Arabidopsis PHOSPHATE TRANSPORTER1 genes PHT1;8 and PHT1;9 are involved in root-to-shoot translocation of orthophosphate

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Lapis-Gaza et al. BMC Plant Biology 2014, 14:334 http://www.biomedcentral.com/1471-2229/14/334 RESEARCH ARTICLE Open Access Arabidopsis PHOSPHATE TRANSPORTER1 genes PHT1;8 and PHT1;9 are involved in root-to-shoot translocation of orthophosphate Hazel R Lapis-Gaza1, Ricarda Jost1 and Patrick M Finnegan1,2* 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. The Arabidopsis thaliana AtPHT1 gene 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 in AtPHT1;8 and AtPHT1;9 knock-out mutants compared to wild-type plants showed that both transporters are involved in the translocation of Pi from the root to the shoot. Upon inactivation of AtPHT1;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 of AtPHT1;5 and AtPHT1;7 was altered in the absence of AtPHT1;8, and the transcript profile of AtPHT1;7 was altered in the Atpht1;9 mutant. 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 of A. thaliana to changes in Pi supply than was previously thought. Keywords: Phosphate transporters, Arabidopsis, Gene expression, Local signaling, Systemic signaling 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 * Correspondence: patrick.finnegan@uwa.edu.au 1 School of Plant Biology, University of Western Australia, 35 Stirling Highway, Crawley (Perth), WA 6009, Australia 2 Institute of Agriculture, University of Western Australia, 35 Stirling Highway, Crawley (Perth), WA 6009, Australia [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]. The Arabidopsis AtPHT1 family has nine members. The family is composed of several high-affinity Pi transporters having Km values 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 the AtPHT1 genes are detected in both roots and shoots [12-15], while AtPHT1;6 transcripts are most abundant in flowers [12]. Transcripts from all AtPHT1 genes except AtPHT1;6 accumulate upon Pi starvation [16]. Transcriptional regulation of AtPHT1 expression seems to be mainly controlled by the internal P status [13,15,17,18]. © 2014 Lapis-Gaza et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Lapis-Gaza et al. BMC Plant Biology 2014, 14:334 http://www.biomedcentral.com/1471-2229/14/334 Sugars and cytokinins can also direct the expression of some AtPHT1 family members [19]. Several strategies have evolved in plants that help them acclimate to variation in Pi availability, including the modulation of PHT1 gene 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 gene AtPHO2 and a family of regulatory, non-coding RNAs encoded by the AtIPS1 and AtAT4 genes [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 AtIPS1 and AtAT4 transcripts 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-responsive AtERF1 transcription factor gene, the metalloproteinase At2-MMP gene, the jasmonateinducible AtGSTU12 and AtLOX4 genes and the AtWRKY75 transcription factor gene, which encodes a modulator of both the Pi-starvation response and root development [25]. Functional characterization of AtPHT1;1 and AtPHT1;4 validated their roles in Pi acquisition from the soil solution under both Pi-sufficient and Pi-deficient growth conditions [26]. AtPHT1;5 plays a role in translocating Pi from source to sink organs [27]. Analysis of a Atpht1;9–1 mutant and pht1;8/pht1;9 silencing 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 two AtPHT1 genes in the pho2 mutant [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 the AtPHT1 gene 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 two AtPHT1 gene 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. Page 2 of 19 Results Transcripts from individual AtPHT1 genes responded differentially to Pi deprivation and re-supply The remodeling kinetics of the AtPHT1 transcript pool in response to both P depletion and Pi re-supply were examined in well-established Arabidopsis plants 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 file 1: 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 the AtPHT1 gene family in root and shoot tissues over the next three days (Figure 1). AtPHT1;6 was 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-ΔCt value of greater than two were considered, corresponding to a four-fold difference in transcript abundance [31]. In Arabidopsis roots, 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 the AtPHT1 genes tested (P ≤0.05), except for AtPHT1;2, when compared to control plants continuously supplied with Pi (Figure 1A, cf. D1). This is in general agreement with what has previously been observed [13,15,32]. The abundance of AtPHT1;7 and AtPHT1;8 transcripts 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 the AtPHT1 genes tested in roots was similar to that in the control plants (Figure 1A). The exceptions were AtPHT1;1, AtPHT1;3 and AtPHT1;4. The repression of AtPHT1 transcript abundance by day 2 of Pi re-supply was generally stronger than after day 1. Transcripts from AtPHT1;3, AtPHT1;4, AtPHT1;5, AtPHT1;7 and AtPHT1;9 were repressed to their lowest levels at this time point. Transcripts from AtPHT1;1, AtPHT1;2 and AtPHT1;8 were repressed to their lowest levels at day 3 of Pi re-supply (Figure 1A). Interestingly, 3 d of Pi re-supply repressed the abundance of AtPHT1 transcripts to levels below those present in control plants that had been Lapis-Gaza et al. BMC Plant Biology 2014, 14:334 http://www.biomedcentral.com/1471-2229/14/334 Page 3 of 19 Figure 1 Responsiveness of AtPHT1 transcript levels to Pi supply. The relative abundance of AtPHT1 transcripts in roots (A) and shoots (B) of 42-day-old Arabidopsis plants 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-ΔCt, 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. continuously supplied with Pi for the entire experiment even though the root Pi concentration was the same as in the control plants (Additional file 1: Figure S1B). The general trends of transcript accumulation in the shoots of Pi-deprived plants were similar to those found in the roots (Figure 1B), except for AtPHT1;7 and AtPHT1;8, Lapis-Gaza et al. BMC Plant Biology 2014, 14:334 http://www.biomedcentral.com/1471-2229/14/334 Page 4 of 19 where transcript abundance remained constant and similar to the control plants. In contrast to roots, 1 d of Pi resupply did not cause a decrease in the abundance of any AtPHT1 transcripts compared to Pi-deprived plants of the same age. Interestingly, the transcript abundance for AtPHT1;8 and AtPHT1;9 was actually higher after 1 d of Pi resupply than in the Pi-deprived plants of the same age. The transcript amount for most of the AtPHT1 genes 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 file 1: Figure S1B). Cluster analysis showed that the transcript responses to changes in Pi supply for the main AtPHT1 transporter genes, AtPHT1;1 and AtPHT1;4, along with AtPHT1;2, were distinct from those of the other AtPHT1 genes in both roots and shoots (Figure 2). In roots, the response patterns for AtPHT1;8 and AtPHT1;7 were similar to each other, while the responses of AtPHT1;9 were most similar to those of AtPHT1;5 and AtPHT1;3. In the shoot, the response patterns for AtPHT1;8 and AtPHT1;9 clustered with those of AtPHT1;7, while the pattern of changes for AtPHT1;3 and AtPHT1;5 clustered discretely. compared to the corresponding Col-0 WT (Figure 3A). This was brought about by a 10% greater shoot biomass combined with a 10% lower root biomass in the mutants (Additional file 5: Figure S5). The decreased root biomass in the P-sufficient Atpht1;8 mutant was at least partly due to a lower primary root length (Figure 3B), although the lateral roots also tended to be shorter in both mutants (Figure 3C). When depleted of P, the root system biomass of Atpht1;8 and Atpht1;9–1 seedlings was not significantly different from the WT (Figure 3, Additional file 5: Figure S5). The Pi concentration in the roots of the mutants was also the same as that in WT (Figure 3D), but it was 20% lower in the mutant compared to WT shoots (Figure 3E). The lower shoot Pi concentration might be expected to enhance P-starvation responses such as anthocyanin production which was indeed the case in the Atpht1;8 mutant (Figure 3F). By contrast, the anthocyanin concentration in the shoots of Atpht1;9–1 seedlings depleted of P was only about half of that in the WT. This lower anthocyanin concentration was not due to a dilution by growth, as shoot biomass in Atpht1;9-1 seedlings depleted of P was about 10% less than that of WT seedlings (Additional file 5: Figure S5). Disruption of AtPHT1;8 or AtPHT1;9 had diverse effects in Arabidopsis seedlings Disruption of AtPHT1;8 or AtPHT1;9 compromised root-to-shoot translocation of Pi There is only a single mutant available for each AtPHT1,8 and AtPHT1;9 that has a predicted T-DNA insertion in the exon region. PCR across the predicted T-DNA leftborder (LB) insertion sites and sequencing of the PCR products confirmed that the putative Atpht1;1-2, Atpht1;8 and Atpht1;9–1 mutants used in this study were homozygous for the presence of T-DNA at sites expected to disrupt gene function (Additional file 2: Figure S2A). At least two T-DNAs have been inserted in a head-to-head orientation in all three mutants. In Atpht1;1–2, two T-DNAs were inserted 42 bp downstream of the start of exon 3, confirming previous results (Additional file 2: Figure S2B) [26]. In the Atpht1;8 mutant, the T-DNAs were located 595 bp downstream of the start of exon 2 (Additional file 2: Figure S2C). The insertion site in the previously characterised Atpht1;9–1 mutant allele [28] was found to be located 77 bp upstream of the start codon in exon 1 (Additional file 2: Figure S2D). These insertions caused the amount 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 file 3: Figure S3). The disruption of AtPHT1;8 or AtPHT1;9 gene function did not cause gross morphological changes in 17-day-old mutant seedlings supplied with sufficient Pi or depleted of Pi (Figure 3, Additional file 4: 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 Forty-eight-day-old Atpht1;8 and Atpht1;9–1 plants 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 the Atpht1;1–2 mutant (Additional file 6: Figure S6). As expected, the Atpht1;1–2 mutation caused a significantly lower rate of Pi removal from the nutrient solution than WT (Table 1). However, the Atpht1;8 and Atpht1;9–1 mutations had a negligible effect on the ability of P-depleted plants to remove Pi from the nutrient solution. Moreover, the Atpht1;8 and Atpht1;9-1 mutants were clearly distinguished from both WT and Atpht1;1–2 in the short-term kinetics of Pi accumulation in the roots and shoots of these P-depleted plants upon Pi re-supply (Figure 4, Additional file 7: Figure S7). The Atpht1;1–2 mutant, although compromised in its ability to acquire Pi from the nutrient solution (Table 1), had a root Pi concentrations similar to that of WT (Figure 4A). The concentration of Pi tended to be somewhat higher in the roots of Atpht1;8 than in WT throughout the time course, while the Pi concentration in the roots of Atpht1;9–1 tended to be marginally lower than in WT. After 300 min of Pi re-supply to the Pi-deprived plants, the roots of the Atpht1;8 mutant had a significantly higher Pi concentration than those of WT, while the roots of Atpht1;9–1 were indistinguishable from those of WT. In sharp contrast to the roots, the shoots of all three P-depleted mutants accumulated dramatically less Pi Lapis-Gaza et al. BMC Plant Biology 2014, 14:334 http://www.biomedcentral.com/1471-2229/14/334 Page 5 of 19 Figure 2 Co-expression analysis of the AtPHT1 gene family in response to Pi supply. A hierarchical cluster analysis of AtPHT1 gene transcript patterns in roots (A) and shoots (B) of the 42-day-old Arabidopsis plants described in the legend to Figure 1 is shown. ΔΔCt values generated from the results shown in Figure 1 using plant organs continuously supplied with Pi as a reference were analysed using Euclidean distance and complete linkage [33]. Lapis-Gaza et al. BMC Plant Biology 2014, 14:334 http://www.biomedcentral.com/1471-2229/14/334 Page 6 of 19 Figure 3 Developmental and biochemical responses of Arabidopsis Col-0 and Atpht1 knock-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) were determined at harvest. Values are means ± 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. than WT during short-term Pi re-supply (Figure 4B). The Atpht1;1–2 mutant 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. Table 1 Capacity of 48-day-old P-depleted Arabidopsis WT and Atpht1 knock-out plants to withdraw Pi from nutrient solution (see Additional file 6: Figure S6) Genotype Rate of Pi removal (nmol Pi plant−1 min−1) ± S.D. R2 Col-0 99 ± 5 0.98 Atpht1;1-2 67 ± 5* 0.99 Atpht1;8 94 ± 12 0.96 Atpht1;9-1 92 ± 7 0.99 *Significantly different means (P <0.05) according to one-way. ANOVA (n = 3 biological replicates with 12 plants each). However, both Atpht1;8 and Atpht1;9–1 plants, 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 the Atpht1;1–2 mutant. After 300-min exposure to Pi, the shoot Pi concentration in both Atpht1;8 and Atpht1;9–1 mutants was less than half of that in WT and 40% lower than in the Atpht1;1–2 mutant that was impaired in the primary uptake of Pi (Figure 4B). The longer-term impact of the Atpht1;1–2, Atpht1;8 and Atpht1;9–1 mutations 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 (Figure 5). For Atpht1;1–2 plants with their compromised Pi uptake capacity (Table 1), 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 Lapis-Gaza et al. BMC Plant Biology 2014, 14:334 http://www.biomedcentral.com/1471-2229/14/334 Page 7 of 19 Figure 4 Short-term accumulation of Pi by P-depleted 48-day-old Col-0 and Atpht1 knock-out Arabidopsis plants grown in hydroponics. Accumulation of Pi in roots (A) and shoots (B) of WT and pht1 mutant 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 file 7: Figure S7. 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 in Atpht1;1–2 roots and shoots under Pi-sufficient conditions. On the other hand, the long-term Pi concentration in the root and the shoot of the Atpht1;8 mutant was indistinguishable from WT and Atpht1;9–1 in both Pi-sufficient and Pi-limited conditions. The Pi concentration was also similar to Atpht1;1–2 but in Pi-limited condition only. The total P concentration in roots of the Atpht1;8 mutant was identical to the WT regardless of Pi supply, while in the shoots the total P concentration was lower and similar to that of the Atpht1;1–2 mutant. This difference in root, but not shoot, total P concentration between these two mutants highlights a fundamental difference between them; the lower shoot P in Atph1;8 Lapis-Gaza et al. BMC Plant Biology 2014, 14:334 http://www.biomedcentral.com/1471-2229/14/334 Page 8 of 19 Figure 5 The effect of Pi supply on the Po and Pi concentrations in 44-day-old Arabidopsis Col-0 WT and Atpht1 plants grown in hydroponics. The P concentrations in the inorganic (Pi) and organic (Po) pools were determined in roots (A & B) and shoots (C & D) of plants grown on nutrient solution containing a 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. compared to WT was not due to a lower P concentration in the root, as may have been the case in Atpht1;1–2, which is impaired in Pi uptake. In the roots of Pi-sufficient Atpht1;9–1 plants, the Pi concentrations were similar to those in the Atpht1;8 mutant, while the Po concentrations were similar to those in the Atpht1;1–2 mutant. However, in the shoots of these plants, both Pi and Po concentrations were similar to those of Atpht1;8, indicating that Atpht1;9 only differs from Atpht1;8 in its ability to allocate Pi to Po in the roots. This difference was also observed in Pi-deficient plants (Figure 5B and D). Loss of AtPHT1;8 and AtPHT1;9 influenced the transcript profiles of other genes repressed by Pi A panel of 17 Pi-responsive genes, including the full set of AtPHT1 genes, was used to assess the interactions of AtPHT1;8 and AtPHT1;9 with other components of the P-starvation response (Additional file 8: Figures S8 and Additional file 9: 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 were AtPHT1;4, AtPHT1;5, AtPHT1;7, AtPHT1;8, AtPHO2, AtMIR399d, At4 and AtIPS1, while those that responded to local P status were AtERF1, AtGSTU12, AtLOX4, At2-MMP and AtWRKY75. In 44-day-old plants, the Atpht1;8 mutation caused changes in the transcript levels of some of the selected Pi-responsive genes (Figure 6, Additional file 8: Figure S8). In the roots of Pi-sufficient Atpht1;8 plants, AtPHT1;3, AtPHT1;5 and AtPHT1;7 were more strongly repressed than in WT, while AtPHT1;2 transcripts were more abundant (Figure 6A). However, transcripts from all the AtPHT1 genes were induced to WT levels upon P depletion. Atpri-MIR399d transcripts were less strongly repressed in Atpht1;8 roots at sufficient Pi than in WT roots, while transcripts 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 the Atpht1;8 mutant resulted in lower AtWRKY75 transcript 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, AtWRKY75 transcript levels were also lower in this tissue than in the WT in the presence of Pi. The shoots of Atpht1;8 plants grown in the presence of sufficient Pi contained less AtPHT1;5 and AtPHT1;7 transcripts than WT (Figure 6B). In P-depleted plants, the abundance of AtPHT1 transcripts, including those from AtPHT1;5, were generally de-repressed to the same levels seen in the WT. The exception was transcripts Lapis-Gaza et al. BMC Plant Biology 2014, 14:334 http://www.biomedcentral.com/1471-2229/14/334 Page 9 of 19 Figure 6 Effect of the Atpht1;8 mutation on the P-responsiveness of transcripts from Pi-responsive genes, including those encoding known P signaling components. Transcript abundance in 44-day-old Atpht1;8 plants is expressed relative to that in WT plants grown under the same conditions (ΔΔCt values). 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). from AtPHT1;7, which were not as strongly de-repressed as in WT. In Atpht1;8 shoots, 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 of Atpri-MIR399d transcripts in the P-depleted plants. Among the genes responsive to local P signals, AtGSTU12 was somewhat more repressed in Pi-sufficient Atpht1;8 shoots compared to WT, but this repression was overcome during P-depletion. By contrast, AtLOX4 transcripts 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-old Atpht1;9–1 plants was similar to, but distinct from, that in the Atpht1;8 mutant (Figure 7, Additional file 9: Figure S9). In the roots of Pi-sufficient Atpht1;9–1, AtPHT1;7 and AtLOX4 transcripts were less abundant than in WT (Figure 7A). During P depletion, AtPHT1;7 was less strongly de-repressed than in WT, while AtPHT1;3 transcripts were more abundant. Atpri-MIR399d and AtAT4 transcripts were less abundant in the P-deprived Atpht1;9–1 mutant. Interestingly, both At2-MMP and AtWRKY75 transcript amounts were lower in response to P depletion in the Atpht1;9–1 mutant, similar to what was observed for AtWRKY75 in the roots of the Atpht1;8 mutant. In the shoot of P-sufficient Atpht1;9–1, AtPHT1;3 was less repressed than in WT (Figure 7B). Transcripts from AtPHO2 were more strongly repressed by Pi in the mutant than in WT which was complemented by a decreased repression by Pi for both Atpri-MIR399d and AtAT4 transcripts. In the shoot of P-depleted Atpht1;9–1, AtPHT1;7 transcripts, along with those from the entire set Lapis-Gaza et al. BMC Plant Biology 2014, 14:334 http://www.biomedcentral.com/1471-2229/14/334 Page 10 of 19 Figure 7 Effect of the Atpht1;9–1 mutation on the P-responsiveness of transcripts from Pi-responsive genes, including those encoding known P signaling components. Transcript abundance in 44-day-old Atpht1;9–1 plants is expressed relative to that in WT plants grown under the same conditions (ΔΔCt values). 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). of genes associated with local signaling events, were less de-repressed than those in the WT. AtPHT1;8 and AtPHT1;9 are necessary for the root-to-shoot translocation of Pi Discussion A clear response in Arabidopsis to changes in P status is the reversible repression of AtPHT1 gene expression [20]. The modulation of AtPHT1 expression alters the Pi transport activity within the plants in response to the prevailing Pi availability [10]. Here we provide evidence that AtPHT1;8 and AtPHT1;9 were 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 with AtPHT1;7 and other genes that are involved in controlling P nutrition (Figure 8). 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 genotypedependent 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 either AtPHT1;8 or AtPHT1;9 severely
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