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ArabidopsisPHOSPHATE TRANSPORTER1 genesPHT1;8andPHT1;9are involved in root-to-shoot translocation of orthophosphate

Abstract

Background

In plants, the uptake from soil and intercellular transport of inorganic phosphate (Pi) is mediated by the PHT1 family of membrane-spanning proton : Pi symporters. TheArabidopsis thaliana AtPHT1gene family comprises nine putative high-affinity Pi transporters. While AtPHT1;1 to AtPHT1;4 are involved in Pi acquisition from the rhizosphere, the role of the remaining transporters is less clear.

Results

Pi uptake and tissue accumulation studies inAtPHT1;8andAtPHT1;9淘汰赛突变体与野生型植物商店wed that both transporters are involved in the translocation of Pi from the root to the shoot. Upon inactivation ofAtPHT1;9, changes in the transcript profiles of several genes that respond to plant phosphorus (P) status indicated a possible role in the regulation of systemic signaling of P status within the plant. Potential genetic interactions were found among PHT1 transporters, as the transcript profile ofAtPHT1;5andAtPHT1;7was altered in the absence of AtPHT1;8, and the transcript profile ofAtPHT1;7was altered in theAtpht1;9突变体。These results indicate that AtPHT1;8 and AtPHT1;9 translocate Pi from the root to the shoot, but not from the soil solution into the root.

Conclusion

AtPHT1;8 and AtPHT1;9 are likely to act sequentially in the interior of the plant during the root-to-shoot translocation of Pi, and play a more complex role in the acclimation ofA. thalianato changes in Pi supply than was previously thought.

Background

Phosphorus (P) is a major essential nutrient for plant growth, development and reproduction. Plants acquire P from the soil in its most oxidized inorganic form, phosphate (Pi) [1]. The uptake of Pi into the plant occurs against a steep electrochemical gradient. While the concentration of Pi in the soil solution is generally less than 2 μM, the Pi concentrations within plant tissues can be greater than 10 mM [2]. However, cytosolic Pi concentrations are tightly controlled, rarely exceeding 60–80 μM Pi [3]. Pi uptake from the soil and transport within the plant against this concentration gradient is mediated by Pi transporters. The first eukaryotic Pi transporter protein to be described was the PHO84p H+: Pi symporter in yeast [4], followed by plant homologs [5],[6]. From the numerous plant sequences now available, four PHOSPHATE TRANSPORTER (PHT) families are recognised: PHT1 (plasma membrane), PHT2 (plastid inner envelope), PHT3 (mitochondrial inner membrane) and PHT4 (mostly plastid envelope and one Golgi-localized transporter) [7],[8].

TheArabidopsisAtPHT1 family has nine members. The family is composed of several high-affinity Pi transporters having Kmvalues in the range of 2.5 μM to 12.3 μM [9] and other members that may have lower affinities for Pi [10],[11]. Transcripts from most of theAtPHT1基因被发现在这两个“根与芽”(12]-[15], whileAtPHT1;6transcripts are most abundant in flowers [12]. Transcripts from allAtPHT1genes exceptAtPHT1;6accumulate upon Pi starvation [16]. Transcriptional regulation ofAtPHT1expression seems to be mainly controlled by the internal P status [13],[15],[17],[18]. Sugars and cytokinins can also direct the expression of someAtPHT1family members [19].

在植物进化了,冥界几个策略p them acclimate to variation in Pi availability, including the modulation ofPHT1gene expression. The deployment of these strategies is modulated by local and systemic signaling networks. The best characterized systemic signaling module involved in the responses to changes in Pi supply includes the phloem-mobile microRNA Atmir399d, its target geneAtPHO2and a family of regulatory, non-coding RNAs encoded by theAtIPS1andAtAT4genes [20],[21]. These functions form a circuit where AtPHO2 activity in the root,which mediates the ubiquitination of AtPHT1 proteins in the post-endoplasmic reticulum compartment [21], is modulated by Atmir399d as the shoot experiences variations in P levels [14],[22]. The activity of Atmir399d in silencing AtPHO2 transcripts is itself antagonistically modulated by AtIPS1andAtAT4transcripts during prolonged periods of Pi starvation [23],[24]. On the other hand, local signaling networks control many of the characteristic changes in root system architecture that accompany changes in Pi availability. Thibaud et al. [18] identified a set of genes that are induced by local signaling networks during Pi starvation. These genes include the ethylene-responsiveAtERF1transcription factor gene, the metalloproteinaseAt2-MMPgene, the jasmonate-inducibleAtGSTU12andAtLOX4genes and theAtWRKY75transcription factor gene, which encodes a modulator of both the Pi-starvation response and root development [25].

Functional characterization ofAtPHT1;1andAtPHT1;4validated their roles in Pi acquisition from the soil solution under both Pi-sufficient and Pi-deficient growth conditions [26].AtPHT1;5plays a role in translocating Pi from source to sink organs [27]. Analysis of aAtpht1;9–1mutant andpht1;8/pht1;9silencing lines suggested a role for AtPHT1;9 and AtPHT1;8 in Pi acquisition at the root-soil interface during prolonged Pi limitation [28]. However, based on the increased transcript abundance from these twoAtPHT1genes in thepho2mutant [22], we hypothesize that AtPHT1;8 and AtPHT1;9 each have a role in translocating Pi from the root to the shoot. In this study we examined the physiological functions of AtPHT1;8 and AtPHT1;9 by characterizing their transcriptional regulation and the phenotypes of corresponding T-DNA insertion mutants in response to changes in Pi supply. Genetic interactions within theAtPHT1gene family were also examined by analyzing the transcript patterns of its members in each mutant in response to Pi availability. Furthermore, the placement of these twoAtPHT1gene functions within the plant response to variations in Pi supply was determined by analyzing the transcript patterns of several genes associated with systemic and local signaling networks in each mutant.

Results

Transcripts from individualAtPHT1genes responded differentially to Pi deprivation and re-supply

The remodeling kinetics of theAtPHT1transcript pool in response to both P depletion and Pi re-supply were examined in well-establishedArabidopsisplants prior to inflorescence emergence. In this and the following experiments, whenever Pi was supplied, the supply was set to be sufficient for non-limited growth, without being luxuriant. Wild-type (WT) plants were grown hydroponically and then deprived of Pi for 12 days until the leaves began to accumulate anthocyanins (Additional file1: Figure S1A), a visible indication that the tissues were beginning to experience P depletion. At this time, plants were transferred to nutrient solution containing either no added Pi (P-deprived) or added Pi (Pi re-supply). Quantitative PCR (qPCR) was used to measure transcript abundance for eight of the nine members of theAtPHT1gene family in root and shoot tissues over the next three days (Figure1).AtPHT1;6was excluded from the analysis because of its low transcript abundance in roots and shoots [15]. Transcript abundance was normalized to the average transcript abundance for a set of reference genes [29],[30]. To conservatively identify genes whose transcript patterns changed with Pi availability, only differences in the 40-ΔCtvalue of greater than two were considered, corresponding to a four-fold difference in transcript abundance [31].

Figure 1
figure1

Responsiveness ofAtPHT1transcript levels to Pi supply.The relative abundance ofAtPHT1transcripts in roots(A)and shoots(B)of 42-day-oldArabidopsisplants that were deprived of Pi for 12 days followed by Pi resupply in hydroponics is shown. Transcript abundance was measured by qRT-PCR and expressed as 40-∆Ct, a log2 measure of the ratio of the transcript amount from the target gene to the average transcript amount from a set of reference genes (see Method). Plants were grown in nutrient solution containing 250 μM Pi for 30 d, transferred to solution without Pi for 12 d to deplete internal P pools, and then transferred to a solution containing either no added Pi (−P) or 250 μM Pi (+P). Tissues were harvested 1, 2 or 3 d (D1, D2, D3) after the start of treatments. Control(C)plants were continuously supplied with 250 μM Pi and were harvested at D1. Data points are means ± S.D. (n = 3 biological replicates of 12 plants each). Different letters indicate significantly different means (P <0.05) between the control and treatment according to one-way ANOVA and Tukey’s Multiple Comparison of Means. The –P plants and + P plants were statistically analysed as separate groups.

InArabidopsisroots, P depletion by growth in the absence of a Pi supply for 13 d resulted in a four-fold to 16-fold greater transcript abundance for all theAtPHT1genes tested (P ≤0.05), except forAtPHT1;2, when compared to control plants continuously supplied with Pi (Figure1A,cf.D1).This is in general agreement with what has previously been observed [13],[15],[32]. The abundance ofAtPHT1;7andAtPHT1;8transcripts were eight-fold higher after 13 d Pi depletion compared to the control plants, but the abundance of these transcripts was lower at D2 and D3, being indistinguishable in abundance to these transcripts in the control plants under continuous Pi supply.

After 1 d of Pi re-supply to Pi-deprived plants, transcript abundance for most of theAtPHT1genes tested in roots was similar to that in the control plants (Figure1A). TheexceptionswereAtPHT1;1, AtPHT1;3andAtPHT1;4.The repression ofAtPHT1transcript abundance by day 2 of Pi re-supply was generally stronger than after day 1. Transcripts fromAtPHT1;3, AtPHT1;4,AtPHT1;5, AtPHT1;7andAtPHT1;9were repressed to their lowest levels at this time point. Transcripts fromAtPHT1;1,AtPHT1;2andAtPHT1;8were repressed to their lowest levels at day 3 of Pi re-supply (Figure1A). Interestingly, 3 d of Pi re-supply repressed the abundance ofAtPHT1transcripts to levels below those present in control plants that had been continuously supplied with Pi for the entire experiment even though the root Pi concentration was the same as in the control plants (Additional file1: Figure S1B).

The general trends of transcript accumulation in the shoots of Pi-deprived plants were similar to those found in the roots (Figure1B), except forAtPHT1;7andAtPHT1;8, where transcript abundance remained constant and similar to the control plants. In contrast to roots, 1 d of Pi re-supply did not cause a decrease in the abundance of anyAtPHT1transcripts compared to Pi-deprived plants of the same age. Interestingly, the transcript abundance forAtPHT1;8andAtPHT1;9was actually higher after 1 d of Pi resupply than in the Pi-deprived plants of the same age. The transcript amount for most of theAtPHT1genes did eventually become repressed; however, the repression was not as strong as in roots, despite the fact that these plants had twice the shoot Pi concentration of the control plants (Additional file1: Figure S1B).

Cluster analysis showed that the transcript responses to changes in Pi supply for the mainAtPHT1transporter genes,AtPHT1;1andAtPHT1;4, along withAtPHT1;2, were distinct from those of the otherAtPHT1genes in both roots and shoots (Figure2). In roots, the response patterns forAtPHT1;8andAtPHT1;7were similar to each other, while the responses ofAtPHT1;9were most similar to those ofAtPHT1;5andAtPHT1;3. In the shoot, the response patterns forAtPHT1;8andAtPHT1;9clustered with those ofAtPHT1;7, while the pattern of changes forAtPHT1;3andAtPHT1;5clustered discretely.

Figure 2
figure2

Co-expression analysis of theAtPHT1gene family in response to Pi supply.A hierarchical cluster analysis ofAtPHT1gene transcript patterns in roots(A)and shoots(B)of the 42-day-oldArabidopsisplants described in the legend to Figure1is shown. ∆∆Ctvalues generated from the results shown in Figure1using plant organs continuously supplied with Pi as a reference were analysed using Euclidean distance and complete linkage [33].

Disruption ofAtPHT1;8orAtPHT1;9had diverse effects inArabidopsisseedlings

There is only a single mutant available for eachAtPHT1,8andAtPHT1;9that has a predicted T-DNA insertion in the exon region. PCR across the predicted T-DNA left-border (LB) insertion sites and sequencing of the PCR products confirmed that the putativeAtpht1;1-2,Atpht1;8andAtpht1;9–1mutants used in this study were homozygous for the presence of T-DNA at sites expected to disrupt gene function (Additional file2: Figure S2A). At least two T-DNAs have been inserted in a head-to-head orientation in all three mutants. InAtpht1;1–2, two T-DNAs were inserted 42 bp downstream of the start of exon 3, confirming previous results (Additional file2: Figure S2B) [26]. In theAtpht1;8mutant, the T-DNAs were located 595 bp downstream of the start of exon 2 (Additional file2: Figure S2C). The insertion site in the previously characterisedAtpht1;9–1mutant allele [28] was found to be located 77 bp upstream of the start codon in exon 1 (Additional file2:图S2D)。这些插入量引起的of the corresponding transcript for each mutant gene to be severely reduced to below the limit of detection by semi-qPCR and just above the limit of detection by qPCR (Additional file3: Figure S3).

The disruption ofAtPHT1;8orAtPHT1;9gene function did not cause gross morphological changes in 17-day-old mutant seedlings supplied with sufficient Pi or depleted of Pi (Figure3, Additional file4: Figure S4). The most striking visible phenotype in both mutants supplied with sufficient Pi was a 20% to 30% reduction in root-to-shoot ratio compared to the corresponding Col-0 WT (Figure3A). This was brought about by a 10% greater shoot biomass combined with a 10% lower root biomass in the mutants (Additional file5: Figure S5). The decreased root biomass in the P-sufficientAtpht1;8mutant was at least partly due to a lower primary root length (Figure3B), although the lateral roots also tended to be shorter in both mutants (Figure3C). When depleted of P, the root system biomass ofAtpht1;8andAtpht1;9–1seedlings was not significantly different from the WT (Figure3, Additional file5: Figure S5). The Pi concentration in the roots of the mutants was also the same as that in WT (Figure3D), but it was 20% lower in the mutant compared to WT shoots (Figure3E). The lower shoot Pi concentration might be expected to enhance P-starvation responses such as anthocyanin production which was indeed the case in theAtpht1;8mutant (Figure3F). By contrast, the anthocyanin concentration in the shoots ofAtpht1;9–1幼苗耗尽P是只有约一半的t in the WT. This lower anthocyanin concentration was not due to a dilution by growth, as shoot biomass inAtpht1;9-1seedlings depleted of P was about 10% less than that of WT seedlings (Additional file5: Figure S5).

Figure 3
figure3

Developmental and biochemical responses ofArabidopsisCol-0 andAtpht1knock-out seedlings to Pi limitation on solid medium.Seedlings were grown for 5 d on solid medium containing 250 μM Pi before transfer to fresh medium containing either 250 μM Pi (High Pi) or 5 μM Pi (Low Pi) for 12 d. Root-to-shoot ratio(A), primary root length(B), lateral root length(C), root Pi concentration(D), shoot Pi concentration(E)and anthocyanin concentration(F)确定在收获。值意味着±S。D. (n = 3 plates of 12 seedlings each). * or different letters indicate significantly different means (P <0.05) compared to the corresponding wild-type or each other according to two-way ANOVA and Tukey’s Multiple Comparison of Means, respectively.

Disruption ofAtPHT1;8orAtPHT1;9compromised root-to-shoot translocation of Pi

Forty-eight-day-oldAtpht1;8andAtpht1;9–1plants depleted of P were assessed for their short-term ability to remove Pi from the external nutrient solution in comparison to the WT and to theAtpht1;1–2mutant (Additional file6: Figure S6). As expected, theAtpht1;1–2mutation caused a significantly lower rate of Pi removal from the nutrient solution than WT (Table1). However, theAtpht1;8andAtpht1;9–1mutations had a negligible effect on the ability of P-depleted plants to remove Pi from the nutrient solution. Moreover, theAtpht1;8andAtpht1;9-1mutants were clearly distinguished from both WT andAtpht1;1–2in the short-term kinetics of Pi accumulation in the roots and shoots of these P-depleted plants upon Pi re-supply (Figure4, Additional file7: Figure S7). TheAtpht1;1–2mutant, although compromised in its ability to acquire Pi from the nutrient solution (Table1), had a root Pi concentrations similar to that of WT (Figure4A). The concentration of Pi tended to be somewhat higher in the roots ofAtpht1;8than in WT throughout the time course, while the Pi concentration in the roots ofAtpht1;9–1tended to be marginally lower than in WT. After 300 min of Pi re-supply to the Pi-deprived plants, the roots of theAtpht1;8mutant had a significantly higher Pi concentration than those of WT, while the roots ofAtpht1;9–1were indistinguishable from those of WT.

Table 1Capacity of 48-day-old P-depletedArabidopsisWT andAtpht1knock-out plants to withdraw Pi from nutrient solution (see Additional file6: Figure S6)
Figure 4
figure4

Short-term accumulation of Pi by P-depleted 48-day-old Col-0 andAtpht1knock-outArabidopsisplants grown in hydroponics.Accumulation of Pi in roots(A) and shoots(B)of WT andpht1mutant plants over a 6-hour time course. Seedlings were grown in nutrient solution containing 250 μM Pi for 30 d, transferred to solution without Pi for 18 d to deplete plant P pools, and then transferred to solutions containing 250 μM Pi. Tissues were harvested every 30 mins for 6 h after the last transfer. Values are means ± S.D. (n = 3 biological replicates with 12 plants each grown at separate times). Error bars have been excluded for clarity, and can be viewed in Additional file7: Figure S7.

In sharp contrast to the roots, the shoots of all three P-depleted mutants accumulated dramatically less Pi than WT during short-term Pi re-supply (Figure4B). TheAtpht1;1–2mutant had noticeably lower shoot Pi concentrations than WT at all time points from 150 min onwards, as might be expected from the decreased ability of this mutant to remove Pi from the nutrient solution. However, bothAtpht1;8andAtpht1;9–1plants, which were not compromised in acquiring Pi from the nutrient solution, accumulated much less of the added Pi in their shoots than either WT or theAtpht1;1–2突变体。After 300-min exposure to Pi, the shoot Pi concentration in bothAtpht1;8andAtpht1;9–1mutants was less than half of that in WT and 40% lower than in theAtpht1;1–2mutant that was impaired in the primary uptake of Pi (Figure4B).

The longer-term impact of theAtpht1;1–2,Atpht1;8andAtpht1;9–1mutations on plant P pools was determined in 30-day-old plants grown in the presence of Pi and then deprived or not of Pi for 14 d (Figure5). ForAtpht1;1–2plants with their compromised Pi uptake capacity (Table1), the long-term total P concentration was lower than in WT in both roots and shoots of plants grown at either Pi supply. This lower total P concentration was due to lower concentrations of both Pi and the P esterified into acid-hydrolyzable organic compounds (Po). The Po was 35% lower in the root and 25% lower in the shoot compared to the WT regardless of the Pi supply, while the Pi concentration was only lower than WT inAtpht1;1–2roots and shoots under Pi-sufficient conditions. On the other hand, the long-term Pi concentration in the root and the shoot of theAtpht1;8mutant was indistinguishable from WT andAtpht1;9–1in both Pi-sufficient and Pi-limited conditions. The Pi concentration was also similar toAtpht1;1–2but in Pi-limited condition only. The total P concentration in roots of theAtpht1;8mutant was identical to the WT regardless of Pi supply, while in the shoots the total P concentration was lower and similar to that of theAtpht1;1–2突变体。这种差异的根源,而不是射击,tal P concentration between these two mutants highlights a fundamental difference between them; the lower shoot P inAtph1;8compared to WT was not due to a lower P concentration in the root, as may have been the case inAtpht1;1–2, which is impaired in Pi uptake. In the roots of Pi-sufficientAtpht1;9–1plants, the Pi concentrations were similar to those in theAtpht1;8mutant, while the Po concentrations were similar to those in theAtpht1;1–2突变体。However, in the shoots of these plants, both Pi and Po concentrations were similar to those ofAtpht1;8, indicating thatAtpht1;9only differs fromAtpht1;8in its ability to allocate Pi to Po in the roots. This difference was also observed in Pi-deficient plants (Figure5B and D).

Figure 5
figure5

The effect of Pi supply on the Po and Pi concentrations in 44-day-oldArabidopsisCol-0 WT andAtpht1plants grown in hydroponics.The P concentrations in the inorganic (Pi) and organic (Po) pools were determined in roots(A & B)and shoots(C & D)植物生长在含有营养解决方案sufficient Pi supply(A & C)or no Pi supply(B & D). Po was calculated as total P minus Pi. Plants were grown in nutrient solution containing 250 μM Pi for 30 d, and then transferred to solution containing 250 μM Pi or no added Pi for 14 d before harvest. For Po and Pi concentrations, values are means ± S.D. (n = 3 biological replicates of 12 plants each grown at separate times). *indicates significantly different means (P <0.05) relative to Col-0 according to one-way ANOVA followed by Tukey’s Multiple Comparison of Means.

Loss ofAtPHT1;8andAtPHT1;9influenced the transcript profiles of other genes repressed by Pi

A panel of 17 Pi-responsive genes, including the full set ofAtPHT1genes, was used to assess the interactions ofAtPHT1;8andAtPHT1;9with other components of the P-starvation response (Additional file8: Figures S8 and Additional file9: Figure S9). The gene panel also included a sub-set of those genes reported to respond to either local or systemic signals generated by plant P status [18]. The genes in the panel that respond to distant systemic P signals wereAtPHT1;4, AtPHT1;5,AtPHT1;7,AtPHT1;8,AtPHO2, AtMIR399d, At4andAtIPS1, while those that responded to local P status wereAtERF1,AtGSTU12,AtLOX4,At2-MMPandAtWRKY75.

In 44-day-old plants, theAtpht1;8mutation caused changes in the transcript levels of some of the selected Pi-responsive genes (Figure6, Additional file8: Figure S8). In the roots of Pi-sufficientAtpht1;8plants,AtPHT1;3, AtPHT1;5andAtPHT1;7were more strongly repressed than in WT, whileAtPHT1;2transcripts were more abundant (Figure6A). However, transcripts from all theAtPHT1genes were induced to WT levels upon P depletion.Atpri-MIR399dtranscripts were less strongly repressed inAtpht1;8根在足够比WT根π,而交易nscripts from the set of genes influenced by local Pi signals were generally more strongly repressed consistent with higher Pi concentrations in these roots. P-depletion of theAtpht1;8mutant resulted in lowerAtWRKY75transcript levels in the roots compared to WT. However, the abundance of these transcripts did not respond to changes in Pi supply in the roots of the mutant. Thus,AtWRKY75transcript levels were also lower in this tissue than in the WT in the presence of Pi.

Figure 6
figure6

Effect of theAtpht1;8mutation on the P-responsiveness of transcripts from Pi-responsive genes, including those encoding known P signaling components.Transcript abundance in 44-day-oldAtpht1;8plants is expressed relative to that in WT plants grown under the same conditions (∆∆Ctvalues). The transcript patterns were examined in both roots(A)and shoots(B). Plants were grown in nutrient solution containing 250 μM Pi for 30 d, and then transferred to solution containing 250 μM Pi or no added Pi for 14 d before harvest. Each panel shows the comparison made using plants grown on nutrient solution containing sufficient Pi or no Pi. Values are means ± S.D. (n = 3 biological replicates of 12 plants each grown at separate times).

The shoots ofAtpht1;8plants grown in the presence of sufficient Pi contained lessAtPHT1;5andAtPHT1;7transcripts than WT (Figure6B). In P-depleted plants, the abundance ofAtPHT1transcripts, including those fromAtPHT1;5, were generally de-repressed to the same levels seen in the WT. The exception was transcripts fromAtPHT1;7,which were not as strongly de-repressed as in WT. InAtpht1;8shoots, the only observed change in transcript profile for genes responsive to systemic signals of P status was a slight decrease in the strength of the de-repression ofAtpri-MIR399dtranscripts in the P-depleted plants. Among the genes responsive to local P signals,AtGSTU12was somewhat more repressed in Pi-sufficientAtpht1;8shoots compared to WT, but this repression was overcome during P-depletion. By contrast,AtLOX4transcripts were not as strongly de-repressed by P depletion in the mutant as in the WT.

The accumulation of transcripts from the target genes in 44-day-oldAtpht1;9–1plants was similar to, but distinct from, that in theAtpht1;8mutant (Figure7, Additional file9: Figure S9). In the roots of Pi-sufficientAtpht1;9–1,AtPHT1;7andAtLOX4transcripts were less abundant than in WT (Figure7A). During P depletion,AtPHT1;7was less strongly de-repressed than in WT, whileAtPHT1;3transcripts were more abundant.Atpri-MIR399dandAtAT4transcripts were less abundant in the P-deprivedAtpht1;9–1突变体。Interestingly, bothAt2-MMPandAtWRKY75transcript amounts were lower in response to P depletion in theAtpht1;9–1mutant, similar to what was observed forAtWRKY75in the roots of theAtpht1;8突变体。

Figure 7
figure7

Effect of theAtpht1;9–1mutation on the P-responsiveness of transcripts from Pi-responsive genes, including those encoding known P signaling components.Transcript abundance in 44-day-oldAtpht1;9–1plants is expressed relative to that in WT plants grown under the same conditions (∆∆Ctvalues). The transcript patterns were examined in both roots(A)and shoots(B). Plants were grown in nutrient solution containing 250 μM Pi for 30 d, and then transferred to solution containing 250 μM Pi or no added Pi for 14 d before harvest. Each panel shows the comparison made using plants grown on nutrient solution containing sufficient Pi or no Pi. Values are means ± S.D. (n = 3 biological replicates of 12 plants each grown at separate times).

In the shoot of P-sufficientAtpht1;9–1,AtPHT1;3was less repressed than in WT (Figure7B). Transcripts fromAtPHO2were more strongly repressed by Pi in the mutant than in WT which was complemented by a decreased repression by Pi for bothAtpri-MIR399dandAtAT4transcripts. In the shoot of P-depletedAtpht1;9–1,AtPHT1;7transcripts, along with those from the entire set of genes associated with local signaling events, were less de-repressed than those in the WT.

Discussion

A clear response inArabidopsisto changes in P status is the reversible repression ofAtPHT1gene expression [20]. The modulation ofAtPHT1expression alters the Pi transport activity within the plants in response to the prevailing Pi availability [10]. Here we provide evidence thatAtPHT1;8andAtPHT1;9were instrumental in the movement of Pi from the root to the shoot. In addition, we show that these genes had overlapping but distinct functions, as well as interactions at the transcript level withAtPHT1;7and other genes that are involved in controlling P nutrition (Figure8).

Figure 8
figure8

Proposed location in the root and genetic interactions of AtPHT1;8 and AtPHT1;9. (A)Proposed root-to-shoot translocation pathway for Pi through the sequential action of AtPHT1;9 and AtPHT1;8. The endodermis and/or pericycle are the most likely locations for AtPHT1;9. AtPHT1;8 appears to act in cell layers deeper in the root than AtPHT1;9, perhaps in the pericycle or xylem. Two potential translocation pathways are envisaged. The location of AtPHT1;9 and/or AtPHT1;8 in the xylem pole pericycle would facilitate the transfer of Pi into the xylem for translocation to the shoot. (Image adapted fromhttp://www.ens-lyon.fr/RDP/SiCE/Resources.html).(B)Summary of the observed genetic interactions of AtPHT1;8 and AtPHT1;9 with other Pi-responsive genes. The transcripts from the indicated genes were either more abundant (black lines) or less abundant (red lines) in the respective PHT1 knock-out lines than in the wild type. The genetic interactions are superimposed onto the relative position proposed for AtPHT1;8 and AtPHT1;9 in the root. The diagram takes into consideration that the P concentration in each mutant is likely to be higher on the exterior side than on the interior side of the missing protein than in the wild-type. For example, since all the genes tested for interactions were repressed by Pi supply, gene transcripts that were expressed at a lower level in a mutant were positioned toward the exterior of the root, where the P concentration was presumed to be higher in the mutant than in the wild type. Such an assignment could not be made for pri-miR399d (red dotted lines) in roots of Pi-deprived seedlings, indicating our imperfect understanding of the regulation of pri-miR399 expression. The repression of AtPHO2 in leaves of Pi-sufficient Atpht1;9-1 may be in response to changes in the expression of the regulatory RNAs mir399d and AT4 (green dotted arrow).

AtPHT1;8andAtPHT1;9are necessary for the root-to-shoot translocation of Pi

Previous work indicated that both AtPHT1;8 and AtPHT1;9 are involved in Pi uptake from the external medium, but that neither gene has a role in translocating Pi to the shoot [28]. This conclusion was based on the observation that the shoots of mutant plants supplied with adequate Pi had the same Pi concentration as wild-type plants. By contrast, plants that were supplied with inadequate Pi had genotype-dependent differences in shoot Pi concentration. These differences in shoot Pi accumulation were concluded to be a direct consequence of the Pi concentration in the roots governing the Pi concentration in the leaves. However, as discussed below, our short-term Pi uptake experiments show that mutations in eitherAtPHT1;8orAtPHT1;9severely compromised the ability ofArabidopsisto translocate Pi to the shoot, explaining the lower total P concentrations that we also observe in the shoots of these mutants compared to WT.

As expected, our results showed that the rate of Pi uptake from solution is markedly reduced in theAtpht1;1–2mutant compared to the WT. SinceAtPHT1;1is one of the main paths for Pi entry into the root during low Pi availability [26], it is not surprising that the lower uptake capacity in this mutant would compromise the amount of Pi translocated to the shoot in the short term. The lower Pi concentrations observed in the shoots in the short term are likely to be due to the roots of P-starved seedlings retaining a higher proportion of the available Pi to satisfy root growth at the expense of Pi translocation to support shoot growth. On the other hand, the lower shoot Pi and Po observed in the longer term in the shoots of this mutant are probably a direct consequence of the decreased P concentrations observed in the roots.

In contrast toAtpht1;1–2, bothAtpht1;8andAtpht1;9–1mutants had the same Pi-uptake rate as WT, which is also not surprising since PHT1;1 would be fully functional in both mutants. However, the short-term accumulation of Pi in the shoots ofAtpht1;8andAtpht1;9–1was much slower than in theAtpht1;1–2mutant, suggesting a role in root-to-shoot translocation. This interpretation is supported by the following. During longer-term growth under P sufficient conditions, roots ofAtpht1;8水平WTπ和阿宝,阿宝才slightly compromised in roots ofAtpht1;9–1. Therefore, the roots ofAtpht1;8were just as capable of extracting Pi and allocating it to Po as those of WT and the roots ofAtpht1;9–1nearly so. On the other hand, Po, but not Pi, concentrations were compromised in the shoots of theAtpht1;8andAtpht1;9–1plants, whereas the concentrations of both these P pools were compromised in the shoots ofAtpht1;1–2. Together, these observations demonstrate thatAtPHT1;8几乎没有,如果有的话,在π的吸收作用,但instead is necessary for the root-to-shoot translocation of Pi. The same is probably true forAtPHT1;9. HoweverAtpht1;9-1has a somewhat lower total P concentration in the root than WT, which is due to a compromised ability to allocate Pi to root Po. While this could be due to decreased Pi uptake or the reduced export of Po from the shoot, it is much more likely given our results to be due to AtPHT1;9 acting more prominently in an outer cell layer of the central cylinder of the root relative to AtPHT1;8 (Figure8). This situation would result in a lower Pi flux into the interior of the root forAtpht1;9–1than forAtpht1;8and a change in the balance of Pi allocation that favours translocation to the shoot over the formation of Po in the root. Thus, AtPHT1;8 and AtPHT1;9 may operate analogous to the rice OsPht1;2 transporter, which also functions mainly in the translocation of Pi from the root to the shoot [34]. However, the twoArabidopsisproteins act sequentially, with AtPHT1;9 acting in cell layers closer toward the root surface than AtPHT1;8 (Figure8).

Differences in the expression profiles ofAtPHT1genes that could lead to differences in function are apparent in a spatiotemporal gene expression map of theArabidopsisroot [35], where the resolution of the original microarray-based map has been substantially increased by additional microarray datasets (see Brady et al. [36] and references therein). The plants used to generate the expression map were grown on solid medium containing 1.25 mM Pi. Therefore, their tissues would have been saturated with Pi, strongly suppressingPHT1gene expression. These seedlings, therefore, contrast with the Pi-deprived seedlings with highPHT1expression that we used to discover that the movement of Pi from the root to the shoot is controlled byAtPHT1;8andAtPHT1;9.

Visualising the root gene expression map using theArabidopsiseFP browser [37],PHT1transcript abundance in seedlings grown under high Pi conditions is especially low in the meristematic region of the root, from the columellar root cap nearly to the elongation zone (columella + sections 1 to 5 in Brady et al. [36], Additional file10: Table S1). This is in contrast to the region extending from the top-most section of the meristematic region through the elongation zone into the maturation zone (sections 6 to 12 in Brady et al. [36], Additional file10: Table S1). The low meristematic expression ofPHT1transcripts holds for all cell layers except the procambium. In the meristematic zone,AtPHT1;8andAtPHT1;9transcripts are generally as abundant, if not more abundant, than the transcripts recognised by the redundant microarray probe sets that detect the sum ofAtPHT1;1 + AtPHT1;2transcripts or the sum ofAtPHT1;4 + AtPHT1;7transcripts (Additional file10: Table S1). This is particularly the case in the endodermal cell layer surrounding the stele [35],[36] (Additional file10: Table S1). This transcript pattern suggests the endodermis of the meristematic region as a potential gateway where the control of Pi transport could be mediated byAtPHT1;8and/orAtPHT1;9upon their de-repression at low Pi availability (Figure8A).

ThePHT1expression patterns within the cells of the stele suggest other potential sites whereAtPHT1;8andAtPHT1;9may regulate Pi transfer into the xylem (Figure8A). Within the stele as a whole, transcripts fromAtPHT1;8andAtPHT1;9do not predominate [35]. However, the higher-resolution gene expression data provided by Brady et al. [36] at the cellular level shows that this is largely due to a dilution effect caused by relatively high levels of transcripts from several otherPHT1genes in the procambium (Additional file10: Table S1).AtPHT1;8and/orAtPHT1;9transcripts are more abundant relative to those from otherAtPHT1genes within the xylem of the meristematic region, at least in these Pi-satiated plants. A similar high relative abundance ofAtPHT1;8and/orAtPHT1;9transcripts also exists in the xylem pole pericycle cell strand. In this case, the high relative abundance ofAtPHT1;8and/orAtPHT1;9transcripts extends from the root tip, through the meristematic and elongation zones into the maturation zone. This expression pattern, then, offers the potential for the xylem pole pericycle cell strand to regulate Pi transport along a large section of the root (Figure8A).AtPHT1;8and/orAtPHT1;9transcripts are also relatively abundant compared to those from otherPHT1genes in the protophloem + metaphloem and in the phloem companion cells within the meristematic region. IfAtPHT1;8and/orAtPHT1;9are involved in controlling Pi transport in these cells, it is likely in response to the flow of Pi back to the root tip from the aerial tissues through the phloem, rather than to the flow from the root to the shoot through the xylem (Figure8A).

From the available results, it seems likely that the cells of the endodermis, the xylem pole pericycle, or the xylem itself, are where AtPHT1;8 and AtPHT1;9 could provide a barrier to Pi transport. This barrier would separate the external solution from the vasculature with AtPHT1;9 acting in a cell layer closer to the root surface than AtPHT1;8 (Figure8A). Confirmation of the cell types in which AtPHT1;8 and AtPHT1;9 regulate the flow of Pi to the shoot awaits a high-resolution gene expression map of the root from seedlings in whichPHT1基因表达是高度由πdepri de-repressedvation. However, a full picture of how AtPHT1;8 and AtPHT1;9 regulate the flow of Pi to the shoot will require a full understanding of the biochemistry of these transporters, including any regulation of their activity by post-translational modification or by interactions with other proteins, including other PHT1 proteins [10].

Absence of AtPHT1;9 modulates the activity of the PHO2 regulon

Our results support and extend the proposed interactions of bothAtPHT1;8andAtPHT1;9with the PHO2 regulatory circuit [14],[22].AtPHO2encodes an ubiquitin conjugating enzyme implicated in repressing Pi uptake when Pi availability is high [14],[21],[22]. Its expression is in turn repressed by the phloem-mobile microRNA Atmir399 [38], a component of the systemic P-signaling system. Atmir399 is synthesised in the shoot and translocated to the roots when shoot P status is low [39],[40]. Atmir399 functions by binding to and providing a substrate for the specific cleavage and degradation ofPHO2transcripts. The activity of Atmir399 is itself modulated by regulatory RNAsAtIPS1andAtAT4, which are thought to mimic theAtPHO2transcript and compete for Atmir399 binding [23]. Together, AtPHO2 and these small RNAs monitor the plant P status [40]-[42]. However, the interactions of theAtPHO2,AtMIR399, AtIPS1andAtAT4gene products are complex, given that the expression of theAtMIR399gene family as well asAtIPS1andAtAT4are all de-repressed by low Pi availability [14],[22],[24]. A decrease in AtPHO2 activity, either by mutating or suppressingAtPHO2, or by ectopic over-expression ofAtMIR399, causes Pi to accumulate in the shoots [14],[22],[43],[44]. Interestingly, bothAtPHT1;8andAtPHT1;9are up-regulated in theAtpho2mutant and inAtMIR399over-expression lines [14],[22]. Therefore, our results indicating that AtPHT1;8 and AtPHT1;9 are necessary for root-to-shoot Pi translocation are consistent with these Pi transport proteins being directly involved in the increased translocation of Pi to the shoots in the transgenics with decreasedAtPHO2expression.

We found that the amounts ofAtpri-MIR399dandAtAT4transcripts in the shoots ofAtpht1;9–1were not as strongly suppressed by Pi as in the WT which may in turn explain the decrease in the amount ofAtPHO2transcript in these shoots (Figure8B). The abundance of transcripts from these three regulatory genes was consistent with the lower organic shoot P status ofAtpht1;9–1. Interestingly, the Pi concentrations in the roots and shoots of these plants were the same as those in WT. Therefore, the modulation of these components of the AtPHO2 regulatory circuit were either being influenced by highly localized Pi concentrations, by an organic P ester, or by the absence of the AtPHT1;9 protein itself. TheAtpht1;8mutation had little impact on the PHO2 regulatory circuit other than a modest modulation in the amounts of theAtMIR399dprimary transcript, further differentiating the activity of AtPHT1;8 and AtPHT1;9.

AtPHT1;8 and AtPHT1;9 activities govern the expression ofAtPHT1genes involved in inter-organ Pi transport

Transcript analysis in theAtpht1;8andAtpht1;9–1mutant backgrounds revealed numerous genetic interactions between these and the otherAtPHT1genes that are repressed by Pi (Figure8B). These findings contradict an earlier report whereAtPHT1;8andAtPHT1;9did not genetically interact with other Pi-starvation induced genes in plants of low P status [28]. However, that earlier study used end-point PCR to quantify transcripts which is a less sensitive method of transcript quantification than real-time PCR. Moreover, the transcript analysis was done on whole seedlings, while in our study roots and shoots were analysed separately and at two Pi supply levels. Together, these methodological differences in the earlier study may have masked the differences in transcript profiles that we report here.

The interactions ofAtPHT1;8andAtPHT1;9with other genes were overlapping (Figure8B),有75%的基因集的商店中身份wed significant changes in gene expression in both mutant lines compared to WT. The differences in effect of these two genes on other genes is a further indication thatAtPHT1;8andAtPHT1;9are functionally distinct, consistent with the view that they act sequentially in Pi translocation. The interactions were apparently independent of organ Pi levels, since the long-term Pi concentrations in theAtpht1;8andAtpht1;9–1mutants were nearly indistinguishable from those in the WT. The interactions did, however, correlate with lower organ Po concentrations, indicating that the plants were not as highly P satiated as WT plants. Thus, a signal other than organ-level Pi concentration was likely to be responsible for the observed co-expression patterns.

The interactions ofAtPHT1;8andAtPHT1;9with otherAtPHT1genes excludedAtPHT1;1,AtPHT1;2andAtPHT1;4, which encode transporters that are important for Pi uptake from the soil solution [12],[26]. Most of the interactions were instead withAtPHT1;3,AtPHT1;5andAtPHT1;7(Figure8B), suggesting that the encoded transporters, like AtPHT1;8 and AtPHT1;9 themselves, are likely important for inter-organ Pi transport processes. This role has already been demonstrated for AtPHT1;5 [27]. Interestingly,AtPHT1;3,AtPHT1;5,AtPHT1;7,AtPHT1;8andAtPHT1;9were also among a very small group of PHR1-regulated genes that displayed a very strong and specific response to Pi availability in the root environment, with only AtPHT1;7 also responding equally well in shoots, as shown previously [32]. There was no evidence for a genetic interaction betweenAtPHT1;8andAtPHT1;9 themselves in our study. This is interesting, given that the two transporters appear to act sequentially in the transport of Pi to the shoot.

The lack of a strong alteration in transcript abundance for otherAtPHT1genes in the roots of theAtpht1;8mutant under Pi deficiency was a further indication that the disruption of AtPHT1;8 function was directly responsible for the lower capacity for Pi translocation to the shoot in this mutant. By contrast, the alterations inAtPHT1;3andAtPHT1;7transcript abundance in the roots ofAtpht1;9–1under Pi deficiency leaves open the possibility that the genetic interaction ofAtPHT1;9with these genes is necessary for Pi translocation in WT. The stronger repression compared to that in WT ofAtPHT1;3,AtPHT1;5andAtPHT1;7inAtpht1;8roots and ofAtPHT1;7inAtpht1;9–1根下足够的π表示,12月供应eases in the amounts of these other AtPHT1 proteins may also have contributed to the lower P accumulation in mutant shoots. A better understanding of the precise location of the various PHT1 proteins within the plant is necessary to more thoroughly interpret these findings.

The genes involved in local Pi signaling were even more strongly repressed by high Pi in theAtpht1;8mutant than in the WT (Figure8B). The stronger repression in roots compared with that in shoots hinted at a possible accumulation of Pi or down-stream metabolites in roots due to reduced Pi transport into the shoot. The observed repression ofAtWRKY75in low-Pi roots andAtLOX4in shoots of the same treatment could indicate that the underlying “local” P regulatory circuits trigger different downstream responses in different organs. For example,AtLOX4repression may lead to reduced shoot growth in low-Pi conditions, given that theAtLOX4gene is involved in the biosynthesis of jasmonic acid, an effector of shoot growth [45].

Integration of PHT1 gene expression and tissue Pi status

The transcriptional responsiveness ofAtPHT1;8andAtPHT1;9to changes in Pi supply was similar to each other, but not identical. Feeding Pi to Pi-deprived plants suppressedAtPHT1;9transcripts in the roots more quickly and more strongly thanAtPHT1;8transcripts. By contrast, the suppression ofAtPHT1;8transcripts in the shoots of these same plants was stronger than that forAtPHT1;9, although the overall response of P-responsive transcripts in the shoots was much weaker than that in the roots.

For bothAtPHT1;8andAtPHT1;9, and indeed nearly all of theAtPHT1genes, the strong repression in the roots upon Pi re-supply brought the transcript levels far below those found in control plants continually fed Pi. This would be a useful adaptation for plants in their natural environment, where the available Pi concentration in the soil rarely exceeds 10 μM [1],[46]. Plants growing under these soil conditions would most certainly not be Pi-limited, but there would be strongPHT1expression and a danger of Pi over-accumulation if a rich supply of Pi was encountered. Thus, should a Pi-limited plant encounter a very Pi-rich soil patch, the rapid and strong systemic suppression ofAtPHT1;9in the roots followed by the suppression ofAtPHT1;8and the otherAtPHT1genes would prevent Pi toxicity through the rapid decrease in the influx of Pi into the shoots. The observed stronger suppression of allAtPHT1成绩单在根π补给可以be due to a systemic signal, given that the nutrient solution is quickly depleted of Pi and Pi accumulation is much stronger in the shoot than in the root itself (Jostet al., personal communication). By contrast, the weaker over-all suppression ofAtPHT1transcript abundance in the shoots could be a response to the lower Pi accumulation in the root, more slowly increasing Po concentrations or to the lower local accumulation of Pi in the cytosol of shoot cells, given that most of the Pi translocated to the shoot would immediately be moved to the vacuole [47]. The observed increase inPHT1transcript abundance in the shoot after 1 d of Pi re-supply was consistent with this proposed delay in Pi signal perception in the shoot.

Loss ofAtPHT1;8andAtPHT1;9没有加强morpho-physiological响应e to Pi limitation

Plants respond to changes in Pi supply by altering their physiology and biochemistry, both of which are modulated by changes in gene expression [48],[49]. Key physiological characteristics associated with low Pi availability are a relative increase in resource allocation to the roots resulting in an increased root-to-shoot ratio as well as increased root-hair proliferation, root branching and lateral root elongation [50]-[52]. The deployment of these traits is often accompanied by an arrest in primary root elongation. In our system, inactivation ofAtPHT1;8andAtPHT1;9had little effect on the establishment of these traits.

Our conclusions regarding the morpho-physiological responses of theAtpht1;8andAtpht1;9–1mutants to changes in Pi status was somewhat contrary to a previous report on these two genes [28]. In the earlier report,Atpht1;9mutants, including theAtpht1;9–1knock-out allele examined here, andAtPHT1;9/AtPHT1;8RNAi co-suppressed线,显示P-starv增加ation responses compared to those in WT. These enhanced responses included effects on primary root length, lateral root length and shoot FW [28]. The disparity between our studies could be due to differences in the plant growth conditions. For example, Remy et al. [28] used a medium containing 100 μM Fe, while our medium contained 40 μM Fe. High Fe concentrations can lead to the precipitation of Pi thus aggravating P limitation responses. Previous work showed that the inhibition of primary root growth during Pi limitation ofArabidopsisgrown on synthetic media was due to Fe toxicity and that the recovery from inhibition was more rapid for larger plants [53]. Thus, our larger plants seem to have been quite immune to the Fe exposure in the medium used here. Other differences in growth conditions that may have led to study-dependent differences in plant root architecture under Pi limitation were the gelling agent [54] and the presence or absence of myo-inositol, a precursor for many secondary messenger molecules and a regulator of plant growth responses [30],[55].

Conclusion

The products of theAtPHT1;8andAtPHT1;9genes are involved in the translocation of Pi from the roots to the shoot inArabidopsis. When supplied with moderate Pi for an extended period, the reduced Pi translocation to the shoots inAtpht1;8andAtpht1;9–1did not affect shoot Pi concentration, but instead lowered the organic P concentration. Some of the observed effects on growth and P allocation in theAtpht1;8andAtpht1;9–1mutants may be indirect, as mutations in these genes caused changes to the transcript abundance of otherAtPHT1genes. The loss of AtPHT1;9 also had an indirect effect on the transcript abundance of genes involved in systemic signalling mediated by the PHO2/mir399 network. Thus, AtPHT1;8 and AtPHT1;9 have a wider role in the control of P nutrition inArabidopsisthan was previously reported.

Methods

Plant material and growth

Arabidopsisecotypes Col-0 and T-DNA insertion lines SALK 088586C (Atpht1;1–2, At5g43350), SALK 056529 (Atpht1;8, At1g20860) and SALK 050730 (Atpht1;9–1, At1g7643) were obtained from theArabidopsisBiological Resource Centre. Seeds were surface-sterilized in 70% (v/v) ethanol for 2 mins and 5% (v/v) sodium hypochlorite for 5 mins followed by five washes in sterile water for 5 mins each. Seeds were stratified at 4°C in the dark for 16 h before sowing. When determining the location of the T-DNA insertion, seeds were sown on solid B5 medium (Phytotechnology Laboratories, Kilsyth, VIC, Australia) containing 3% (w/v) sucrose. Seedlings were grown with the plates held in a vertical position.

For hydroponic growth of plants, a modified nutrient solution approximating one-half strength Hoagland’s solution [56],[57] was used. The solution contained 2 mM Ca(NO3), 2 mM KNO3, 0.5 mM NH4NO3, 0.5 mM MgSO4, 0.25 mM KH2PO4, 50 μM KCl, 40 μM of Fe-EDTA, 25 μM of H3BO3, 2 μM MnCl2, 2 μM ZnSO4, 0.5 μM CuSO4, 0.15 μM of CoCl2and 0.075 μM NH4Mo7O24. The seeds were sown on rock wool as described [58].

For the initial Pi re-supply experiments, Col-0 plants were grown in hydroponics. In the first four weeks, the nutrient solution was exchanged three times. In the depletion phase, nutrient solutions were exchanged twice a week for both the control and experimental plants. The controls were on nutrient solution containing 250 μM Pi while the experimental plants were on nutrient solution lacking Pi. After 12 days (d) of Pi deprivation on nutrient solution without Pi, experimental plants were transferred either to solutions containing 250 μM Pi or to solutions lacking Pi. To ensure a stable osmotic environment the solutions were exchanged daily during this period.

For the long-term Pi depletion study, the standard nutrient solution was changed once within the first four weeks of hydroponic growth, then alternating on every 2 d or 3 d for plants grown on Pi-containing nutrient solution, or every 7 d for plants grown on nutrient solution lacking in Pi. Tissues were harvested after 14 d of treatment.

For the experiment to measure the short-term rate of Pi depletion from the medium the seedlings were grown in nutrient solution containing 250 μM Pi, exchanged three times within 30 d. The seedlings were then transferred to solutions without Pi to deplete plant P pools, where the nutrient solution was changed every 7 d. After 18 d of Pi deprivation plants were then transferred to solutions containing 250 μM Pi. Tissues were harvested every 30 mins for 6 h after the last transfer.

For root-growth assays plants were grown on vertical plates of solid one-half-strength Hoagland’s nutrient solution containing 0.5% sucrose and 8 g/L of agar (Plant Micro Agar, Phyto Technologies Laboratory LLC, Shawnee Mission, Kansas, USA). Seedlings were grown for 5 d on solid medium containing 250 μM Pi before transfer to fresh medium containing either 250 μM Pi (High Pi) or 5 μM Pi (Low Pi) for 12 d.

All plants were grown with 10 h light (160 μmol m−2s−1PAR) at 21°C and 14 h dark at 18°C. Plant tissues were harvested with appropriate staggering starting 3 h after the beginning of the light period to ensure that plants were at a comparable physiological and metabolic state for each harvest.

For seed production, seedlings were transferred to pots with soil after 3 weeks and grown for a month under 10 h/14 h short-day conditions as above, before transfer to long-day conditions (16 h light [100 μmol m−2s−1PAR] at 22°C and 8 h dark at 19°C) until harvest.

Characterization of T-DNA insertion mutant lines

Genomic DNA was isolated from leaves of 4-week-old plants using a cetyl trimethylammonium bromide (CTAB)-based method [59]. PCR genotyping was performed using primers specific for the T-DNA left border and gene-specific primers corresponding to the regions of At5g43350 (AtPHT1;1), At1g20860 (AtPHT1;8) and At1g76430 (AtPHT1;9) reported to be flanking the T-DNA inserts (see Additional file11: Table S2).

RNA isolation and quantitative reverse-transcription PCR

Poly-A+RNA was isolated from up to 40 mg of plant tissue using oligo-dT magnetic beads (Dynabeads, Invitrogen Life Technologies, Grand Island, NY), followed by cDNA synthesis (Bioscript RT, Bioline, Alexandria, NSW) on the beads [60]. Primers for quantitative reverse-transcription PCR (qRT-PCR) (see Additional file11: Table S2) were designed against candidate cDNA sequences using two software packages (Primer Express, Applied Biosystems, Life Technologies; BLAST Primer3,http://www.ncbi.nlm.nih.gov/tools/primer-blast). PCR primer efficiencies were determined using LinRegPCR [61]. Efficiencies did not change significantly between different cDNA samples (Additional file11: Table S2).AtPDF2[29],AtYLS8[30] andAtACT7(At5g09810) were used as reference genes. qRT-PCR was carried out using a commercial reagent kit according to the manufacturer’s instructions (Power SYBR Green Master Mix, Applied Biosystems, Life Technologies). Reactions (10 μl) were performed in 96-well format and contained approx. 0.5 ng cDNA, 2.5 μl of a mixture containing 1.2 μM each of the forward and reverse primers, and 5 μl of master mix. The PCR conditions were one cycle of 50°C for 20 sec and 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min (7500FAST Sequence Detection System, Applied Biosystems, Life Technologies) followed by a melt curve analysis to detect non-specific amplification products. The cycle threshold (Ct) and normalized fluorescence values were determined for each sample during the quantitative PCR cycling reaction (Prism Sequence Detector Software v. 2.0, Applied Biosystems, Life Technologies). The threshold fluorescence value for determining Ctwas set to 0.2 relative units. A comparative Ctapproach was applied, where the transcript level of a target gene was normalized to the average signal from theAtPDF2,AtACT7andAtYLS8reference genes [29]. Expression levels were given on a log2scale expressed as 40-∆Ct,where ∆Ctis the difference between the Ctof the target gene and the average Ctreference genes [22]. Therefore, 40-∆Ctvalue equal to 40 represents a transcript amount that is equal to the average transcript abundance for the reference genes. The threshold 40-∆Ctvalue in this experiment was 25, using a theoretical lowest target Ctvalue of 40. According to the manufacturer’s instructions, a Ctvalue of 35 (corresponding to a 40-∆Ctvalue of 30) marks the lower detection limit of the PCR instrument used. Changes in transcript levels compared to the corresponding tissues from plants continuously supplied with Pi are given on a log scale expressed as ∆∆Ct [62],[63].

P determinations and elemental analysis

Pi was measured using a modified ammonium molybdate method [64]. Fresh tissues were homogenized at a ratio of 1 mg sample fresh weight to 10 μl 1% (v/v) acetic acid. The homogenate was centrifuged twice at 12,000 × g for 15 min at 4°C. Samples were diluted and a 90 μl aliquot was combined with 210 μl 0.35% (w/v) NH4MoO4, 1.4% (w/v) ascorbic acid in 1 N H2SO4and incubated in the dark for 60 min at 37°C. A standard curve was constructed using dilutions of KH2PO4. The absorbance of the reaction products was measured at 820 nm and the Pi concentration of the unknown sample extrapolated from the standard curve.

Total P was extracted from approx. 10 to 20 mg of dried tissue by digesting in 3 ml HNO3at 100°C for 10 min. After cooling for 5 min, 1 ml of HClO4was added. The acid digests were heated to 150°C until the vigorous reaction that takes place between the HClO4and the organic residue had run to completion. Samples were heated to 180°C for 10 min to dehydrate any silica present. After the sample had cooled, 2 ml of de-ionized water were added. Samples were diluted as necessary before combining 150 μl with 50 μl malachite green and polyvinyl alcohol mixture [65] and allowed to stand for 8 min. The absorbance of the reaction was measured at 650 nm and the total P concentrations of the samples were extrapolated from a standard curve constructed using standard solutions of KH2PO4. Po was calculated from these data as total P minus Pi.

Root length measurement

For measuring root growth of seedlings on vertical plates, the position of the root tip was marked daily on the plate. At the end of the experiment, the plates were scanned and the incremental changes in root length were measured (LSM Image Browser, Carl Zeiss Microscopy GmbH, Jena, Germany).

Anthocyanin measurement

Anthocyanins were measured using a pH differential method [66]. Fresh tissue was homogenized at a ratio of 1 mg fresh weight to 10 μl 1% (v/v) acetic acid. The homogenate was centrifuged twice at 12,000 × g for 15 min at 4°C. A 60 μl portion of the final supernatant was diluted with 240 μl of 0.025 M potassium chloride solution, pH 1.0 and another 60 μl aliquot diluted with 240 μl 0.4 M sodium acetate buffer, pH 4.5. The two dilutions, which exhibit the pH-dependent structural transformation of the chromophore of anthocyanins, were equilibrated for 15 min. Sample absorbance was measured at 520 nm and at 700 nm to correct for overall background absorbance from other pigments. The concentration of monomeric anthocyanin pigments was calculated using the formula: Anthocyanin (μmol/g FW) = (A × MW × DF × 1000)/(ε × 1 cm) where: A is the absorbance of diluted sample, MW is the molecular weight of cyanidin-3-glucoside (MW = 449.2), DF is the dilution factor and ε is the molar absorption coefficient for cyanidin-3-glucoside (ε = 26,900 L mol−1cm−1) [66].

Statistical analyses

Unpaired T-tests were done on the qPCR data (GraphPad Quick Calcs, GraphPad Software, Inc., San Jose, CA, USA). Analysis of Variance (ANOVA) followed by Tukey’s Multiple Comparison of Means was done for the root-to-shoot ratio, anthocyanin and Pi measurements, lateral root density, and primary and lateral root lengths (R software, R Foundation for Statistical Computing, Vienna, Austria).

Availability of supporting data

The data sets supporting the results of this article are included within the article as additional files.

Additional files

Abbreviations

ANOVA:

Analysis of variance

cDNA:

Complimentary deoxyribonucleic acid

CTAB:

Cetyl trimethylammonium bromide

Ct:

Cycle threshold

Col-0:

Columbio-0

d:

Days

DF:

Dilution factor

DNA:

Deoxyribonucleic acid

FW:

Fresh weight

MW:

Molecular weight

LB:

Left-border primer

LP:

Left gene-primer

P:

Phosphate

Pi:

Inorganic phosphate

PHT:

Phosphate transporters

qRT-PCR:

Quantitative reverse-transciption polymerase chain reaction

RB:

Right-border

RP:

Right gene primer

SD:

Standard deviation

WT:

Wild-type

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Acknowledgements

We thank Dr Khalil Kariman (University of Western Australia), Dr Xing Wang (University of Western Australia) and Dr Made Pharmawati (Undayana University, Bali, Indonesia) for their technical advice and assistance.

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Correspondence toPatrick M Finnegan.

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The authors declare that they have no competing interests.

Authors’ contributions

HRLG participated in the design of the study, performed experiments, analysed the data and drafted the manuscript. RJ participated in the conception of the study, contributed to its design, performed experiments, and helped draft the manuscript. PMF conceived and coordinated the study, contributed to its design, and helped draft the manuscript. All authors read and approved the final manuscript.

Electronic supplementary material

(A)

Additional file 1: Figure S1.: Plants used in the phosphate (Pi) re-supply experiment. Induction of anthocyanin production by Pi deprivation. Red arrows show areas where anthocyanins accumulated.(B)Pi concentration in the roots and shoots of control plants and plants re-supplied with Pi for 3 days. Control plants were continuously supplied with 250 μM Pi in nutrient solution for 43 days. For Pi re-supply, plants were continuously provided with 250 μM Pi for 30 d, transferred to solution without Pi for 12 days to deplete internal P pools, and then transferred to a solution containing 250 μM Pi for three days before harvest. Values are means ± S.E. (n = 3 biological replicates). * indicates significantly different means (P <0.05) within a tissue according to Student’s t-test. (PDF 90 KB)

(A)

Additional file 2: Figure S2.: Confirmation ofAtpht1mutant genotypes. Genomic analysis of the homozygous mutants compared to WT(A). Using the left gene primer (LP, LP = PXR in panelsBtoDand in the Additional file11: Table S2, where X = 1, 8 or 9) and the right gene primer (RP, RP = PXF in panelsBtoDand in the Additional file11: Table S2, where X = 1, 8 or 9) produced a gene specific fragment in the wild-type, but not in the knock-out line. Schematic representations of the T-DNA insertion sites in theAtpht1;1–2(B),Atpht1;8(C)andAtpht1;9–1(D)mutants. Untranslated regions (gray boxes), exons (black boxes), introns (thick black lines), T-DNAs (white boxes) and segments lost during the insertion process (hatched boxes) are shown. DNA sequencing of the fragments amplified using the indicated primers (arrows) confirmed the location of the T-DNA left border (LB) in the genome. The underlined sequence belongs to the T-DNA insert, while the non-underlined sequence belongs to the target gene and identifies the insertion site. Two T-DNAs were tandemly inserted in an inverted orientation in each mutant; hence, the right borders (RB) were presumed to be next to each other. For theAtpht1;8mutant, the LB of T-DNA #1 lost 95 bp, while the LB of T-DNA #2 lost 79 bp during insertion. ForAtpht1;9–1mutant, the LB of T-DNA #1 lost 106 bp, while the LB of T-DNA #2 lost 77 bp during insertion. The seedlings were grown on solid media containing 250 μM Pi for five days before transfer to the same media containing 5 μM Pi for 12 days. (PDF 196 KB)

(A)

Additional file 3: Figure S3.: Relative abundance ofAtPHT1;1–2, AtPHT1;8 and AtPHT1;9–1transcripts in the roots of their respectiveAtpht1knock-out lines (KO) and Col-0 WT described in Additional file2: Figure S2. RT-PCR(A)and qPCR(B)analysis ofAtPHT1transcript abundance in the WT and knock-out mutants. Seedlings were grown in hydroponic medium containing 250 μM Pi for 30 d before transfer to media lacking added Pi for 14 d to maximiseAtPHT1transcript abundance. Transcript abundance was determined in whole seedlings using gene specific primers (Additional file11: Table S2) and compared toAtACT2transcript abundance as a control for equal loading of cDNA. Transcript abundance ofAtPHT1;1,AtPHT1;8andAtPHT1;9was determined in the root of phosphate deprived wild-type plant and their respective KO lines using gene specific primers (Additional file11: Table S2). The limit of detection for our assay was equivalent to a 40-∆Ctvalue of 30. The residual signal observed for each mutant may represent intact or fragmented transcripts, or may have arisen from spurious products formed in the absence of abone fidetarget. Values are means ± S.D., n = 3 biological replicates. (PDF 56 KB)

12870_2014_334_MOESM4_ESM.pdf

Additional file 4: Figure S4.: Morphological response of WT andAtpht1knock-out lines to Pi supply. Seedlings were grown for 5 d on solid medium containing 250 μM Pi before transfer to fresh medium containing either 250 μM Pi (+P) or 5 μM Pi (−P) for 12 d. (PDF 36 KB)

12870_2014_334_MOESM5_ESM.pdf

Additional file 5: Figure S5.: Root and shoot fresh weight ofAtpht1mutant and Col-0 seedlings. These seedlings were those described in Figure3. Values are means ± S.D. (n = 3 plates of 12 seedlings each). * indicates significantly different means (P <0.05) according to Student’s t-test compared to the WT grown under the same conditions. (PDF 57 KB)

12870_2014_334_MOESM6_ESM.pdf

Additional file 6: Figure S6.: Pi depletion of the growth medium by Pi-deprived plants re-supplied with 250 μM Pi. Plants were grown in 150 ml nutrient solution containing 250 μM Pi for 30 d and transferred to solution lacking added Pi for 18 d before a final transfer to solution containing 250 μM Pi. The final nutrient solution was sampled every 30 mins after the last transfer. Values are means ± S.D. (n = 3 boxes with 12 plants each). Trend lines are the best approximation of the initial rates of Pi withdrawal from the solution. The slope of the trend line is given on each graph. The slope (rates) may differ from the values in Table1, where the average slope from three independent experiments is recorded. (PDF 141 KB)

(A)

Additional file 7: Figure S7.: Accumulation of Pi in the WT andAtpht1knock-out lines. Time course of Pi accumulation in root(A)and shoot(B)of WT andAtpht1mutant seedlings. Values are means ± S.D. (n = 3 biological replicates with 12 plants each grown at separate times). Data are the same as shown in Figure4except that error bars (± S.D.) are shown. (PDF 61 KB)

(A)

Additional file 8: Figure S8.: Relative transcript abundance for a panel of 17 Pi-responsive genes in the root and shoot(B)tissues ofAtpht1;8. The horizontal black line represents the theoretical detection limit of the qPCR instrument (see Methods). All bars below the line represent transcripts that were below the detection limit. The plants were supplied with 250 μM Pi (sufficient Pi) or no added Pi (no Pi) for 14 d and were the same plants as those described in Figure5. Values are means ± S.D. (n = 3 biological replicates grown at separate times). * indicates that the 40-∆Ct value in the mutant was significantly different (P <0.05) according to Student’s t-test, compared to the WT grown under the same conditions. (PDF 153 KB)

(A)

Additional file 9: Figure S9.: Relative transcript abundance from a panel of 17 Pi-responsive genes in the root and shoot(B)tissues of Atpht1;9–1. The horizontal black line represents the theoretical detection limit of the qPCR instrument (see Methods). All bars below the line represent transcripts that were below the detection limit. The plants were supplied with 250 μM Pi (sufficient Pi) or no added Pi (no Pi) for 14 d and were the same plants as those described in Figure5. Values are means ± S.D. (n = 3 biological replicates grown at separate times). * indicates that the 40-∆Ct value in the mutant was significantly different (P <0.05) according to Student’s t-test, compared to the WT grown under the same conditions. (PDF 153 KB)

12870_2014_334_MOESM10_ESM.xlsx

Additional file 10: Table S1.: Expression levels of AtPHT1 genes derived from microarray experiments using root cells and tissues isolated by fluorescence activated cell sorting of protoplasts from marker lines. (XLSX 307 KB)

Additional file 11: Table S2.: Gene specific primers used in the experiment. (XLSX 15 KB)

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Lapis-Gaza, H.R., Jost, R. & Finnegan, P.M.ArabidopsisPHOSPHATE TRANSPORTER1 genesPHT1;8andPHT1;9are involved in root-to-shoot translocation of orthophosphate.BMC Plant Biol14,334 (2014). https://doi.org/10.1186/s12870-014-0334-z

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Keywords

  • Phosphate transporters
  • Arabidopsis
  • Gene expression
  • Local signaling
  • Systemic signaling