Acclimation responses of Arabidopsis thaliana to sustained phosphite treatments

Abstract

Phosphite () induces a range of physiological and developmental responses in plants by disturbing the homeostasis of the macronutrient phosphate. Because of its close structural resemblance to phosphate, phosphite impairs the sensing, membrane transport, and subcellular compartmentation of phosphate. In addition, phosphite induces plant defence responses by an as yet unknown mode of action. In this study, the acclimation of Arabidopsis thaliana plants to a sustained phosphite supply in the growth medium was investigated and compared with plants growing under varying phosphate supplies. Unlike phosphate, phosphite did not suppress the formation of lateral roots in several Arabidopsis accessions. In addition, the expression of well-documented phosphate-starvation-induced genes, such as miRNA399d and At4, was not repressed by phosphite accumulation, whilst the induction of PHT1;1 and PAP1 was accentuated. Thus, a mimicking of phosphate by phosphite was not observed for these classical phosphate-starvation responses. Metabolomic analysis of phosphite-treated plants showed changes in several metabolite pools, most prominently those of aspartate, asparagine, glutamate, and serine. These alterations in amino acid pools provide novel insights for the understanding of phosphite-induced pathogen resistance.

Introduction

Phosphorus is an essential macronutrient for plants and is taken up and assimilated in the form of inorganic phosphate (, Pi). The Pi concentrations in most soils are very low, and Pi availability is further decreased by immobilization, such as by the formation of insoluble complexes of Fe and Al oxides (Gerke, 1992). Plants have developed mechanisms to cope with Pi limitation. The inducible developmental and metabolic adaptations that enhance uptake and utilization are together referred to as the phosphate-starvation response (PSR) (Raghothama, 1999; Chiou and Lin, 2011). Uptake of Pi from the soil can be enhanced by secretion from the roots of organic acids, such as citrate or malate, and phosphatases to mobilize Pi from mineralized and organic forms (Duff et al., 1994; Ryan et al., 2001). Morphological alterations of root architecture, such as decreased primary root growth and increased lateral root and root-hair production, enhance the capacity to forage Pi from the richer topsoil layers (Lynch and Brown, 2001; Peret et al., 2011). Some plant species are able to generate specialized root structures composed of dense clusters of lateral roots or form associations with mycorrhizal fungi to form a symbiotic partnership to enhance Pi acquisition (Shane and Lambers, 2005; Bucher, 2007). The developmental and physiological processes controlled by Pi availability are under complex control through local and systemic signalling as well as external and internal Pi concentrations (Thibaud et al., 2010).

The PSR includes the transcriptional induction of genes such as high-affinity P_i transporters or phosphatases to increase P_i acquisition, remobilization from storage compounds such as phytate and phospholipids, and redistribution between tissues and/or subcellular compartments. At the post-transcriptional level, the miRNA399/PHO2/PHR1 regulon has been identified as an important mechanism to control P_i homeostasis. Under P_i deficiency, miRNA399 transcription is highly upregulated by the transcription factor PHR1 to direct the degradation of the mRNA for the E2 ubiquitin conjugase PHO2 (Bari et al., 2006; Chiou et al., 2006). The regulated degradation of PHO2 transcripts by miRNA399 is in turn attenuated by the regulatory RNAs IPS1 and At4 (Franco-Zorrilla et al., 2007). PHO2 controls P_i concentrations by regulating protein turnover through ubiquitination of a set of target proteins that include PHO1 and probably also P_i transporters (Liu et al., 2012). As a consequence, a pho2 mutant, as well as miRNA399 overexpressing Arabidopsis lines, hyperaccumulate P_i (Aung et al., 2006; Chiou et al., 2006). Furthermore, the phloem mobility of miRNA399 has been interpreted to indicate that miRNA399 is a signal in shoot-to-root communication (Pant et al., 2008). Transcriptional regulation has also been found for genes that are part of the acclimation to limited P_i supply, for example for SQD2 and NMT3 involved in the remodelling of membrane lipid composition (Essigmann et al., 1998; Hartel et al., 2000).

It is not clear how P_i levels are perceived in plants. In yeast, a phosphate transporter (PHO84) has been identified that signals P_i status through a conformational change induced by P_i binding that is independent of the transport of P_i across the membrane (Popova et al., 2010). A similar transceptor function has also been identified for the nitrate transporter NRT1.1/CHL1 in Arabidopsis, indicating that similar sensing mechanisms could exist for P_i in plants (Ho et al., 2009).

Post-translational regulation of the PSR involves metabolic adjustments to cope with the changed availability of P_i for enzymatic reactions and their regulation (Plaxton and Tran, 2011). P_i is a structural component of many organic components, such as DNA, RNA, phospholipids, and phosphorylated sugars. Therefore, P_i deficiency has profound effects on plant metabolism, such as leading to the accumulation of starch in the shoot (Ghosh and Preiss, 1966; Fliege et al., 1978). Another example is the remodelling of membrane lipid composition by replacing phospholipids with sulfo- and galactolipids to release P_i from this membrane component and conserve P_i (Essigmann et al., 1998; Hartel et al., 2000; Lambers et al., 2012). With P_i being a substrate in many more important pathways of primary metabolism, the metabolic pools of P_i in the various subcellular compartments need to be regulated, with the vacuole being the major storage compartment releasing P_i under a low external supply (Lee and Ratcliffe, 1993; Mimura et al., 1996; Danova-Alt et al., 2008; Pratt et al., 2009). Recent results from ³¹P-NMR studies gave estimates of subcellular P_i pools with concentrations in the range of 60–80 μM in the cytosol and 7mM in organellar mitochondrial and plastidal pools combined (Pratt et al., 2009). This same study provided evidence for considerable fluctuation of cytosolic but not of organellar concentrations under a P_i supply, which could be used to sense changes in cellular levels and trigger the signalling cascade of the PSR.

Phosphite (, Ph_i) is an analogue of phosphate in which a hydroxyl group is replaced by a hydrogen atom. It enters the cell via P_i transporters, therefore competing with P_i for uptake, and is mobile within the plant (Ouimette and Coffey, 1989; Carswell et al., 1996; Danova-Alt et al., 2008). On the other hand, Ph_i is used in the control of plant pathogens, especially oomycetes such as Phytophthora or Pythium species; it has been used for decades as the active agent in biostats such as Fosetyl-Al (Cohen and Coffey, 1986; Guest and Grant, 1991; Hardy et al., 2001). High concentrations of Ph_i directly inhibit growth of oomycete mycelia, and this has been attributed to interference of Ph_i with key reactions by competing with P_i for the enzyme catalytic sites, leading to altered abundances of metabolites such as ATP, NAD, polyphosphates, and pyrophosphate (Griffith et al., 1990; Barchietto et al., 1992; Niere et al., 1994). In addition to this direct effect of Ph_i on pathogens, it also activates plant defence responses (Molina et al., 1998; Friedrich et al., 2001; Eshraghi et al., 2011). This Ph_i-induced resistance depends on the signalling component NPR1 (non-expressor of PR1) and the accumulation of salicylic acid (Molina et al., 1998; Friedrich et al., 2001). Recently, it has been shown that Ph_i pre-treatment primes plants for a stronger defence response when subsequently challenged with Hyaloperonospora arabidopsidis and this priming involves the mitogen-activated protein kinase MPK4 (Massoud et al., 2012).

The chemical resemblance of Ph_i to P_i has made it an attractive tool to investigate P_i sensing. Ph_i is metabolically inert in plants, does not enter the organic P pool and thus accumulates in the tissues (Carswell et al., 1996, 1997; Danova-Alt et al., 2008; Berkowitz et al., 2011). The treatment of severely P_i-starved plants with Ph_i suppresses the activation of many PSR genes and it is therefore assumed that Ph_i is recognized by the same sensing mechanism as P_i. The Ph_i-dependent downregulation of the PSR then leads to an accentuation of P_i deficiency, for example by decreasing P_i uptake capacity. This interference with the P_i regulatory network has been suggested to cause the plant growth-inhibiting effect of Ph_i (Carswell et al., 1996, 1997; Ticconi et al., 2001; Varadarajan et al., 2002). ³¹P-NMR studies using tobacco BY2 cells have shown that Ph_i accumulates in the cytoplasm of P-deprived cells and that resupply of P_i leads to efflux of Ph_i from the cell. In P_i-sufficient cells, however, Ph_i accumulates in the vacuole, illustrating the complex interactions of P_i and Ph_i with respect to cell compartmentation, sensing, and physiology (Danova-Alt et al., 2008).

To understand the specific effects of Ph_i on plant growth and metabolism, it is necessary to compare directly the effects of Ph_i-treated plants with plants under P_i limitation. In this study, Arabidopsis plants grown on medium containing Ph_i and P_i in varying ratios but at a constant combined concentration were investigated. Growth parameters, root development, and gene expression patterns of Ph_i-treated plant were compared with those of plants grown with various P_i supplies. In addition, Ph_i-induced metabolic adjustments with potential relevance for plant defence mechanisms were identified.

Materials and methods

Plant material

Seeds of accessions of Arabidopsis thaliana (L.) Heynh. used in this study were obtained from the European Arabidopsis stock centre (Col-0, N1093) and the Versailles Resource Centre (Col-4, AV187; Jea, AV25; Nd-1, AV220; Sha, AV236; Tsu-1, AV239). Arabidopsis plants were grown on a modified MS medium (Murashige and Skoog, 1962) in a vertical plate system for 10 d. The medium contained 2mM NH₄NO₃, 1.5mM CaCl₂, 0.5mM MgSO₄, 3mM KNO₃, 3mM MES (pH 5.8), 0.5% (w/v) sucrose and 0.8% (w/v) agar. The agar contributed ~10 μM Pi. Phosphate was added as KH₂PO₄ (pH 5.8). KCl was substituted for KH₂PO₄ to maintain a constant osmotic potential. A freshly prepared 0.1M potassium phosphite stock solution was prepared from phosphorous acid by adjusting to pH 5.8 with KOH and was added to the medium as required. Micronutrients were used according to Murashige & Skoog (1962) and obtained from Austratec (Bayswater, Australia). After sterilization [2min in 70% (v/v) ethanol, 5min in 5% (v/v) sodium hypochlorite], the seeds were resuspended in 0.1% (w/v) agarose and stratified for 2 d at 4 °C. The seeds were transferred to 10×10cm square plates, which were then sealed with microporous tape, and the plants were grown in a controlled-environment growth chamber with a 10h photoperiod at ~100 μmol m^–2 s^–1. For metabolomic analyses, plants were grown for 3 weeks in horizontal 90mm Petri dishes sealed with microporous tape under the same growth conditions as described above. For analysis of growth responses, all experiments were repeated in five independent replicates consisting of eight seedlings each.

Phosphate and phosphite quantification

Phosphate and phosphite were extracted from ~10mg of shoot or root tissue with 1 % (v/v) acetic acid at 1:10 (w/v) using a tissue lyser (Qiagen, Doncaster, Australia) for 1min at 25 Hz. After clearing the lysate by centrifugation at 14 000g and 4 °C for 15min, the supernatant was assayed for phosphate and phosphite as described previously (Ames, 1966; Berkowitz et al., 2011).

Root growth measurements

For measurements of root growth, the position of the root tip was marked every 24h for the 10-day growth period. Plates were then scanned for analysis of root length, growth rate, and lateral root number using ImageJ software with the NeuronJ plug-in (Meijering et al., 2004).

Gene expression analyses

For each treatment, RNA was extracted from five independent biological replicate samples and reverse transcribed using Plant RNA Mini and Tetro cDNA synthesis kits according to the manufacturer’s instructions (Bioline, Alexandria, Australia). Quantitative real-time PCR on a Rotor Gene 6000 platform (Qiagen) was performed with 2ng of cDNA and a SensiFast SYBR No-ROX kit (Bioline) as described previously (Jost et al., 2007). Relative expression levels were calculated using the comparative ΔΔC _T method (Livak and Schmittgen, 2001) and normalized to the internal reference genes PDF2, UBC9 and UPL7 (Czechowski et al., 2005). For evaluation of reference genes and amplification efficiencies, the geNORM and LinRegPCR algorithms, respectively, were used (Vandesompele et al., 2002; Ramakers et al., 2003). Primers were designed using Primer Express software (Applied Biosystems, Mulgrave, Australia) and the QuantPrime tool (Arvidsson et al., 2008). Primer sequences and Arabidopsis Genome Initiative (AGI) gene identifiers are given in the supplementary data (Table S1 at JXB online).

Metabolomic analysis by gas chromatography–mass spectrometry (GC-MS)

Polar metabolites were extracted from shoot tissue harvested from eight biologically independent experiments 3h after the onset of light and immediately frozen in liquid nitrogen. After homogenization in a tissue lyser (Tissue Lyser II; Qiagen), 30mg of frozen tissue powder was extracted with 500 μl extraction solution (20:3 methanol:water including 10 μg ml^–1 of ribitol as an internal standard) for 20min at 75 °C with shaking at 1200rpm in a thermomixer (Thermo mixer compact; Eppendorf, North Ryde, Australia). After centrifugation at 16 100g for 3min at room temperature, 60 μl of the supernatant was dried under vacuum and the polar metabolites redissolved by incubation in 20 μl of methoxyamine hydrochloride (20mg ml^–1) in pyridine at 37 °C with shaking at 750rpm for 90min. For derivatization, 30 μl of N-methyl-N-(trimethylsilyl)trifluoroacetamide was added and the mixture was incubated at 37 °C with shaking at 750rpm for 30min. A retention time standard (10 μl) containing 140 nl ml^–1 each of n-dodecane, n-pentadecane, n-nonadecane, n-docosane, n-octacosane, n-dotriacontane and n-hexatriacontane in pyridine was added to each sample. GC-MS analysis was performed by injecting 1 μl of derivatized sample using the splitless mode on an Agilent 7890GC/5975MSD system (Agilent, Mulgrave, Australia) equipped with a Varian Factor 4 column and operated in scan mode (range: 50–650 m/z). Metabolites were identified by comparing retention times and ionization patterns with standards and the National Institute of Standards and Technology mass spectral library. The integrated signal of the total ion current for each peak was compared between samples after normalization. Metabolites were normalized first using the internal standard and then the sample weight.

Statistical analyses

For statistical analyses, the SPSS Statistics Base software package (version 17; IBM, New York, USA) was used. Analysis of variance with a Tukey HSD test was performed for multiple comparisons.

Results

Growth responses to sustained Ph_i supply

Because detailed reports on the acclimation of plants under sustained Ph_i supply that also take into account internal P_i and Ph_i concentrations are lacking, this study analysed the responses of Arabidopsis Col-0 seedlings to varying P_i and Ph_i concentrations in the growth medium at a constant combined total concentration of 0.5mM. Seeds were germinated directly on medium containing differing Ph_i:Pi ratios ranging from 1:1 to 50:1. Growth was compared with that of control seedlings grown on medium containing P_i only at concentrations over the same range (Fig. 1, Table 1). For the Ph_i-treated seedlings, the P_i and Ph_i concentrations of the shoots and roots varied according to the external supply. Interestingly, the total concentration of P_i+Ph_i in both tissues was remarkably constant, despite the substantial variation in the concentrations of the two anions in the growth medium (Fig. 1).

Fig. 1.

Fresh weights and Pi and Phi concentrations of Arabidopsis (Col-0) shoots and roots under varying Pi and Phi supplies. Plants were grown in a vertical plate system for 10 d before harvesting the shoot and root tissues. Numbers in bars represent the corresponding ratios of Phi and Pi concentrations in the tissue. For each sample, eight plants grown on one plate were pooled, and for each treatment five independent biological replicates were performed (n=5 pools of eight per treatment). Results are given as means ±standard error (SE). Significant differences between Phi treatments and their corresponding controls grown on the same Pi concentration are indicated (ANOVA Tukey: *P <0.05; ** P < 0.01; *** P < 0.001).

Table 1.

Comparison of fresh weights and Pi/Phi tissue concentrations of Ph_i-treated and non-treated plants of Arabidopsis thaliana (see also Fig. 1)Fresh weights (FW) and P_i tissue concentrations of Ph_i-treated plants are expressed relative to P_i-sufficient plants grown on 0.5mM P_i or control plants with the same P_i concentration in the medium but lacking Ph_i. Results are given as means of five independent biological replicates with eight seedlings each.

	Leaves				Roots
	% of 0.5mM Pi		% of Pi control		% of 0.5mM Pi		% of Pi control
Pw/Phi treatment	FW	[Pi]	FW	[Pi]	FW	[Pi]	FW	[Pi]
0.25mM/0.25 mM	93	88	98	115	74	78	80	107
0.1mM/0.4 mM	74	71	81	85	48	67	63	110
0.05mM/0.45 mM	57	62	75	66	28	42	56	151
0.01mM/0.49 mM	36	20	66	190	21	22	52	229

Shoot fresh weights for seedlings grown on 0.25mM P_i+0.25mM Ph_i did not differ significantly from seedlings grown on 0.25mM P_i alone. Quantification of P_i and Ph_i tissue concentrations revealed that the seedlings supplied with Ph_i had the same shoot P_i concentration as plants supplied with P_i but not Ph_i. However, despite the equimolar supply of P_i and Ph_i, the Ph_i accumulation was only 20% of that of P_i (Fig. 1). Higher Ph_i supplies led to a decrease in shoot biomass that was accentuated compared with that of control seedlings grown at the same external P_i. Interestingly, the seedlings treated with 0.1mM P_i+0.4mM Ph_i and 0.05mM P_i had equal shoot P_i concentrations and similar shoot fresh weights. Thus, for this Ph_i treatment, the reduced biomass was explained mainly by a reduction in the P_i concentrations in the shoot tissue, and the accumulation of Ph_i seemed to have little direct effect on shoot biomass. However, the seedlings that contained the highest concentration of Ph_i in the shoots had an accentuated reduction in shoot biomass compared with seedlings grown on the lowest P_i supply. Surprisingly, in the two treatments containing 0.01mM P_i, the seedlings that were supplied with Ph_i showed a higher P_i concentration in the shoots.

Phosphite accumulation had a more pronounced effect on decreasing the fresh weight of the roots than on that of the shoots (Fig. 1, Table 1), which resulted in an increase in shoot:root ratio (Fig. S1 at JXB online). Across all treatments, seedlings grown on Ph_i-containing medium had a lower root biomass than seedlings that had a similar P_i tissue concentration in the absence of Ph_i. For example, the roots of seedlings exposed to the 0.05mM P_i+0.45mM Ph_i and the 0.1mM P_i treatments showed similar P_i concentrations, but the Ph_i-treated seedlings had a much lower root biomass. The Ph_i concentration in the roots was only comparable to the P_i concentration when Ph_i was in a ten- to 50-fold external excess (Fig. 1), and was only equimolar in the 0.01mM P_i+0.49mM Ph_i treatment. At this highest Ph_i:Pi ratio, the root fresh weight was reduced by about 80% compared with seedlings in the 0.5mM P_i treatment, whilst the shoot fresh weight was only reduced by ~25% (Table 1).

Across all Ph_i treatments tested, the Ph_i:Pi tissue ratio was consistently about twofold higher in the shoots than in the roots, indicating preferential transport and accumulation of Ph_i in the shoot. However, this did not lead to an accentuated impact on shoot biomass, as the relative decrease in biomass with increasing external Ph_i was higher in the roots. This discrepancy might indicate a higher Ph_i tolerance of the shoot tissue. A surprising observation was the consistently higher P_i concentration in roots of Ph_i-treated seedlings compared with seedlings grown at the same P_i supply. A similar effect was observed for shoot tissue but only at the highest external Ph_i:Pi ratios.

Phosphite does not suppress increases in lateral root density (LRD) under P_i limitation

Next, the effect of Ph_i on root development under our growth conditions was examined. Plants grown at lower P_i supplies showed the characteristic reduction in primary root length (Fig. 2A), as reported previously (Peret et al., 2011). Ph_i only had an effect on primary root length at the highest concentration tested, with a 68% reduction in primary root length compared with seedlings grown at the same external P_i supply (Fig. 2A). The primary root growth rate in the 0.25mM Ph_i treatment was reduced by ~1mm d^–1 from 4 d after germination compared with the control (Fig. 2B, C). Interestingly, primary roots exposed to either 0.1mM P_i+0.4mM Ph_i or 0.05mM P_i+0.45mM Ph_i initially had decreasing growth rates, which recovered from 4 d after germination and increased steadily for the rest of the experiment, leading to root lengths comparable to roots grown on the same P_i but without Ph_i in the medium. Only primary roots of seedlings growing at the lowest P_i supplies had a constant deceleration in growth that ultimately caused the shortest primary roots across treatments. This effect was stronger in the presence of Ph_i.

Fig. 2.

Lengths and growth rates of Arabidopsis (Col-0) primary roots in response to external Ph_i and P_i supply. (A) Primary root lengths of plants grown in a vertical plate system for 10 d with varying Ph_i and P_i concentrations in the growth medium. Means with different letters are significantly different (ANOVA Tukey, P <0.05). (B, C) Root growth rates for plants grown with decreasing P_i (B) and variation of Ph_i:Pi ratios (C) in the medium. Data points are means ±SE of five biological replicates with eight seedlings each. Medium concentrations in (B) and (C): solid line, 0.5mM P_i; long-dashed line, 0.25mM P_i (+0.25mM Ph_i); medium-dashed line, 0.1mM P_i (+0.4mM Ph_i); short-dashed line, 0.05mM P_i (+0.45mM Ph_i); dotted line, 0.01mM P_i (+0.49mM Ph_i).

LRD (the number of lateral roots per cm of primary root) for seedlings grown in the absence of Ph_i increased with decreasing P_i supply, as expected (Fig. 3). Intriguingly, Ph_i treatment had no effect on LRD at the lowest Ph_i:Pi ratio tested, whilst plants growing on 0.1mM Ph_i+0.4mM P_i had a higher LRD than plants on 0.1mM P_i alone (Fig. 3). This was surprising, as the combined total concentration of P_i+Ph_i in the root and shoot tissues was unchanged, and also across all treatments the combined concentration of P_i and Ph_i in the growth medium was constant at 0.5mM (Fig. 1). Thus, Ph_i did not seem to have a suppressing effect on LRD similar to that of P_i. Only for the two highest Ph_i concentrations was the increase in LRD reduced compared with the corresponding control seedlings, but these Ph_i-treated seedlings also had a higher P_i concentration than the control seedlings grown on the same P_i concentration in the absence of Ph_i (Fig. 1). Therefore, even at these higher concentrations, Ph_i had very little impact on suppressing lateral root formation, and instead had rather an indirect effect leading to elevated P_i in the root tissue, which subsequently resulted in a decreased LRD when compared with the corresponding control plants.

Fig. 3.

LRD of Arabidopsis (Col-0) plants grown on medium with different Ph_i and P_i concentrations (mM). Primary root lengths and the numbers of lateral roots (>100 μm) were determined from five biological replicates with eight seedlings each. Results are given as means ±SE. Means with different letters are significantly different (ANOVA Tukey, P <0.05).

As a first step to analyse the natural genetic variation of Arabidopsis accessions in the response to Ph_i, the LRD of another five accessions in addition to the Col-0 accession used in our other experiments was analysed. All of these accessions showed an increase in LRD on 0.1mM P_i when compared with growth on 0.5mM P_i (Fig. 4). Interestingly, we observed an even higher LRD for plants grown on 0.1mM P_i+0.4mM Ph_i, especially for the accessions Col-4, Jea, Sha and Tsu-0 with a 1.5- to twofold increase in LRD, and a less pronounced increase for Col-0 and Nd-0. Thus, Ph_i did not suppress lateral roots in a similar fashion to P_i but instead led to an accentuated increase in LRD.

Fig. 4.

Comparison of LRD for six Arabidopsis accessions grown on growth medium containing 0.5mM P_i, 0.1mM P_i, or 0.1mM P_i+0.4mM Ph_i. For each accession, at least 16 seedlings per treatment were analysed (four independent replicates with four seedlings each). Results are given as means ±SE. Statistically significant differences between the 0.1mM P_i and the 0.1mM P_i+0.4mM Ph_i treatments are indicated (ANOVA Tukey, *P < 0.05; **P < 0.01; ***P < 0.001).

Gene expression analyses

As Ph_i was not able to suppress an increase in LRD under P_i limitation, a known PSR, the effects of Ph_i treatment on the expression of genes involved in the PSR and others associated with P_i metabolism were examined next. For these experiments, shoots from seedlings exposed to 0.1mM P_i+0.4mM Ph_i and 0.05mM P_i were compared. These two shoot sets had comparable P_i concentrations, which were lower than those of seedlings supplied with 0.5mM P_i (Fig. 1). The shoots of these Ph_i-treated seedlings also accumulated similar concentrations of both Ph_i and P_i. The transcript levels for a set of genes involved in the primary P_i regulatory network (At4, miRNA399d, PHR1, PHO1, and PHO2) and downstream genes involved in the adaptation to low-P_i levels (PHT1;1, PHT1;4, PAP1, NMT3, SQD2, and ASK11) were quantified. As expected, transcripts for the well-documented P_i-starvation-induced genes At4 and miRNA399d showed strong induction (~60-fold) under P_i limitation (Fig. 5). Transcripts of PHT1;1, PHT1;4, SQD2, PAP1 and PHO1 showed a milder induction (between 2.5- and eightfold), whilst NMT3 expression was repressed (approx. twofold). These results confirmed that these seedlings were sensing and responding to the reduced availability and tissue accumulation of P_i (Bari et al., 2006; Chiou et al., 2006). Surprisingly, At4, miRNA399d, PHO1 and PHT1;4 transcripts had a very similar response in the Ph_i-treated seedlings (Fig. 5). The expression of these genes was not affected by the substantial accumulation of Ph_i in the leaf tissue. Thus, under these growth conditions, seedlings were still able to sense and respond to the depletion of P_i and this was not suppressed by Ph_i. However, Ph_i suppressed the induction and repression of genes important for the remodelling of membrane lipid composition, SQD2 and NMT3, respectively. By contrast, the observed induction in gene expression of PHT1;1, PAP1 and ASK11 under P_i limitation was accentuated in the presence of Ph_i. These results demonstrated a multifaceted impact of Ph_i on gene regulation beyond the direct mimicking of P_i in sensing mechanisms.

Fig. 5.

Gene expression analysis for leaf tissue of 10-d-old Arabidopsis (Col-0) seedlings. Shown are quantitative RT-PCR results for P_i-deficient plants grown on 0.05mM P_i (–P) and 0.1mM P_i+0.4mM Ph_i (+Ph_i). For both treatments, expression levels are given relative to control plants with a sufficient P_i supply (0.5mM P_i) and normalized to the reference genes PDF2, UBC9 and UPL7. Results are given as means ±SE (n=5). Statistically significant differences between the –P and +Ph_i treatments are indicated (ANOVA Tukey, *P < 0.05; **P < 0.01).

Metabolic changes induced by phosphite treatment

Current evidence suggests that plants do not assimilate Ph_i into organic compounds and that oxidation of Ph_i to P_i as occurs in microbes is unlikely (Carswell et al., 1997; Danova-Alt et al., 2008; Berkowitz et al., 2011). However, this does not rule out the possibility that Ph_i affects metabolism by mimicking P_i, for example by competing for catalytic sites or in the allosteric regulation of enzymatic reactions.

An untargeted approach to measure metabolite levels in Ph_i-treated and untreated control plants by GC-MS was carried out to probe for Ph_i-induced metabolomic changes. To differentiate between P_i-dependent and Ph_i-specific differences in metabolite pools, control plants were supplied with either high (0.5mM) or low (0.05mM) P_i in the growth medium. Accordingly, these growth conditions resulted in high and low P_i concentrations, respectively, in the harvested shoot tissues after 3 weeks of growth (Fig. 6A). In parallel, and on the basis of earlier experiments, the Ph_i treatment was exposure of plants to 0.5mM P_i+2.5mM Ph_i in the growth medium. This treatment yielded plants that had accumulated shoot P_i to concentrations that were intermediate compared with those obtained in the shoots of the control plants exposed to either high or low P_i supply. The Ph_i concentrations at the time of harvest were below the threshold where Ph_i indirectly affects shoot growth. The fresh weight of the Ph_i-treated plants was reduced by only 13% compared with the high-Pi-treated plants, whereas the fresh weight of plants supplied with low P_i was reduced by 36% (Fig. 6B). The plants exposed to low P_i, however, had not accumulated anthocyanins (data not shown) and were therefore not severely P limited. This conclusion was supported by the still substantial P_i concentration in the shoots. Therefore, after being exposed to either Ph_i or low P_i, these plants were not severely P limited, and any differences in metabolite abundance were expected to represent metabolic adjustments rather than a stress response brought on by severe P_i deficiency.

Fig. 6.

Metabolomic profiling. Arabidopsis plants (Col-0) were grown for 3 weeks on three different growth media to allow comparison of Ph_i-treated (0.5mM P_i+2.5mM Ph_i) and P_i-limited (0.05mM P_i) plants with P_i-sufficient (0.5mM P_i) control plants. (A) Shoot P_i and Ph_i concentrations. (B) Shoot fresh weights. Results are given as means ±SE from eight independent biological replicates.

In total, we were able to robustly quantify 112 polar metabolites by GC-MS. Of these, 84 metabolites could be identified through peak comparison with mass spectra libraries. For quantitative comparison of the metabolites present in the shoots of plants exposed to the low-P_i and Ph_i treatments, we normalized the data to the metabolite levels obtained from shoots of plants supplied with high (0.5mM) P_i. A total of 28 metabolites showed a significant difference (ANOVA Tukey, P <0.05) in abundance of more than 20% in at least one comparison with the control (Fig. 7).

Fig. 7.

Effect of Ph_i accumulation and P_i limitation on levels of polar metabolites in 3-week-old Arabidopsis (Col-0) shoots. Changes in metabolite pools of Ph_i-treated (0.5mM P_i+2.5mM Ph_i, open bars) and P_i-limited (0.05mM P_i, filled bars) plants are given in as percentages of the results for the P_i-sufficient (0.5mM P_i) control plants. Shown are only metabolites identified by GC-MS analysis as having a greater than 20% change for at least one comparison are shown (ANOVA Tukey, P <0.05). Results are given as means ±SE of eight independent biological replicates per treatment.

The most prominent group of primary metabolites for which differences across treatments were observed were the amino acids asparagine, glutamic acid, aspartic acid, and serine, which showed contrasting changes in the two treatments. The abundance of each of these amino acids was 40–60% lower in the shoots of the Ph_i-treated plants, but slightly higher (10–20%) in the shoots of plants exposed to low P_i when compared with the control plants.

The largest differences in metabolite abundance in the shoots of plants exposed to low P_i were found for the organic acids citrate and threonate, which were about twofold more abundant than in control tissues. For the Ph_i treatments, both metabolites were about 25% more abundant. Organic acids such as citrate have frequently been found to be elevated under P deficiency, probably to increase pools to allow for excretion and improve P_i uptake from the soil (Koyama et al., 2000; Narang et al., 2000). An increase in myo-inositol and inositol-3-phosphate in the low-P_i samples and to a lesser degree for Ph_i treatments might indicate the mobilization of P_i from, or a reduced flux into, storage pools. Both these metabolites are intermediates in the biosynthesis and degradation of the phosphate-storage compound phytate (inositol hexakisphosphate) (Raboy, 2003). Similarly, replacement of phospholipids by galacto- and sulfolipids is an important means of releasing P_i from membrane lipids and remodelling plant lipid composition by providing precursors for lipid biosynthesis (Essigmann et al., 1998; Hartel et al., 2000; Li et al., 2006). Correspondingly, we were able to observe changes for the major fatty acids linolenic and linoleic acid. For both, levels decreased in the low-P_i but not in the Ph_i-treated samples. Palmitic acid on the other hand shows a decreased abundance in the Ph_i-treated shoots only, whilst stearic acid levels dropped in both treatments. A slight decrease in glucose-6-phosphate in the P_i-limited plants and an increase in the Ph_i treatments was also detected. Several metabolomic studies have reported decreases in levels of phosphorylated sugars under P deficiency (Rychter and Randall, 1994; Morcuende et al., 2007; Huang et al., 2008).

Overall, 11 and one metabolite(s) were specifically more abundant in the low-Pi and Ph_i treatments, respectively, whilst four metabolites increased in abundance in both treatments (Fig. S2A at JXB online). For the Ph_i-treated plants, nine metabolites, most notably the central amino acids asparagine, aspartic acid, serine, and glutamic acid and its degradation product pyroglutamic acid were less abundant and six metabolites differed in their abundance between both treatments (Fig. S2A).

Discussion

Impact of phosphite accumulation on plant growth

Understanding the consequences of Ph_i accumulation in plants is complicated by the different molecular processes that Ph_i may impact on and their possible interactions. Several studies have reported an inhibitory effect of Ph_i on the growth of plants (Carswell et al., 1996, 1997; Ticconi et al., 2001; Varadarajan et al., 2002; Thao et al., 2008a, b). However, knowledge on the cause of the inhibitory effect of Ph_i on plant growth is limited, and detailed studies on the interaction of P_i and Ph_i on root architecture are not available. Ph_i competes with P_i for uptake by the same membrane transport system and should therefore, at least at high external concentrations, reduce the amount of P_i entering the cells and lead to P_i depletion of the plant (Carswell et al., 1996; Danova-Alt et al., 2008). In addition, Ph_i influences subcellular compartmentation of P_i (Danova-Alt et al., 2008; Pratt et al., 2009) and both processes will impinge on P_i homeostasis with downstream effects on P_i-dependent metabolism. At the same time, the pseudo-pyramidal Ph_i closely resembles tetrahedral P_i in its geometry, which suggests a potential for Ph_i to mimic P_i and impair P_i-sensing mechanisms or P_i-dependent enzyme reactions and their allosteric regulation. In this study, a constant combined tissue concentration of Ph_i+P_i in Ph_i-treated plants across a wide range of external concentration ratios was observed, suggesting a mechanism integrating P_i and Ph_i concentrations and regulating the homeostasis in both shoot and root tissues. Intriguingly, a higher P_i concentration in roots of seedlings grown in the presence of Ph_i was detected when compared with those grown on the same medium lacking Ph_i. A correspondingly higher shoot P_i concentration with Ph_i treatment was apparent only for the highest Ph_i supply. As plants are unable to oxidize Ph_i to P_i and the shoot accumulated Ph_i to a higher extent than the roots, this increased root P_i concentration is probably caused by an increased uptake of P_i from the medium and/or retention in the root. As the expression of miRNA399d was not suppressed by Ph_i, the uptake of P_i by high-affinity transporters might not be downregulated under sustained Ph_i treatments. Inhibition of P_i assimilation into the organic P pool as a consequence of Ph_i accumulation could also lead to increased P_i concentrations in the roots. This could occur either through forcing more P_i into the vacuolar storage pool, thereby decreasing P_i availability in the cytosol and organelles, or by directly inhibiting metabolic processes. These results highlight the necessity to determine Ph_i and P_i concentrations in the tissues to be able to differentiate between direct effects of Ph_i, for example on the PSR or metabolism, and indirect effects on P_i homeostasis.

Suppression of the PSR by phosphite

The perception of Ph_i by the plant has the potential to suppress the PSR. In support of this hypothesis, several studies have shown the capacity of Ph_i to suppress the PSR in severely P_i-starved plants when transferred to medium containing high concentrations of Ph_i, for example suppression of P_i-transporter genes and the inhibition of root-hair growth or anthocyanin accumulation (Carswell et al., 1996; Ticconi et al., 2001; Varadarajan et al., 2002). Another characteristic developmental pattern influenced by P_i availability is the alteration of root architecture as part of the PSR. Plants respond to reduced P_i supply by slowing primary root growth and increasing LRD, which is regulated locally by the external P_i concentration (Thibaud et al., 2010). Surprisingly, under acclimation to Ph_i, seedlings showed an increase in LRD comparable to P_i-deficient plants in this study. This was the case even though the combined concentrations of both anions in the external medium were constant and overall tissue concentrations were also the same, i.e. there were no changes in external or internal [Pi+Ph_i] across all treatments. These results suggest that plants are able to differentiate between the two anions at least to some extent and sense the depletion of P_i even in the presence of Ph_i. This was not only the case for the accession Col-0 but also for several other Arabidopsis accessions we tested for changes in LRD, which responded even more strongly. Similar studies have also shown a varying sensitivity of Arabidopsis accessions to P_i deficiency (Chevalier et al., 2003; Reymond et al., 2006).

In addition, our gene expression studies also indicated that plants accumulating Ph_i can still sense P_i deficiency. P_i-starvation-induced (PSI) genes of the primary regulatory network (At4, miRNA399d, PHO1, and PHT1;4) remained upregulated, although Ph_i accumulation resulted in a combined P_i+Ph_i tissue concentration that was comparable to plants on a high P_i supply. By contrast, several other studies have reported repression of several PSI genes (e.g. At4, PAP1, PHT1;1 and IPS1) by Ph_i treatment in severely P_i-starved plants (Ticconi et al., 2001; Varadarajan et al., 2002; Ribot et al., 2008), but Ribot et al. (2008) also found no suppression of PHO1 by Ph_i. A possible explanation for these differences in response might be a distinct subcellular localization of Ph_i and/or P_i under different experimental conditions. In P_i-sufficient plants, Ph_i preferentially accumulates in the vacuole, whereas in P_i-starved plants, Ph_i accumulates in the cytoplasm (Danova-Alt et al., 2008). Such differential compartmentation might explain differences in the ability of Ph_i to interfere with P_i-sensing mechanisms and subsequently the regulation of gene expression. In our experiments, the induction and repression of SQD2 and NMT3, respectively, was attenuated by Ph_i, whilst the induction of PHT1;1, PAP1 and ASK11 was accentuated. At the same time, the induction of miRNA399d, At4 and PHT1;4 was not affected by Ph_i. These results indicate alternative sensing mechanisms of the P_i (and Ph_i) status within the plant. A possible mechanism allowing the plant to differentiate between P_i and Ph_i could be the additional sensing of a P-containing or a derived component as a measure of flux of free P_i into the organic P pool. Members of the NMT gene family have already been implicated in a stress signalling pathway involving the phospholipid-derived second messenger phosphatidic acid (Jost et al., 2009). Such regulation would be reminiscent of other two major macronutrients nitrate and sulfate, for which downstream assimilation products, i.e. glutamate/glutamine and glutathione, respectively, are part of the nutrient-sensing network (Lappartient et al., 1999; Gutierrez et al., 2008).

Metabolomic response to phosphite accumulation

Several enzymes, such as glyceraldyde-3-phosphate dehydrogenase and phosphorylases such as starch phosphorylase, use P_i as a substrate and many enzymes of primary metabolism are regulated by P_i, such as phosphofructokinase (PFK), sucrose phosphate synthase, ADP-glucose pyrophosphorylase, and glutathione synthetase (Sowokinos and Preiss, 1982; Doehlert and Huber, 1984; Hausler et al., 1989; Jez and Cahoon, 2004). Because of its steric similarity to P_i, Ph_i probably mimics and competes with P_i for binding in catalytic or regulatory sites of enzymes, causing competitive or allosteric inhibition. Indeed, for ATP-dependent PFK, but not PPi-dependent PFK, of Brassica nigra, Ph_i inhibits in vitro enzyme activity (Carswell et al., 1996). Similarly, several glycolytic enzymes from Phytophthora palmivora and yeast are also inhibited by Ph_i (Stehmann and Grant, 2000), indicating that the regulation of enzyme activity by Ph_i may be of wider significance.

Metabolomic analyses identified alterations in a number of metabolite pools. These results showed the ability of Ph_i to induce specific shifts, which for some metabolites contrasted with those of P_i-limited plants. We found that Ph_i specifically decreased levels of the central amino acids asparagine, aspartate, glutamate, and serine (Figs 7 and S2). Alterations in amino acid levels have also been observed for severely P-limited Arabidopsis and barley plants (Morcuende et al., 2007; Huang et al., 2008).

Although these results do not allow the underlying specific targets of Ph_i causing these changes to be identified, they reveal that even moderate accumulation of Ph_i influences plant metabolic processes. This may provide clues to increase our understanding of the mechanisms of Ph_i-induced resistance of plants to oomycete pathogens such as Phytophthora and Pythium species (Guest and Grant, 1991). The molecular mode of action of Ph_i in inducing resistance has not yet been identified, but studies in Arabidopsis have implicated salicylic acid and its major signalling component NPR1, as well as a MAP kinase regulatory network (Molina et al., 1998; Friedrich et al., 2001; Eshraghi et al., 2011; Massoud et al., 2012). Several recent studies have identified a role of primary metabolites, especially amino acids, in the establishment of resistance against plant pathogens (Song et al., 2004; Chanda et al., 2008; van Damme et al., 2009; Liu et al., 2010; Park et al., 2010; Brauc et al., 2011, 2012; Chanda et al., 2011; Hwang et al., 2011; Stuttmann et al., 2011; Voll et al., 2012). These studies show that the alteration of specific metabolite levels by feeding or genetic manipulation of plant metabolic pathways can lead to resistance against pathogens. Interestingly, alterations in the abundance of amino acids derived from the aspartate pathway have been identified to be important for resistance against the oomycete pathogen H. arabidopsidis and the bacterial pathogen Pseudomonas syringae (van Damme et al., 2009; Stuttmann et al., 2011; Navarova et al., 2012). As we found Ph_i-induced changes in levels of aspartate and the related amino acids asparagine and glutamate (Fig. S2B), we surmise that these are similarly important for Ph_i-induced resistance against oomycete pathogens. Currently, it is not clear how these metabolic changes induce resistance. It has been suggested that, due to the evolutionary relationship between plants and oomycetes, metabolic pathways are conserved and metabolic perturbations in the host decrease the colonization ability of the pathogen (Stuttmann et al., 2011). These results indicate the possibility for a mode of Ph_i action by inducing alterations in primary metabolite pools leading to pathogen resistance.

Supplementary data

Supplementary data are available at JXB online.

Supplementary Fig. S1. Shoot:root ratios of plant under +Ph_i and –Pi treatments as in shown in Fig. 1.

Supplementary Fig. S2. (A) Representation of metabolites with overlapping and contrasting changes in Ph_i-treated and P_i-limited Arabidopsis plants. (B) Simplified metabolic pathways showing interconnections of metabolites with altered abundance (in bold) under Ph_i treatment or P_i limitation.

Supplementary Table S1. Sequences of the primers used in gene expression experiments.

Glossary

Abbreviations:

GC-MS

gas chromatography–mass spectrometry

LRD

lateral root density

PFK

phosphofructokinase

Phi

phosphite; inorganic phosphate

PSR

phosphate-starvation response

SE

standard error.