;1Δ4Δ
Analysis of the Arabidopsis genome sequence suggests that there are at least nine members of the Pht1 Pi transporter family, eight of which are expressed in roots (Mudge et al., 2002) (Figure S2). Two genes, Pht1;2 and Pht1;3 have spatial expression patterns that overlap partially with Pht1;1 and Pht1;4. Before embarking on analysis of the Pi transport characteristics of the mutants, we monitored the transcript levels of the eight Pht1 family members expressed in roots to determine whether their expression was significantly altered in pht1;1-1, pht1;4-1 and pht1;1Δ4Δ relative to wildtype. Northern blot analyses indicated that there were no significant differences in the transcript levels of the Pht1;4, 1;5, 1;7, 1;8 and 1;9 genes in the pht1;1-1 and pht1;4-1 mutants relative to wildtype (Figure S3). Pht1;1, Pht1;2 and Pht1;3 transcripts cannot be individually resolved in this analysis but the combined transcript level in the Pht1;4-1 mutant was marginally higher than wildtype. For analysis of the double mutant, pht1;1Δ4Δ,mutant and wildtype lines were grown in media under two different Pi regimes and transcript levels of the eight Pht1 family genes were monitored by RT-PCR (Figure 2). Consistent with previous studies, transcript levels of all members of the Pht1 family increased during growth in low-Pi (2 μm Pi) conditions but levels were not significantly different in pht1;1Δ4Δ relative to wildtype. In high-Pi (500 μm Pi) conditions, the At4 transcript level was significantly higher in the double mutant relative to wildtype, suggesting that the double mutant may be experiencing Pi-starvation even during growth with high Pi. At4 transcripts were elevated, although to a lesser extent, in the single mutants also (Figure 1c,d).
Pi uptake rates are altered in the Pht1;1, Pht1;4 and pht1;1Δ4Δ mutants
To investigate the contribution of Pht1;1 and Pht1;4 to total Pi uptake by Arabidopsis roots, plants were grown in low-Pi (2 μm) or high-Pi (500 μm) media and Pi uptake rates were measured at the same Pi concentration (Figure 3a). Following growth at 2 μm Pi, the Pi uptake rates in pht1;1-1 and pht1;1-2 were reduced to 82 ± 9% and 77 ± 3%of the wildtype rate, while the Pi uptake rates in pht1;4-1 and pht1;4-2 were reduced to 78 ± 6% and 57 ± 7% of the wildtype rate. At 500 μm Pi, the pht1;1-1 and pht1;1-2 Pi uptake rates were only 66 ± 7% and 59 ± 4% of the wildtype rate, respectively, a greater reduction than that observed at low-Pi. By contrast, Pi uptake rates in the Pht1;4 mutants had increased slightly. In pht1;4-1, the Pi uptake rate was 107 ± 4% of the wildtype rate, which is not statistically different to the wildtype; however, the Pi uptake rate of pht1;4-2 was 125 ± 10% of the wildtype rate, suggesting a compensation mechanism by other Pi transporters. Interestingly, pht1;4-2 (Col-O background) showed a greater reduction than pht1;4-1 (Ws background) at low-Pi and a greater increase at high-Pi, pointing to differences in the relative contribution of Pht1;4 to total Pi uptake in the different ecotypes.
RNA was isolated from the plants grown under the same conditions as above and RT-PCR analysis was used to monitor the expression levels of the Pht1;1 and Pht1;4 genes (Figure 3b and Table 1). In plants grown with high Pi, Pht1;1 transcript levels were higher in the Pht1;4 single mutants, pht1;4-1 and pht1;4-2, compared with wildtype. This suggests that the increases in Pi uptake rates of the Pht1;4 mutants at high Pi may be the result of compensation by Pht1;1. The transcript levels of the Pht1;2 and Pht1;3 genes, the two family members, with spatial expression patterns that overlap partially with Pht1;1 and Pht1;4 were not altered (Figure S4).
Table 1. Expression of Pht1;1 and Pht1;4 in different genetic backgrounds
Mean relative expression levels (% ± SE) in different genetic backgrounds
Relative semi-quantitative RT-PCR analysis. Data are the expression levels as a percentage of expression of that gene in the appropriate wildtype. Wildtype background Wsa and Colb were designated 100%. PCR experiments were replicated at least three times for each gene to obtain mean values ± SE.
2.4 ± 1.1
73.4 ± 1.2
1.2 ± 0.3
92.1 ± 2.4
3.6 ± 2.1
154.7 ± 6.6
2.0 ± 1.5
169.2 ± 7.3
89.6 ± 4.7
101.5 ± 5.7
87.2 ± 10.6
97.8 ± 7.2
131.6 ± 7.5
165.8 ± 5.7
170.6 ± 9.1
112 ± 4.8
134.4 ± 6.3
216.1 ± 12.1
172.6 ± 10.5
282.9 ± 11.2
169.2 ± 7.1
201.7 ± 8.9
The loss of both Pht1;1 and Pht1;4 functions had a significant impact on Pi uptake under both high and low-Pi conditions. The Pi uptake rate of the double mutant was reduced to 57 ± 6% of the wildtype rate at low Pi, and was only 29 ± 6% of the wildtype rate at high Pi. This suggests that the effects of the mutations are additive. The drastic reduction at high Pi implies that Pht1;4 functions under high-Pi conditions also, a finding that was not apparent from analysis of the Pht1;4 single mutants because of compensation by Pht1;1.
The double mutant shows a reduced capacity to exploit a high Pi source following a period of Pi-starvation
The uptake analyses demonstrated that the Pi transporter mutants showed a defect in uptake when grown under two Pi regimes, 2 and 500 μm Pi. To further examine Pi uptake in the double mutant, pht1;1Δ4Δ, we extended the analysis to additional growth conditions in which plants were grown initially at very high Pi levels (2 mm) and then subjected to Pi-starvation by growth in media without additional Pi. Pi uptake was measured from solutions containing 2 or 500 μm, which are the same conditions used in the previous experiment. Consistent with the data in Figure 3, the double mutant showed a significant reduction in Pi uptake compared with wildtype regardless of the Pi growth condition (Table 2). Following growth at 2 mm Pi, the Pi uptake rate of pht1;1Δ4Δ was 20.9 or 24.1% of the wildtype rate when determined at 2 or 500 μm, respectively. Following Pi-starvation, the Pi uptake rates were 44.3 and 22% of the wildtype rates at 2 or 500 μm, respectively. This is consistent with the initial uptake data and suggests that Pht1;1 and Pht1;4 are responsible for a significant portion of the total Pi uptake capacity of Arabidopsis roots under both low- and high-Pi conditions.
Table 2. Pi-uptake in the pht1;1Δ4Δ mutant is reduced during growth in either Pi-sufficient (Pi+) or Pi-starved (Pi−) conditions
Pi uptake rate
2 μm Pi
500 μm Pi
Pi uptake rates are nmol Pi g−1 FW h−1.
0.162 ± 0.009
17.03 ± 2.62
0.034 ± 0.004
4.11 ± 0.38
4.81 ± 0.61
257.08 ± 40.21
2.13 ± 0.03
56.64 ± 6.11
0.022 ± 0.003
4.85 ± 0.61
0.011 ± 0.001
2.20 ± 0.38
0.15 ± 0.05
21.40 ± 1.66
0.08 ± 0.01
4.19 ± 0.63
As expected, the Pi uptake rates of both the wildtype and pht1;1Δ4Δ lines were higher following Pi-starvation, which demonstrates that other Pi-starvation-inducible Pi transporter activities are functional in pht1;1Δ4Δ. More interestingly, however, the measurements of Pi uptake from the two Pi solutions revealed significant differences between wildtype and pht1;1Δ4Δ in their capacity to exploit a high Pi source and to rapidly accumulate Pi in both roots and shoots following a period of starvation. Following growth in Pi-starvation conditions, the uptake rates at 500 μm Pi were greater than at 2 μm Pi and wildtype rates were always higher than those of pht1;1Δ4Δ. Notably, the difference in the root Pi uptake rates between wildtype and pht1;1Δ4Δ was approximately 2.7 nmol Pi g−1 FW h−1 at 2 μm and 200 nmol Pi g−1 FW h−1 at 500 μm Pi, indicating the significance of Pht1;1 and Pht1;4 in exploiting a high Pi source after Pi starvation. Furthermore, a comparison of uptake rates from the 500 and 2 μm Pi solutions showed that the wildtype line has the capacity to increase Pi uptake rates in the root 53-fold, while pht1;1Δ4Δ shows only a 25-fold increase. These data further indicate that Pht1;1 and Pht1;4 comprise a significant component of the Pi-starvation-inducible Pi uptake capacity of the root system and contribute to Pi uptake from both low- and high-Pi environments.
Loss of Pht1;1 and Pht1;4 functions impact Pi content and plant growth
To determine whether the defect in the Pi uptake capacity affects Pi content and plant growth, we measured the Pi content and fresh weights of shoots from plants grown on agar media containing a range of Pi concentrations. The pht1;1-1 mutant showed a reduction in Pi content of the shoots relative to wildtype with the most significant differences at 0.5 and 1.0 mm Pi (Figure 4a). As shown in Table 3, in the F2 progeny obtained from a cross between pht1;1-1 and wildtype, the low-Pi content phenotype co-segregated with the T-DNA insertion.
Table 3. The reduction in shoot Pi content co-segregates with a T-DNA insertion in the Pht1;1 gene
No. of plants
Shoot Pi content (nmol mg−1 fresh weight)
pht1;1-1 was backcrossed to wildtype and the resulting F1 were selfed. Sixty-nine F2 progeny were grown for 17 days on agar-solidified medium containing 1 mm Pi. Genotypes were determined by PCR. Shoot Pi content was determined for 10 plants of each genotype. Values represent mean ± SE for whole shoot tissue from each plant.
9.80 ± 0.33
9.13 ± 0.19
6.37 ± 0.17
The Pi content of the pht1;4 mutants was not significantly different to wildtype (Figure 4a and data not shown). In contrast, pht1;1Δ4Δ showed highly significant reductions in Pi content at 0.5 and 1 mm Pi with levels between 56 and 83% of those seen in wildtype (Figure 4a).
Differences in the growth of wildtype and the Pht1;1 and Pht1;4 mutants were not consistently significant under these conditions, however, the double mutant, pht1;1Δ4Δ exhibited significantly slower growth at Pi concentrations over 0.2 mm. As the Pi concentration increased from 0.1 to 1 mm, the mass of wildtype shoots increased 79.8%, while that of pht1;1Δ4Δ increased only 34.1% (Figure 4b). This reduction in growth rate was clearly visible (Figure 5a). Other visible phenotypic differences between pht1;1Δ4Δ and wildtype could be seen following prolonged growth under extremely low-Pi conditions, below the levels assessed in Figure 4. The double mutant displayed more severe Pi-starvation symptoms than wildtype, including a visible change in leaf color from green to yellowish purple, probably associated with anthocyanin accumulation and senescence (Figure 5b). Together, these results indicate that the double mutant has lost a major portion of the total Pi uptake capability, and other Pi transporters cannot compensate for this, particularly during growth under high-Pi conditions.
Root hair and lateral root growth rates are higher in the pht1;1Δ4Δmutant
The root-to-shoot ratios of the double mutant and wildtype plants were determined following growth on media containing 500 μm Pi (Table 4). The results are consistent with the suggestion that the double mutant is experiencing Pi deprivation even during growth in high-Pi conditions, and the root-to-shoot ratio of the pht1;1Δ4Δ was 0.46 ± 0.02 compared with 0.39 ± 0.02 in the wildtype. In this experiment, the Pi content of pht1;1Δ4Δ shoots and roots were 30 and 51% of wildtype content, respectively.
Table 4. Root-to-shoot (R:S) ratio and Pi content in pht1;1Δ4Δ
Growth (mg FW)
Pi content (nmol mg−1 FW)
Data represent mean ± SE (n = 20 from duplicate experiments).
19.09 ± 1.30
7.44 ± 0.59
0.39 ± 0.02
10.97 ± 0.53
6.90 ± 0.30
11.52 ± 0.87
5.33 ± 0.50
0.46 ± 0.03
3.36 ± 0.08
3.55 ± 0.30
To examine the effect of Pi availability on the growth of root hairs, plants were grown initially at high Pi (1 mm) and then transferred to low Pi, which accelerates the growth of root hairs. Under these conditions, we were able to detect a visible difference in the elongation of root hairs between the wildtype and mutants, especially the double mutant pht1;1Δ4Δ (Table 5 and Figure 5c). The average length of root hairs on the double mutant was 34% longer than those on wildtype roots. Pht1;1-1 showed a similar level of root hair elongation, while pht1;4-1 showed only 9.2% increase compared with wildtype root hairs. By contrast, there were no differences in the length of root hairs on plants grown continuously on high-Pi media.
Table 5. Pi transporter mutants show alterations in root hair and lateral root growth
Root hair length (mm)
LR length (mm)
LR growth rate (mm day−1)
High to low Pi
Root hair length, 24 h following transfer to media containing no additional Pi. Lateral root (LR) length and growth rate 4 and 7 days after the primary roots have entered the high Pi section of the agar plate.
0.33 ± 0.01
0.65 ± 0.02
1.47 ± 0.18
6.53 ± 0.83
1.69 ± 0.26
0.36 ± 0.01
0.87 ± 0.01
2.24 ± 0.24
10.59 ± 0.88
2.78 ± 0.23
Several classical studies have shown that a local application of high Pi stimulates the proliferation of lateral roots within that region (Drew, 1975; Robinson, 1994). We examined the effects of a local Pi supply on lateral root growth in the wildtype and double mutant by growing the plants on agar plates in which only the middle section of the plate contained high Pi (Table 5 and Figure 5d). In response to the local Pi source, wildtype and mutant plants initiated the same number of lateral roots but the elongation rate of lateral roots was 1.65-fold greater in pht1;1Δ4Δ relative to wildtype.
The pht1;1Δ4Δ mutant displays arsenate resistance
Arsenate, the oxyanion of arsenic, is a structural analog of Pi that is probably transported into plant roots via the high-affinity Pi transport system(s). This conclusion was drawn from a series of arsenate and Pi transport studies and the observation that suppression of high-affinity Pi uptake decreased the uptake of arsenate also (Clark et al., 2000; Lee et al., 2003; Meharg and Hartley-Whitaker, 2002; Meharg and Macnair, 1990, 1992). However, a causal relationship with one or more of the root Pi transporters has not been established. Here, we examined the Pht1 mutants to determine whether they are more resistant to arsenate than wildtype plants. As shown in Figure 6, growth of the wildtype plants was impaired significantly in response to increasing levels of arsenate. Growth of pht1;4-1 was not significantly different to wildtype but pht1;1-1 displayed some resistance and growth was greater than wildtype (Figure 6). The double mutant, pht1;1Δ4Δ, displayed significant resistance to arsenate and even in the presence of 200 μm arsenate, growth was reduced by only 50% relative to 0 μm arsenate controls. By contrast, growth of wildtype was reduced by 94% (Figure 6). These results indicate that Pht1;1 and Pht1;4 mediate a significant proportion of the arsenate uptake in Arabidopsis.
Phosphorous is one of the mineral nutrients essential for plant growth and development. It constitutes up to 0.2% of the dry weight of the plant cell and consequently is required in significant quantities (Bieleski and Ferguson, 1983; Schachtman et al., 1998). P assimilation is initiated by the transport of Pi into the roots and the number of Pi transporters present in the roots is perhaps a reflection of the complexity and significance of the process. In Arabidopsis, eight of the nine members of the Pht1 Pi transporter family are expressed in roots and it is assumed that collectively, they mediate Pi uptake from the soil and transport to the shoots (Bucher et al., 2001; Mudge et al., 2002; Okumura et al., 1998). However, the roles of the individual Pi transporters and their contributions to this process are unknown. Four of the eight Pi transporters are expressed in cells at the root–soil interface, including the epidermis, root hair cells and the root cap and of these four, Pht1;1 and Pht1;4 are the most highly expressed. Two other Pi transporters, Pht1;2 and Pht1;3, are expressed at lower levels but have spatial expression patterns that overlap partially with Pht1;1 and Pht1;4. Following Pi-starvation, Pht1;2 is expressed in the epidermal cells but not in the root cap, while Pht1;3 is expressed strongly in the stele with lower levels of expression in the epidermal cells near the tips of the lateral roots (Mudge et al., 2002). Here, via analysis of insertion mutants, we provide direct evidence that Pht1;1 and Pht1;4 play a central role in the acquisition of Pi from the external environment. Furthermore, they play a crucial role in Pi acquisition under both low- and high-Pi conditions.
The members of the Pht1 transporter families from a range of plant species share sequence similarity with the yeast and N. crassa high-affinity Pi transporters, and as the genes encoding these transporters are up-regulated by Pi-starvation it was generally assumed that the transporters would play a more important role during growth in low-Pi conditions (Raghothama, 1999; Rausch and Bucher, 2002). The analysis of the activities of the plant Pi transporters was undertaken in yeast where the results were variable. Consistent with the designation as high-affinity transporters some of these transporters showed Km values in the low micromolar range (Daram et al., 1998); however, others showed Km values of over 200 μm (Leggewie et al., 1997; Liu et al., 1998b). Pht1;4 has not been analyzed but Pht1;1 was expressed in tobacco cells and showed a Km value of 3 μm (Mitsukawa et al., 1997).
Our investigations revealed that the Pht1;1 mutants showed a reduction in Pi uptake capacity under both low- and high-Pi conditions. Given the plasma membrane location of the Pht1;1 protein and expression patterns of Pht1;1 gene, a role in Pi uptake in low-Pi conditions might have been expected (Karthikeyan et al., 2002; Mudge et al., 2002). However, a role in Pi uptake under high-Pi conditions was not predicted. Interestingly, it appears that the other Pi transporters do not compensate for the loss of Pht1;1. By contrast, analysis of the Pht1;4 mutant reveals that under high-Pi conditions, Pht1;1 compensates for the loss of Pht1;4 and this is reflected in the Pi uptake rate of the Pht1;4 mutant lines. Consequently, the role of Pht1;4 in Pi uptake under high-Pi conditions is not apparent from analysis of the single Pht1;4 mutant but is revealed from studies of the double mutant, pht1;1Δ4Δ, where it is apparent that loss of both Pht1;1 and Pht1;4 functions are additive. Under low-Pi conditions, Pi uptake in the double mutant is reduced more than 50% relative to wildtype, while under high-Pi conditions, the reduction is close to 75% relative to wildtype. The Pi content of the shoots and the growth of shoot tissues reflect this significant reduction in Pi uptake. The biomass of the double mutant is reduced significantly and on defined media, visible differences in growth are apparent. These data suggest that under low-Pi conditions, Pht1;1 and Pht1;4 contribute to Pi acquisition but that other transporters, potentially Pht1;2 and Pht1;3, make a significant contribution also. However, the majority of the Pi transport under high-Pi conditions appears to be mediated by the Pht1;1 and Pht1;4 proteins. This interpretation is consistent with the expression patterns. Pht1;2 and Pht1;3 are expressed at very low levels under high-Pi conditions but expression is enhanced significantly under low-Pi conditions (Mudge et al., 2002). However, an alternate interpretation is that the four Pi transporters present in the epidermal cells, may act together in higher order oligomeric structures. Thus, the loss of one protein would have the potential to impact the transport activity of the whole complex. It was shown recently that sucrose transporters located in the sieve elements have the capacity to interact with each other and to form oligomeric complexes (Reinders et al., 2002). Furthermore, studies of the human glucose transporters have demonstrated that they form dimers and oligomers and that the higher order form influences transport (Hebert and Carruthers, 1992). While we currently do not have the appropriate tools to examine potential Arabidopsis Pi transporter oligomers, there is evidence that MtPT1, a Pht1 family transporter from M. truncatula, forms higher order structures, potentially dimers or tetramers (Chiou et al., 2001). Regardless of whether the Arabidopsis Pht1;1 and Pht1;4 proteins are operating collectively or independently, these data clearly indicate their central role in Pi acquisition.
Following transfer from low to high-Pi conditions, plants display a significant increase in Pi uptake capacity, which results from an increase in expression of Pi transporter genes and a concomitant increase in the levels of Pi transporter proteins in the membrane (Chiou et al., 2001; Clarkson and Scattergood, 1982; Cogliatti and Clarkson, 1983; Dunlop et al., 1997). Excess Pi acquired under such conditions is stored in the vacuole and is essential for the plant to survive short-term periods of Pi deprivation (Schachtman et al., 1998). Analysis of the double mutant reveals that the ability to exploit a high-Pi source following a period of Pi-starvation is severely reduced with loss of Pht1;1 and Pht1;4 functions. Following transfer from low to high-Pi conditions, the mutant shows only a 52-fold increase in Pi transported to the shoots, while the wildtype plants show a 140-fold increase. These results indicate that Pht1;1 and Pht1;4 proteins comprise a significant proportion of the Pi-starvation inducible Pi transport capacity of the root and in particular enable the plant to take advantage of a high-Pi supply following a period of Pi-starvation.
Consistent with a reduction in Pi content of the tissue resulting from an impaired Pi uptake capacity, the double mutant displays symptoms of Pi deficiency even during growth under high-Pi conditions. The expression of the At4 gene, a gene induced by Pi-starvation, is higher in the double mutant even under high-Pi conditions. The root-to-shoot ratio and elongation rate of the lateral roots are both higher in the double mutant, and finally, the rate of root hair growth following transfer to low-Pi media is enhanced. Interestingly, significant increases in the transcript levels of the other Pi transporters were not seen. However, this probably reflects the Pi threshold at which these genes respond. Previous studies have indicated that At4 and its orthologs in other species are the most sensitive molecular markers of Pi- starvation and transcriptional increases occur before alterations in the Pi transporter transcript levels (Burleigh and Harrison, 1998, 1999; Liu et al., 1997, 1998a; Versaw and Harrison, 2002).
Collectively, these data illustrate that Pht1;1 and Pht1;4 contribute to Pi uptake by roots under low-Pi conditions and are required also for Pi uptake into roots under higher Pi conditions, particularly above 0.2 mm. While Pi levels in the soils are generally less than 10 μm, nutrient patches with higher concentrations exist (Bieleski, 1973). Thus, transporters such as Pht1;1 and Pht1;4 contribute both to the acquisition of Pi under low-Pi availability and provide a mechanism to rapidly exploit high-Pi containing regions. This is similar to the situation for both potassium and nitrate acquisition by plants. Dual-affinity potassium transporters that function in both low- and high-affinity potassium transport have been identified (Fu and Luan, 1998; Hirsch et al., 1998; Kim et al., 1998). In addition, a nitrate transporter, CHL1, mediates nitrate uptake under low and high-nitrate environments, and shows a dual affinity for nitrate controlled by phosphorylation of the transporter (Liu and Tsay, 2003; Liu et al., 1999; Wang et al., 1998). Our findings are in line with recent studies in yeast that showed that PHO84, a high-affinity, Pi-starvation-inducible Pi transporter, also makes the major contribution to Pi uptake under high-Pi conditions (Wykoff and O'Shea, 2001). Interestingly, as seen here with the pht1;1Δ4Δ mutant, the PHO84 knockout mutant displays symptoms of continual Pi deprivation even under high-Pi conditions.
Our data provide the first insights into the in planta roles of two root Pi transporters and reveal contributions to P acquisition not envisaged previously. These experiments pave the way for the dissection of the complex mechanisms underlying P assimilation in plants.
Plant material and growth conditions
In all experiments, seeds were surface-sterilized and then refrigerated at 4°C in sterile water for 2–4 days to synchronize germination. All plants were grown in growth rooms or chambers at 21–23°C with a light intensity of 100–130 μE m−2 sec−1. If not mentioned specifically, all experiments were carried out under a short-day light cycle (10 h light/14 h dark). For general propagation, plants were grown on Metro Mix 350 (Scotts, Marysville, OH, USA) with a 16-h light/8-h dark cycle.
The defined nutrient media for plant growth was based on 0.5 × Hoagland nutrient solution (Arnon and Hoagland, 1940), and Pi was varied by the addition of KH2PO4. K2SO4 was altered accordingly to maintain a constant K+ concentration in media. The solution was buffered to pH 6.1 with 0.5 mm MES and where appropriate, solidified with 0.8% agar-agar (Sigma, St. Louis, MO, USA), which contributed approximately 9 μm Pi to the media. Media contained sucrose 1% (w/v) but this was omitted from the solution used to fertilize sand cultures.
Plants were analyzed under a wide range of growth conditions. For growth on sand (Figure 1c), plants were grown for 14 days on 0.5 × Hoagland, 1 mm Pi agar plates and then transferred to pots (eight plants per pot) containing acid-washed river sand. During the first 3 weeks after transfer, plants were fertilized weekly with 0.5 × Hoagland, 1 mm Pi solution. For the next 3 weeks, Pi concentration was reduced to 0.02 mm to impose Pi starvation conditions, or it was maintained at 1 mm for high-Pi conditions.
For Pi uptake experiments (Figure 3), plants were grown in semi-solid agar media. Approximately 50 sterile seeds were sown on a 300-μm nylon mesh (2 × 2 cm2) placed in a Magenta box on the top of 0.5 × Hoagland, 0.3% agar with 500 μm (high) or 2 μm (low) Pi. Four meshes of seeds were grown per 100 ml of media. Plants were grown for 12 days prior to Pi uptake measurements. In the second experiment (Table 2), plants were grown for 10 days in semi-solid media containing 2 mm Pi (Pi-sufficient), and then transferred to liquid media containing 2 mm Pi or no additional Pi (Pi-starved) for another 7 days before harvest. Differences noted between the wildtype and mutants were significant at a >95% confidence level (Student's t-test).
For plant growth determination (Figure 4), plants were grown on 0.5 × Hoagland agar plates with 0.1, 0.2, 0.5, and 1 mm Pi. Each plate contained 90 ml of media and approximately 120 seeds were planted per plate at equal spacing. To minimize plate to plate variation, four genotypes were planted per plate. The plants were grown for 3 weeks. The fresh weight of at least 30 plants per genotype per Pi condition was measured individually. The Pi content of individual plants was measured using at least 10 plants per genotype per Pi condition. The experiment was replicated.
Identification of insertion mutants
Pht1;1 and Pht1;4 insertion mutants, subsequently named pht1;1-1 and pht1;4-1, were identified from the T-DNA-transformed lines available from the Arabidopsis Knockout Facility (University of Wisconsin). PCR was carried out on pooled genomic DNA with a T-DNA left-border primer, JL202 and gene-specific primers 1a or 1b for the Pht1;1 gene and 4a or 4c for the Pht1;4 gene. Pools were deconvoluted and individual lines identified by PCR. A second line with an insertion in the Pht1;1 gene (pht1;1-2) was identified from the Salk T-DNA lines (Alonso et al., 2003) through the analysis of the SIGnAL Database at http://www.signal.salk.edu/cgi-bin/tdnaexpress. The T-DNA insertion in pht1;1-2 was confirmed by genomic PCR and sequencing. The T-DNA left-border primer LB-b1 was used in combination with the gene-specific primers 1a and 1b. A second Pht1;4 insertion mutant, pht1;4-2, was isolated from the Arabidopsis transposon-tagged (dSpm) lines (Sainsbury Laboratory, John Innes Center). The individual line carrying pht1;4-2 was identified by PCR with a transposon-specific primer, 3S, and either of two Pht1;4 gene-specific primers, 4a or 4c. The junction regions flanking the insertions were amplified by PCR using additional primers, 1c and 4b, shown in Figure 1. Primer sequences are available in Table S1 (supplemental data).
To generate a double mutant containing insertions in both the Pht1;1 and Pht1;4 genes, a pht1;1-1 homozygote was crossed with a plant homozygous for the pht1;4-1 allele. The F1 plants were allowed to self and a double mutant, designated pht1;1Δ4Δ, was identified from the F2 population by PCR with primers pairs 1b and 1e, and 4a and 4b. Forty-five F2 plants were screened, and three lines homozygous for both the Pht1;1 and Pht1;4 insertions were identified.
Expression of the Pht1 gene family members
Total RNA was isolated by using TRI reagent. For RT-PCR, first-strand cDNA was synthesized from 0.5 μg of total RNA using Superscript II reverse-transcriptase (Invitrogen, CA, uSA) in a total reaction volume of 20 μl. One microliter of the first strand synthesis mixture was used as template in a 50-μl PCR containing primers and Ex-Taq DNA polymerase (Takara, Otsu, Japan).
cDNAs for eight members of the Pht1 gene family were cloned by RT-PCR using RNA from wildtype roots with the primers shown (Table S2, supplemental data). Primers for Pht1;1 and Pht1;4 were designed to the 5′ and 3 untranslated regions, but all the others were designed to the coding regions because sequence information for the untranslated regions was not available. As Pht1;1 and Pht1;2 share 98% identity at the nucleotide level, primers designed to Pht1;2 (pht2-f and pht2-r) amplify Pht1;1 also.
For the semi-quantitative RT-PCR shown in Table 1, total RNA was isolated from the root tissues grown under the same conditions used in Figure 3 Pi-uptake experiment. The first-strand cDNAs synthesized from duplicate RT reactions were mixed, and the yield of the mixed cDNAs was measured based on the PCR signal generated from the amplification of a house-keeping gene eIF-4A (GeneBank ID: AC005287) after 25 PCR cycles. From this, the cDNAs were adjusted to give the same amplification signal for eIF-4A with 1 μl of cDNA stock. To double-check this standardization of cDNAs, all PCR mixtures included a primer pair for eIF-4A internal control. The numbers of PCR cycles were 25–30 depending on the specific gene.
The amplified products were separated on 1% agarose gel, stained with ethidium bromide (10 μg ml−1), and captured as digital images under UV light. The band signals were quantified from digital images using LabWorks software. The gene expression levels in different mutant backgrounds were calculated relatively to that of wildtype PCR product (100%).
Lateral root and root hair growth rates
Plants were grown for 6 days on agar plates containing 1 mm Pi and then transferred to agar plates containing no additional Pi. Twenty-four hours after transfer, images of the root hairs were captured and root hairs growing in the region 3–4 mm above the root tips were measured. Data are based on the measurements of root hairs on six seedlings, 10 root hairs per seedling.
To measure the response of lateral roots to localized Pi supply, agar plates containing 0.5 × Hoagland media containing no additional Pi were separated into three parts, top/middle/bottom, by removing thin strips of agar from the plate. Eighty microliters of 100 mm KH2PO4 solution was spread evenly over the middle section of the plate to provide a Pi concentration of approximately 1 mm. Plates were oriented vertically and plants were grown on the top section of the agar plate for 3 days. The root tips of the seedlings were moved gently to the middle section of the plate containing 1 mm Pi. Images of lateral roots were captured 7 and 10 days later and lateral root length was measured. Data are based on three to four lateral roots per seedling and eight seedlings per genotype. Images of root hairs and lateral roots were analyzed using Scion Image (Scion Corporation, Frederick, MD, USA) computer program.
Pi uptake and Pi content analyses
Pi uptake was measured according to Poirier et al. (1991) with minor modifications. Plants growing on the nylon meshes were incubated in 0.5 × Hoagland solution containing either 2 or 500 μm KH2PO4 for 1 h. Pools of approximately 50 plants (one mesh) were transferred to 6 ml of Pi uptake solution containing 33Pi at 1 μCi ml−1. Except for 33Pi, the Pi uptake solution was the same as the nutrient solution used in the previous step. Plants were harvested at hourly intervals over the subsequent 4 h, then rinsed briefly in the ice-cold nutrient solution and blotted dry. The root and shoot fresh weights of the pooled plants were measured and tissues were dried overnight at 60°C prior to scintillation counting. 33Pi uptake by the root was linear for 3 h. The Pi uptake rate was calculated as nmol Pi h−1 g−1 fresh weight and mean values ± SE were based on five to seven replicates per genotype.
Plants were grown with a 16-h light/8-h dark cycle on 0.5 × Hoagland agar plates containing 500 μm Pi and arsenate at 0, 125, 150, 175, and 200 μm. Plants were grown either for 2 weeks (Figure 6a,b) or 3 weeks (Figure 6c,d). For growth analysis, approximately 60 plants were grown per agar plate. Plates contained 30 ml media. Ten to 12 plants per genotype per condition were sampled to measure individual shoot fresh weights. The mean values for the fresh weights were obtained from measurements of at least 20 plants harvested from duplicate plates.
Funding for this work was provided by The Samuel Roberts Noble Foundation (SRNF). The authors thank members of the Harrison laboratory at Noble Foundation and Boyce Thompson Institute, and members of the Plant Biology division, for helpful discussions.
Figure S1. Pht1;1-GFP fusion proteins are localized in the plasma membrane. Confocal images of N. benthamiana leaf cells expressing free GFP (a), Pht1;1 with GFP fused to the carboxy terminus of the Pht1;1 protein (Pht1;1-GFP) (b), and Pht1;1 with GFP fused to the amino terminus of the Pht1;1 protein (GFP-Pht1;1) (c). Confocal image of a tobacco cell expressing Pht1;1-GFP after plasmolysis with 0.1 m NaCl treatment (d). The labeled Hechtian strands are a further indication that the fusion protein is located in the plasma membrane (Oparka, 1994; Oparka et al., 1994). Fusion constructs were created by cloning Pht1;1 in frame to the 5′ SalI or 3′ BsrGI site of green fluorescent protein (GFP) in the plasmid CaMV35S-sGFP(S65T)-nos/pUC18 (Chiu et al., 1996), generating Pht1;1-GFP or GFP-Pht1;1, respectively. Bars equal 20 μm.
Figure S2. A phylogenetic diagram (ClustalW) for the nine members of the Arabidopsis Pht1 gene family.
Figure S3. Northern blot analysis with RNA from the wildtype and pht1;1-1 and pht1;4-1 mutants. Plants were grown in hydroponic culture. 200 seeds were germinated and grown for 2 weeks in 100 ml 0.5 × Hoagland, 1 mm Pi solution in Magenta GA-7 boxes with vented lids. The media was replaced with 0.5 × Hoagland, 0.02 mm Pi and plants were grown for an additional 2 weeks before harvest. During the experiment, the media was replaced weekly. For preparation of Pi transporter gene probes, the RT-PCR products amplified as in Table S2 were cloned into TA cloning vector and sequenced to confirm the product. 32P-labeled probes were created by PCR with the gene-specific primers using the plasmid clones as template DNA (Table S2). As indicated in Figure S1, Pht1;1, Pht1;2, Pht1;3 share high levels of sequence identity and the Northern blots hybridized with either Pht1;1, Pht1;2, or Pht1;3 probes show the same result. One representative hybridization is shown.
Figure S4. Pht1;2 and Pht1;3 gene expression in the pht1;1 and pht1;4 and pht1;1Δ4Δ mutants following growth under 2 and 500 μm Pi. Note that Pht1;1 and Pht1;2 share 98% identity at the nucleotide level and primers designed to Pht1;2, (pht2-f and pht2-r) amplify Pht1;1 also. Therefore, in the wildtype and pht1;1-4 mutants, the product of the Pht1;2 primers is a mixture of Pht1;1 and Pht1;2 transcripts. Expression of the Pht1;2 and Pht1;3 genes is not altered significantly in the mutants.
Table S1 Primers used for identification and verification of T-DNA and transposon insertions in the Pht1;1 and Pht1;4 genes
Table S2 Primers used for cloning Pht1 gene family members used in Figure 2 and Table 1. Label a represents the PCR products from the pht1;1-1 mutant