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Plant Physiol. Jul 2006; 141(3): 1000–1011.
PMCID: PMC1489903

pho2, a Phosphate Overaccumulator, Is Caused by a Nonsense Mutation in a MicroRNA399 Target Gene1,[W]

Abstract

We recently demonstrated that microRNA399 (miR399) controls inorganic phosphate (Pi) homeostasis by regulating the expression of UBC24 encoding a ubiquitin-conjugating E2 enzyme in Arabidopsis (Arabidopsis thaliana). Transgenic plants overexpressing miR399 accumulated excessive Pi in the shoots and displayed Pi toxic symptoms. In this study, we revealed that a previously identified Pi overaccumulator, pho2, is caused by a single nucleotide mutation resulting in early termination within the UBC24 gene. The level of full-length UBC24 mRNA was reduced and no UBC24 protein was detected in the pho2 mutant, whereas up-regulation of miR399 by Pi deficiency was not affected. Several characteristics of Pi toxicity in the pho2 mutant were similar to those in the miR399-overexpressing and UBC24 T-DNA knockout plants: both Pi uptake and translocation of Pi from roots to shoots increased and Pi remobilization within leaves was impaired. These phenotypes of the pho2 mutation could be rescued by introduction of a wild-type copy of UBC24. Kinetic analyses revealed that greater Pi uptake in the pho2 and miR399-overexpressing plants is due to increased Vmax. The transcript level of most PHT1 Pi transporter genes was not significantly altered, except PHT1;8 whose expression was enhanced in Pi-sufficient roots of pho2 and miR399-overexpressing compared with wild-type plants. In addition, changes in the expression of several organelle-specific Pi transporters were noticed, which may be associated with the redistribution of intracellular Pi under excess Pi. Furthermore, miR399 and UBC24 were colocalized in the vascular cylinder. This observation not only provides important insight into the interaction between miR399 and UBC24 mRNA, but also supports their systemic function in Pi translocation and remobilization.

In addition to assimilating carbohydrates in photosynthetic tissues, plants have to acquire mineral nutrients from soil to build up basic components and maintain functional machinery to support their normal growth, development, and reproduction. Among these essential mineral nutrients, phosphorus (P) is one of the least available nutrients in soil. Although the total P content in soil is high, most of it is unavailable for uptake. Concentration of the available P source, inorganic phosphate or orthophosphate (Pi), in the soil solution is usually below that of many other micronutrients (Barber et al., 1963) and seldom exceeds 10 μm (Bieleski, 1973). The low availability of Pi in the soil solution is mainly caused by the rapid adsorption of Pi to clay and organic substances, precipitation of Pi with cations, or conversion of Pi into organic forms by soil microbes (Marschner, 1995; Holford, 1997; Raghothama, 1999). To reduce Pi deficiency to ensure crop productivity and the impact on the environment because of overfertilization, development of crops with improved Pi acquisition has been a focus of research. To achieve this goal, understanding the physiological, biochemical, and molecular changes under Pi deficiency in regulating Pi homeostasis within plants is necessary.

Plants have developed a series of adaptive responses to enhance Pi acquisition under Pi-limited conditions. These responses include changes in root morphology and architecture, increased Pi uptake activity, secretion of organic acids or phosphatase, and association with mycorrhizal fungi (for review, see Harrison, 1999; Raghothama, 1999; Poirier and Bucher, 2002; Lopez-Bucio et al., 2003; Ticconi and Abel, 2004). Moreover, conservation and remobilization of internal P is also crucial for maintenance of Pi homeostasis within plants. In the recent decade, both genetic and molecular approaches have been used to explore the genes involved in these adaptive responses. With the advantage of genomics tools, a large number of Pi-responsive genes involved in various physiological functions and metabolic pathways have been reported (Hammond et al., 2003; Wasaki et al., 2003; Wu et al., 2003; Misson et al., 2005), which is consistent with the roles of Pi involved in different aspects of plant growth and development. Among them, specific genes involved in phospholipid degradation, galactolipid and sulfolipid synthesis, and anthocyanin pathways during Pi deficiency were identified (Misson et al., 2005).

In addition, forward and reverse genetics approaches have identified several Arabidopsis (Arabidopsis thaliana) mutants with changes in internal Pi concentration (Poirier et al., 1991; Delhaize and Randall, 1995), Pi uptake (Misson et al., 2004; Shin et al., 2004), root morphology (Li et al., 2006; Sánchez-Calderón et al., 2006), or altered responses under Pi-deficient conditions (Chen et al., 2000; Zakhleniuk et al., 2001; Ticconi et al., 2004; Tomscha et al., 2004; Lopez-Bucio et al., 2005). From a reporter system driven by a Pi starvation-induced promoter, PHOSPHATE STARVATION RESPONSIVE 1 (PHR1), encoding a MYB transcription factor, was identified to up-regulate a specific group of Pi-responsive genes through the GNATATNC cis-element (Rubio et al., 2001; Franco-Zorrilla et al., 2004). Interestingly, characterization of a SUMO E3 ligase mutant, siz1, suggested that sumoylation is involved in modification of PHR1 function (Miura et al., 2005). A mutant defective in plasma membrane targeting of a Pi transporter (PHT1;1) was recently identified and revealed that the PHOSPHATE TRANSPORTER TRAFFIC FACILITATOR 1 (PHF1) protein mediates this process (González et al., 2005). Furthermore, cross-talk of Pi signaling pathways with the cytokinin and sugar-sensing pathways was proposed by the analyses of several other mutants (Franco-Zorrilla et al., 2002, 2005).

By screening the Pi content of shoots, two Arabidopsis mutants, pho1 and pho2, defective in Pi homeostasis, were identified (Poirier et al., 1991; Delhaize and Randall, 1995). Shoots of the pho1 mutant displayed a Pi-starvation phenotype because loading Pi into the xylem was impaired (Poirier et al., 1991). In contrast, the shoots of the pho2 mutant accumulated excessive amounts of Pi and exhibited Pi toxic symptoms because of increased Pi uptake and translocation of Pi from roots to shoots (Delhaize and Randall, 1995; Dong et al., 1998). Nevertheless, normal Pi concentration was maintained in the roots of the pho1 and pho2 mutants. pho1 was shown to be epistatic to pho2 because the double mutation displayed the pho1 phenotype (Delhaize and Randall, 1995). Consistent with its role in xylem loading, PHO1 was later identified to encode a membrane protein located in the stellar cells of the root (Hamburger et al., 2002). However, the PHO2 gene has not been reported but is suspected to be involved in phloem transport of Pi between shoots and roots or in regulating leaf Pi concentration (Dong et al., 1998).

Recently, we reported that microRNA399 (miR399) controls Pi homeostasis by regulating the expression of a ubiquitin-conjugating E2 enzyme (assigned as UBC24; Kraft et al., 2005) in Arabidopsis (Chiou et al., 2006). Accumulation of UBC24 mRNA was suppressed by the targeting of miR399, whose expression is up-regulated by Pi starvation (Fujii et al., 2005; Chiou et al., 2006). Significantly, overexpression of miR399 or loss of function of the UBC24 gene led to accumulation of high Pi content to a toxic level in leaves, which resulted from increased uptake of Pi from roots, increased translocation of Pi from roots to shoots, and retention of Pi in the leaves (Chiou et al., 2006). Moreover, impairment of Pi remobilization from old to young leaves accelerates toxicity in old leaves (Chiou et al., 2006). These observations suggest that interaction between miR399 and UBC24 regulates Pi homeostasis at the systemic level through long-distance communication.

It is interesting to note that the pho2 mutant, miR399-overexpressing, and UBC24 loss-of-function plants all displayed similar phenotypes, except that the defect in Pi remobilization within leaves has not been described in pho2. Moreover, PHO2 was mapped to chromosome 2 (Delhaize and Randall, 1995) near UBC24. In this study, we systematically compared the phenotypes of pho2, miR399-overexpressing, and UBC24 loss-of-function plants and demonstrated that PHO2 is indeed the UBC24.

RESULTS

pho2 Mutant Plants Resemble miR399-Overexpressing and UBC24 Loss-of-Function Plants

The pho2 mutant was obtained from an ethyl methylsulfonate mutagenesis pool as a Pi overaccumulator (Delhaize and Randall, 1995). To verify the physiological resemblance, pho2, miR399-overexpressing, and UBC24 T-DNA knockout plants (SAIL_47_E01; designated as ubc24-1) were compared. In Pi-sufficient soil, they all displayed the Pi toxic symptom as chlorosis and necrosis in the mature leaves (Fig. 1A). In agreement with this phenotype, the Pi concentration in the shoots of these plants was more than 4-fold that of wild-type plants (Fig. 1B). Moreover, these plants showed increased Pi uptake activity from roots (Fig. 1C) and greater Pi distribution from roots to shoots as compared with wild-type plants (Fig. 1D).

Figure 1.
Resemblance of Pi toxicity in miR399-overexpressing, pho2, and UBC24 loss-of-function (ubc24-1) plants. A, Pi toxic phenotype shown as chlorosis and necrosis in the leaves of 24-d-old miR399b-overexpressing (b), pho2 (c), and ubc24-1 (d) plants. Pi toxicity ...

The defect in Pi remobilization from old leaves observed in miR399-overexpressing and ubc24-1 plants (Chiou et al., 2006) was examined in the pho2 mutant. The Pi distribution among different leaves was inspected during growth (Fig. 2A). As expected, Pi concentration in leaves of pho2 plants was higher than that of wild-type plants; however, pho2 mutant plants and wild-type plants exhibited different Pi distribution patterns. Pi concentration in the old leaves (e.g. cotyledons and the first two leaves) of wild-type plants declined over time, which indicates movement of Pi out of the old leaves. In contrast, Pi concentration increased in the old leaves of pho2 (Fig. 2A). The pulse-chase experiment further verified the ability of Pi remobilization (Fig. 2B). After 10 d of growth in 33P-free nutrient solution, 33P radioactivity was mobilized out of the old leaves and distributed into the newly developed leaves in wild-type plants, but remained in the old leaves of pho2 and miR399-overexpressing plants (Fig. 2B). Retention of 33P radioactivity in these old leaves was associated with the emergence of Pi toxic symptoms (Fig. 2B, asterisks). These data indicate that, like miR399-overexpressing plants, pho2 mutant plants are also defective in remobilization of Pi out of the old leaves. Taken together, these data show that pho2, miR399-overexpressing, and ubc24-1 plants all show Pi overaccumulation resulting from increased Pi uptake from roots and Pi translocation to shoots, retention of Pi in the leaves, and impaired Pi remobilization within the leaves.

Figure 2.
Impairment of Pi remobilization in the pho2 mutant. A, Changes in Pi concentration in the leaves of wild-type plants (dotted lines) or pho2 mutants (solid lines) grown under Pi-sufficient (1 mm KH2PO4) conditions. Individual leaves were collected at the ...

pho2 Is Mutated in UBC24, a miR399 Target Gene

Initial mapping of pho2 suggested that the pho2 locus was linked to the as1 marker on chromosome 2 (Delhaize and Randall, 1995). The as1 marker (At2g37630) is close to UBC24 (At2g33770), about 1.5 Mb apart. cDNA and genomic fragments of UBC24 in pho2 were sequenced and a single nucleotide mutation from G to A, causing early termination in the 671 amino acid, was found (Fig. 3A). The termination is located in exon 6 at the beginning of the UBC domain, which suggests loss of ubiquitin conjugation activity of UBC24 in pho2.

Figure 3.
A, Mutation of the UBC24 gene in the pho2 mutant. An early termination (indicated as the asterisk) at the W671 position caused by a single nucleotide change in the sixth exon of UBC24 was identified in the pho2 mutant. The translation initiation site ...

Expression of UBC24 and miR399 in pho2 was further investigated by RNA gel-blot analysis. Content of UBC24 mRNA was reduced in both roots and shoots of pho2, especially under Pi-sufficient conditions (Fig. 3B). A decreased amount of UBC24 mRNA may reflect the instability of mRNA encoding a truncated nonfunctional protein, a phenomenon called nonsense-mediated mRNA decay (Conti and Izaurralde, 2005). miR399 was up-regulated by Pi deficiency and its accumulation in both roots and shoots was not changed in pho2 (Fig. 3B).

Protein gel-blot analysis using the anti-UBC24 antibody revealed an approximately 100-kD protein in the Pi-sufficient roots of wild-type plants, but not in those of pho2, miR399-overexpressing, or ubc24-1 plants (Fig. 3C). No corresponding signal was observed when probing with the preimmune antibody (Fig. 3C), which suggests that this antibody is specific to UBC24. Detection of UBC24 in Pi-sufficient but not Pi-deficient roots of wild-type plants is consistent with the expression pattern of UBC24 mRNA (Fig. 3B; Chiou et al., 2006), which suggests that expression of UBC24 may be controlled mainly at the transcriptional or posttranscriptional level. Because of the suppression of UBC24 mRNA by the overexpression of miR399, no UBC24 protein was detected. Furthermore, a truncated UBC24 (approximately 74 kD) undetectable in the pho2 mutant could be explained by the reduced amount of UBC24 mRNA and its translational ability or stability of the truncated UBC24.

To confirm that the pho2 phenotype is indeed caused by a nonsense mutation in UBC24, functional complementation was carried out by introducing a 16.8-kb wild-type genomic copy of UBC24 into pho2. The corresponding entire genomic DNA fragment from the pho2 mutant was sequenced and showed no other mutation, except the point mutation in the UBC24 gene. Phenotypes of pho2 were rescued by wild-type UBC24, as observed by lack of Pi toxic symptoms (Fig. 1A), a similar Pi concentration in shoots (Fig. 4A), and distribution of Pi within the leaves to wild-type plants (Fig. 4B). The similarity of pho2 phenotypes with miR399-overexpressing plants and the ubc24-1 mutant and the complement of pho2 phenotypes by UBC24 strongly demonstrated that the pho2 locus is UBC24.

Figure 4.
Complementation of pho2 phenotypes by UBC24. A, Pi concentration in the shoots of 25-d-old wild-type (Wt), miR399b-overexpressing (miR399b), pho2, and two rescued transgenic-line (pho2-C1 and pho2-C2) plants grown under Pi-sufficient soil. Error bars ...

Increased Pi Uptake Activity in pho2 Is a Result of Elevated Vmax

The initial cause for Pi toxicity in pho2 or miR399-overexpressing plants is the increased uptake of Pi by the root. Because increased Pi uptake activity in pho2 was more obvious under Pi-sufficient conditions (Dong et al., 1998), plants were grown under 250 μm KH2PO4 medium and subjected to kinetic analysis. Pi uptake rate was measured in the uptake solution, with Pi concentrations from 2 to 2,000 μm. The activities were drawn as Eadie-Hofstee plots (Hofstee, 1952; McPharlin and Bieleski, 1987) to reveal different Pi uptake systems. When the transport activity (V) was plotted against V/[S] ([S] is the external Pi concentration in the uptake solution), two distinct lines were derived (Fig. 5A), which suggests two uptake systems with two different affinities (e.g. Km). Interestingly, the Km for either the low- or high-affinity uptake system in pho2 and miR399-overexpressing plants was not changed compared with that in wild-type plants. Km values were 120 to 130 μm and 10 to 11 μm for low- and high-affinity uptake systems, respectively (Fig. 5B). However, pho2 and miR399-overexpressing plants showed higher Vmax (about 50% increase) than wild-type plants in both uptake systems (Fig. 5B). Thus, we concluded that the increased Pi uptake activity observed in pho2 (Fig. 1C; Dong et al., 1998) and miR399-overexpressing plants (Chiou et al., 2006) is attributed to elevated Vmax.

Figure 5.
Kinetics analysis of Pi uptake activity. A, Eadie-Hofstee plots of Pi uptake rate for wild-type (Wt), pho2, and miR399b-overexpressing (miR399b) plants with 2 to 2,000 μm Pi concentration. V indicates the Pi transport activity and [S] the external ...

One possibility for enhanced uptake activity with increased Vmax without changing Km is the increase in the molecules of transport proteins. Therefore, we further examined the expression of all members in the Pi transporter (PHT) family (Poirier and Bucher, 2002). Root and shoot samples isolated under Pi-sufficient and Pi-deficient conditions were subjected to reverse transcription (RT)-PCR analyses (Fig. 6A). Expression of PHT1;6, which is mainly expressed in the anthers (Mudge et al., 2002), was not detected in our samples. Other members of the PHT1 family were up-regulated by Pi starvation in various degrees in roots and/or shoots, and no differences in expression levels were observed among wild-type, miR399-overexpressing, and pho2 plants grown under Pi-sufficient or Pi-deficient conditions except for PHT1;8 (Fig. 6A). Moreover, expression of a plastid Pi transporter, PHT2;1 (Versaw and Harrison, 2002), and two mitochondrial Pi transporters, PHT3;2 and PHT3;3, was altered in the Pi-sufficient shoots of pho2 or miR399-overexpressing plants (Fig. 6A). Quantitative PCR was carried out to verify changes in the expression of these Pi transporters (Fig. 6B). Accumulation of PHT1;8 mRNA was not detected in Pi-sufficient roots of wild-type plants; however, a substantial amount of PHT1;8 mRNA was detected in Pi-sufficient roots of pho2 or miR399-overexpressing plants (Fig. 6B). Quantitative PCR experiments also confirmed the down-regulation of PHT2;1 and the up-regulation of PHT3;2 and PHT3;3 in the Pi-sufficient shoots of pho2 or miR399-overexpressing plants (Fig. 6B). Up-regulation of PHT3;2 was much greater than that of PHT3;3. This observation may be associated with the redistribution of intracellular Pi under excess Pi. Interestingly, induction of PHT3;2 and PHT3;3 mRNA by Pi deprivation was reduced in the shoots of pho2 or miR399-overexpressing plants (Fig. 6B), which was not shown by previous RT-PCR analysis (Fig. 6A).

Figure 6.
Expression of members of PHT1, PHT2, and PHT3 families and PHO1 in wild-type, miR399b-overexpressing, and pho2 plants. A, RT-PCR analyses of mRNA levels in the root and shoot samples collected from Pi-sufficient (+Pi) or Pi-deficient (−Pi) ...

Additionally, we examined the expression of PHO1, which is involved in loading Pi into the xylem of roots. No detectable differences in the accumulation of PHO1 transcripts suggests that PHO1 may not be associated with increased translocation of Pi from roots to shoots in the miR399-overexpressing or pho2 mutant plants. However, we did not rule out changes in the protein level of PHO1 in these plants.

miR399 and UBC24 Are Colocalized in the Vascular Cylinder

The prerequisite for miRNA action is colocalization of miRNA and target transcripts. Although targeting the UBC24 transcript by miR399 was demonstrated by RACE experiments (Allen et al., 2005), no information was provided for their tissue or cellular locations. Here, the tissue and cellular localization patterns of miR399 and UBC24 were determined by promoter::reporter analyses. Staining of β-glucuronidase (GUS) activity driven by the UBC24 promoter was observed in the vascular tissues of cotyledons, true leaves, and roots (Fig. 7A, a–e). Interestingly, staining was also observed in the vascular tissue of sepals, filaments, anthers, and receptacles (junction between the inflorescence stem and silique; Fig. 7A, f and g). Expression of UBC24 in the reproductive organs suggests the regulation of Pi transportation by UBC24 in these organs. Close examination of the GUS signal suggests the localization of UBC24 in the vascular cylinder of the root, including pericycle, vascular tissue, and intervascular regions (Fig. 7A, h).

Figure 7.
Tissue and cellular localization of UBC24 and miR399 by promoter::reporter analyses. A, GUS staining in the vascular tissues of UBC24 promoter::GUS transgenic plants grown under Pi-sufficient conditions. a, Whole seedling; b, cotyledon; c, the third true ...

Consistent with the expression pattern of UBC24, GUS or green fluorescent protein (GFP) expression driven by promoter sequences of miR399a, b, c, d, e, and f genes was also observed in the vascular tissues of roots or leaves subjected to Pi starvation (Fig. 7B, d–q and s), which indicates that these six miR399 genes are all functional and induced by Pi deficiency. Under Pi-sufficient conditions, no reporter signal was detected in most plants (Fig. 7B, a), which suggests that up-regulation of miR399 by Pi deficiency is predominantly contributed by transcriptional regulation. However, weak GUS activity was occasionally detected in the cotyledons of transgenic lines harboring the miR399b or miR399f promoter grown under Pi-sufficient conditions (Fig. 7B, b and c). This observation may reflect the low cloning efficiency of miR399b and miR399f in Pi-sufficient tissues (Sunkar and Zhu, 2004). Reporter activity was not detected in the root tip, except in the miR399b construct (Fig. 7B, h). The GFP or GUS signal in the root driven by other miR399 promoters (Fig. 7B, o and p) or the UBC24 promoter (Fig. 7A, d) became visible about 250 μm away from the tip, where the vascular tissue starts to differentiate (Zhu et al., 1998; Stadler et al., 2005). Similar to UBC24, the cross section of root also revealed GUS activity driven by miR399b in the vascular cylinder (Fig. 7B, m). In some cases, the GFP signal driven by miR399f was found specifically in the phloem of roots (Fig. 7B, s). Taken together, these results show that miR399 and UBC24 are colocalized in the vascular cylinder, which further supports their interaction in vivo.

Other than in vascular tissues, GUS or GFP signals were also detected in the mesophyll cells of seedlings driven by miR399e, d, and f promoters (Fig. 7B, j, k, and n; data for expression driven by miR399d not shown), where expression of UBC24 was not detected. This observation may explain our previous finding that even miR399 was accumulated in both roots and shoots in similar amounts and targeting miR399 to the UBC24 transcript in the shoots was not as efficient as that in the roots (Chiou et al., 2006). Nevertheless, the function of miR399 in the mesophyll cells remains to be studied.

DISCUSSION

UBC24 Is the PHO2 Gene

From genetic screening, the pho2 mutant was identified as a Pi overaccumulator (Delhaize and Randall, 1995; Dong et al., 1998). Recently, we observed a phenotype similar to pho2 when miR399 was overexpressed or its target gene, UBC24, was knocked out (Chiou et al., 2006). Although remobilization of Pi within the leaves was not previously described in pho2, here we showed that pho2, miR399-overexpressing, and ubc24-1 plants all display Pi toxic symptoms (Fig. 1, A and B) as a consequence of enhanced Pi uptake (Fig. 1C), facilitated translocation of Pi from roots to shoots (Fig. 1D), and impaired Pi remobilization from old to young leaves (Fig. 2). Because of these similarities and the initial mapping of pho2 close to UBC24, we reasonably hypothesized that pho2 could be defective in the expression of UBC24. Sequence and expression analyses of UBC24 in pho2 (Fig. 3) and complementation of the pho2 phenotype by the wild-type copy of UBC24 (Figs. 1A and and4)4) demonstrated that UBC24 is the pho2 gene.

Based on the 32Pi feeding experiment on a single leaf, Dong et al. (1998) showed that translocation of 32Pi from shoots to roots was partially defective in pho2 under both Pi-sufficient and Pi-deficient conditions. pho2 appeared to retain Pi in shoots because the excess Pi in the shoots of pho2 was not translocated to roots during Pi deprivation (Dong et al., 1998). These results support our observations concerning the ineffectiveness of Pi mobilization out of old leaves of pho2 or miR399-overexpressing plants (Fig. 2). The opposite result was noted by Delhaize and Randall (1995), who measured Pi concentrations in the fully expanded leaves before and after 4 d of Pi deprivation and found that most of the Pi can be mobilized out of the leaves of pho2. We also observed the reduction of Pi concentration in the old leaves of pho2 or miR399-overexpressing plants under Pi deficiency; however, unlike wild-type plants in which the oldest leaf contained the lowest concentration of Pi, the oldest leaf of pho2 or miR399-overexpressing plants accumulated Pi at the highest concentration among all leaves (Chiou et al., 2006). Abnormal distribution of Pi in the leaves of pho2 or miR399-overexpressing plants implies that translocation of Pi out of the old leaves is not as efficient as wild-type plants even under Pi-deficient conditions. Moreover, we observed that the defect in mobilization of Pi out of the old leaves of pho2 was less severe when plants were grown under Pi-deficient, rather than Pi-sufficient, conditions (data not shown). Induction of an alternative pathway to enhance Pi remobilization under Pi deprivation is thus necessary to compensate the situation.

Altered Expression of Specific PHT Genes in pho2

The dual transport system with low and high affinity for nutrient uptake (Epstein, 1976) has been widely accepted. A single-phase or multiphase Pi transport system has been described in many plant species or cultured cells (Ullrich-Eberius et al., 1984; McPharlin and Bieleski, 1987; Nandi et al., 1987; Mimura et al., 1990; Furihata et al., 1992; Shimogawara and Usuda, 1995; Dunlop et al., 1997; Narang et al., 2000). Our data suggest that Arabidopsis possesses a dual Pi uptake system under the conditions we analyzed: a low-affinity system operating at high Pi concentration (Km = 120–130 μm) and a high-affinity system operating at low Pi concentration (Km = 10–11 μm; Fig. 5). This finding is different from the previous observation of only one Pi uptake system in Arabidopsis (Narang et al., 2000). We suspect that a narrow Pi concentration range (2.5–100 μm) in the kinetic assay may overlook this dual-affinity system. With a concentration range from 0.5 μm to 5 mm, Dunlop et al. (1997) also reported two mechanisms operating Pi uptake in Arabidopsis, which is consistent with our results. The kinetic analysis suggests that increased Pi uptake activity by overexpressing miR399 or loss-of-function UBC24 is caused by elevated Vmax in both low- and high-affinity transport systems without alteration of Km (Fig. 5B). However, we did not observe differences in the value of Vmax and Km between wild-type and pho2 mutant plants grown under Pi-deficient conditions (data not shown). This finding is consistent with a previous report that increased Pi transport activity was not detected in pho2 under Pi-deficient conditions (Dong et al., 1998).

In our system, the transport activity was conducted in whole seedlings and the Vmax and Km were measured as a sum of various transporters, which is much more complicated than the reaction operated by a single protein. We postulate two possibilities for increased Vmax without a change in substrate affinity: the increased amount of transporter molecules per se and the increased catalytic activity, such as the translocation of substrate across membrane. The up-regulation of PHT1;8 transcripts in Pi-sufficient roots of miR399-overexpressing and pho2 plants (Fig. 6) suggests that UBC24 may act upstream of PHT1;8 to suppress its expression under Pi-sufficient conditions. Because the UBC-E2 enzyme does not normally participate in regulating transcriptional activity, the reduction of PHT1;8 transcripts by UBC24 may be mediated through an intermediate, possibly a transcription factor that is the target of ubiquitination mediated by UBC24. However, the increased level of PHT1;8 transcripts in Pi-sufficient roots of pho2 or miR399-overexpressing plants was only 10% of that induced by Pi deficiency (Fig. 6B). Whether increased expression of PHT1;8 transcripts is responsible for the increased number of transporter molecules, thus resulting in greater Vmax (Fig. 5), or enhancing Pi translocation into shoots (Fig. 1D) remains to be investigated. It is intriguing to find out where PHT1;8 is expressed. Unfortunately, promoter::reporter analysis of PHT1;8 failed to identify tissue localization (Mudge et al., 2002). Where PHT1;8 is expressed and how it functions in concert with UBC24 located in the vascular tissue are topics for further study. In addition to expression of PHT1;8, that of other intracellular Pi transporter genes, such as PHT2;1, PHT3;2, and PHT3;3, was altered (Fig. 6B). Although efflux or influx of these Pi transporters is not clearly defined yet, changes in their expression indicate the adjustment of Pi partitioning across different organelles when intracellular Pi concentration is too high. Fujii et al. (2005) reported that accumulation of PHT1;1 mRNA was slightly increased in the miR399-overexpressing plants under Pi-sufficient conditions, but we did not observe this. Differences in plant stages or growth and treatment conditions may not reproduce this marginal change. Consistent with our results, Smith et al. (1997) did not observe differences in the transcript level of APT1 (PHT1;2) and APT2 (PHT1;1) in roots of pho2 and wild-type plants.

That the transcript level of PHT1 transporters did not change markedly under Pi-deficient conditions (Fig. 6A) is in agreement with unaffected transport activity (Dong et al., 1998) and could be due to the down-regulation of UBC24 by Pi deficiency. Although most of the Pi transporters examined did not show changes in their transcript level, it is likely that they are regulated at the posttranslational step via ubiquitination-mediated protein degradation through UBC24. Specific antibodies directed against these Pi transporters are needed to prove this hypothesis. Recently, PHF1 was identified to be required for localization of the PHT1;1 Pi transporter to the plasma membrane (González et al., 2005). It is of interest to know whether enhanced Pi uptake activity in pho2 or miR399-overexpressing plants is the consequence of enhanced PHF1 activity, resulting in targeting more Pi transporters to the plasma membrane.

Vascular Localization and Systemic Effects on Whole-Plant Pi Homeostasis

Colocalization of miR399 and UBC24 in the vascular cylinder (Fig. 7) supports targeting UBC24 mRNA by miR399. UBC24 is predominantly expressed in vascular tissue, whereas miR399 is also expressed in the root tip and mesophyll cells in addition to vascular tissue (Fig. 7B, h, j, k, and n). However, it does not seem logical that miR399 is expressed in these cells where its target gene, UBC24, is not expressed. We suspect that other unidentified target genes of miR399 may be expressed in these cells or that the expression of miR399 in these cells is just a remnant during evolution.

It is of interest to note that GUS staining driven by the miR399 promoter was initiated in the vascular tissues of leaf tips (Fig. 7B, b and c) and gradually moved toward the base upon Pi starvation (Fig. 7B, g and n). This observation coincides with the trend of leaf maturation and the emergence of chlorosis and necrosis symptoms of Pi toxicity. In contrast to this basipetal expression pattern, GUS staining driven by the UBC24 promoter was acropetal. The signal was strongly observed at the petiole and leaf base and gradually diffused toward the leaf margins (Fig. 7A, a–c). These opposite expression patterns between miR399 and UBC24 may be correlated with their antagonistic effects.

The vascular location of miR399 and UBC24 is particularly fascinating because it correlates well with the systemic effects observed in pho2, miR399-overexpressing, and ubc24-1 plants, such as Pi translocation between roots and shoots and Pi mobilization within leaves. Pi uptake by roots might be regulated by Pi recycling in the phloem (Drew and Saker, 1984; Schjørring and Jensén, 1987). Increased Pi uptake and reduced Pi recycling by overexpression of miR399 or loss of function of UBC24, which are all expressed in the vascular tissues, support this hypothesis. Enhanced Pi uptake activity did not occur in the detached pho2 roots without shoots (Dong et al., 1998), which suggests that a systemic signal derived from the shoot controls the increased uptake of Pi. Moreover, ineffective down-regulation of a Pi starvation-induced gene, At4, in pho1 roots grown under Pi-sufficient conditions, supports translocation of a shoot-derived signal molecule to roots (Burleigh and Harrison, 1999). Transmission of this shoot-derived signal to roots requires passage through the phloem, which may depend on coordination between miR399 and UBC24, also located in the vascular tissues. Further studies focusing on the regulatory pathways of miR399/UBC24 are necessary to uncover this systemic signal molecule.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

Arabidopsis (Arabidopsis thaliana) wild-type plants (Col 0), miR399-overexpressing (Chiou et al., 2006) and UBC24 T-DNA knockout plants (ubc24-1, SAIL_47_E01), and pho2 mutant plants (Delhaize and Randall, 1995) requested from the Arabidopsis Biological Resource Center (ABRC) were used for this study. Seeds were surface sterilized and germinated on agar plates with one-half modified Hoagland nutrient solution containing 1 mm KH2PO4 (sufficient Pi; +Pi), 1% Suc, and 1% Bactoagar. For hydroponic culture, Pi starvation was initiated by transferring 2-week-old seedlings originally grown in high-Pi solution (250 μm instead of 1 mm KH2PO4 was used to reduce Pi toxicity) into Pi-free solution. After 11 d, root and shoot samples were collected separately for Pi assay or RNA isolation. For liquid culture, 20-d-old seedlings grown in high-Pi B5 liquid medium were subjected to Pi starvation treatment by transferring them into Pi-free medium for 5 d. Root samples were collected for protein isolation. All growth conditions were at 22°C under a 16-h photoperiod with cool fluorescent light at 100 to 150 μE m−2 s−1.

RNA Gel-Blot and PCR Analyses

RNA isolation and RNA gel-blot analyses were performed as described previously (Lin et al., 2005). Total RNA was treated with DNase I (Ambion) prior to RT-PCR analyses to eliminate genomic DNA contamination. cDNA was synthesized from total RNA by SuperScript III RNase H reverse transcriptase (Invitrogen). Quantitative RT-PCR was performed by using the FastStart DNA Master SYBR Green I kit on the LightCycler machine (Roche), following the manufacturer's instructions. The amplification program was performed at 95°C for 10 s, 60°C for 5 s, and 72°C for 10 s. Relative quantitative results were calculated by normalization to Actin2. All PCR products were verified by sequencing. PCR conditions and sequences of primers are listed in Supplemental Tables I and II.

Complementation of the pho2 Mutant

A 16.8-kb KpnI- and NcoI-digested genomic DNA fragment containing the wild-type copy of the UBC24 gene was isolated from a T1B8 bacterial artificial chromosome clone (requested from the ABRC) and subsequently constructed into the pCAMBIA 1,300 binary vector. DNA was introduced into the pho2 mutant by the floral-dip method (Clough and Bent, 1998).

Phosphorous Content Analysis

Pi content was determined as described (Ames, 1966) with minor modifications (Chiou et al., 2006).

Pi Uptake Analysis and Pulse-Chase Labeling

Twelve-day-old seedlings grown on one-half modified Hoagland medium supplemented with 250 μm of KH2PO4 were used for Pi uptake assay. The uptake assay was performed as previously described (Chiou et al., 2006). Kinetic analysis was carried out over a broad range of external Pi concentrations (2, 5, 10, 20, 50, 100, 200, 500, 1,000, and 2,000 μm KH2PO4, indicated as substrate concentration [S]). To reveal different transport systems, Eadie-Hofstee plots (Hofstee, 1952) were applied whereby the transport activity (V) was drawn as the function of V/[S]. Km was calculated from V = −Km × (V/[S]) + V and V = Vmax when V/[S] = 0. Pulse-chase labeling was performed as described (Chiou et al., 2006).

Antibody Production and Protein Gel-Blot Analysis

The recombinant protein corresponding to the 468 to 642 amino acids of UBC24 protein was expressed, purified, and used as an antigen to immunize rabbits. Total proteins of roots from liquid culture were extracted with Tris-HCl buffer (60 mm, pH 8.5) containing 2% SDS, 2.5% glycerol, 0.13 mm EDTA, and 1% protease inhibitor cocktail (Roche). The protein amount was measured by use of DC protein assay reagents (Bio-Rad) with bovine serum albumin as a standard. An amount of 100 μg of total protein was separated in 10% SDS-PAGE and then transferred to polyvinylidene difluoride membrane (Millipore). The membrane was probed with the preimmune antibody or antibody directed against the UBC24 protein, followed by secondary antibody conjugated with horseradish peroxidase (Pierce). The signal was subsequently detected by use of SuperSignal West Dura Extended Duration substrate (Pierce). Afterward, the membrane was stained with 0.1% (w/v) amido black to ensure a compatible amount of loading.

Localization of miR399 and UBC24

The upstream sequences from the initiation site of UBC24 and the precursor fold-back structure of six miR399 genes (miR399a, b, c, d, e, and f) were cloned and used to drive expression of GUS or mGFP reporter genes. The primer sequences and promoter lengths are listed in Supplemental Table III. The 5′-untranslated region of UBC24, consisting of the target sequences of miR399, was not included in the UBC24 promoter construct. These promoter sequences were cloned by PCR, verified by sequencing, and subcloned into pMDC204 (Curtis and Grossniklaus, 2003) to drive expression of an mGFP reporter gene or into pKGWFS7.0 (Karimi et al., 2005) to drive expression of GUS reporter genes. These constructs were introduced into Arabidopsis by the floral-dip method (Clough and Bent, 1998). Transgenic T2 plants grown under Pi-sufficient and Pi-deficient conditions underwent reporter analysis. GUS activity was detected according to Lin et al. (2005) and the signal was observed under an Olympus SZX12 or a Zeiss AxioSkop microscope. The GFP signal was observed under a Zeiss AxioSkop fluorescence microscope or a Zeiss LSM 510 Meta confocal microscope.

Supplementary Material

[Supplemental Data]

Acknowledgments

We thank Dr. Kuo-Chen Yeh and Dr. Yee-Yung Charng for critically reading the manuscript. We also thank Wen-Chien Lu, Su-Fen Chiang, and Ya-Ni Chen for technical assistance, and Hsing-Yu Huang for plant propagation.

Notes

1This work was supported by Academia Sinica (grant no. AS91IBAS2PP) and the National Science Council of the Republic of China (grant no. NSC94–2311–B–001–057) to T.-J.C.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Tzyy-Jen Chiou (wt.ude.acinis.etag@uoihcjt).

[W]The online version of this article contains Web-only data.

Article, publication date, and citation information can be found at www.plantphysiol.org/cgi/doi/10.1104/pp.106.078063.

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