Effect of elevated CO₂ on phosphorus nutrition of phosphate-deficient Arabidopsis thaliana (L.) Heynh under different nitrogen forms

Abstract

Phosphorus (P) nutrition is always a key issue regarding plants responses to elevated CO₂. Yet it is unclear of how elevated CO₂ affects P uptake under different nitrogen (N) forms. This study investigated the influence of elevated CO₂ (800 µl l^–1) on P uptake and utilization by Arabidopsis grown in pH-buffered phosphate (P)-deficient (0.5 µM) hydroponic culture supplying with 2mM nitrate (NO₃ ⁻) or ammonium (NH₄ ⁺). After 7 d treatment, elevated CO₂ enhanced the biomass production of both NO₃ ⁻- and NH₄ ⁺-fed plants but decreased the P amount absorbed per weight of roots and the P concentration in the shoots of plants supplied with NH₄ ⁺. In comparison, elevated CO₂ increased the amount of P absorbed per weight of roots, as well as the P concentration in plants and alleviated P deficiency-induced symptoms of plants supplied with NO₃ ⁻. Elevated CO₂ also increased the root/shoot ratio, total root surface area, and acid phosphatase activity, and enhanced the expression of genes or transcriptional factors involving in P uptake, allocation and remobilization in P deficient plants. Furthermore, elevated CO₂ increased the nitric oxide (NO) level in roots of NO₃ ⁻-fed plants but decreased it in NH₄ ⁺-fed plants. NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) inhibited plant P acquisition by roots under elevated CO₂. Considering all of these findings, this study concluded that a combination of elevated CO₂ and NO₃ ⁻ nutrition can induce a set of plant adaptive strategies to improve P status from P-deficient soluble sources and that NO may be a signalling molecule that controls these processes.

Introduction

Concentrations of atmospheric CO₂ have risen from about 270 µl l^–1 in pre-industrial times to over 380 µl l^–1 at present, and are predicted to reach between 530 and 970 µl l^–1 by the end of this century (IPCC, 2007). It has been demonstrated that increasing atmospheric CO₂ concentration has a profound impact on the growth and development of plants. Many studies have investigated plant responses to elevated CO₂ on ecosystem, community, population, physiological, and molecular scales (Gibeaut et al., 2001; Teng et al., 2006; Jin et al., 2009; Niu et al., 2011a ). However, relatively little information exists on the effects of elevated CO₂ on nutrition acquisition and metabolism. Previous studies have shown that elevated CO₂ generally stimulates the rates of carboxylation but suppresses the rate of oxygenation of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) in C3 plants, thereby increasing plant growth and crop yield (Stitt et al., 1991; Drake et al., 1997). However, sustaining these increased rates requires increased nutrient uptake. Consequently, the role of nutrition availability is a key unresolved issue regarding plant responses to elevated CO₂.

Phosphorus is an essential element for plant growth and development. It plays important roles in energy transfer, signal transduction, photosynthesis, enzyme activation/inactivation, membrane synthesis and stability, and respiration, and is a structural component of nucleic acids and phospholipids (Abel et al., 2002; Vance et al., 2003; Chiou and Lin, 2011; Péret et al., 2011). However, the low solubility and high sorption capacity of phosphate in soils make it relatively unavailable to plant roots. In soil solution, concentrations of available P (mainly H₂PO₄ ⁻ and HPO₄ ²⁻) are generally less than 10 µM, which is well below the critical level needed for the optimal performance of crops (Schachtman et al., 1998; Batjes, 1997). The problem of P deficiency is mitigated by the application of concentrated fertilizers that provide soluble P to plants. This practice, however, is inherently inefficient due to chemical immobilization and surface runoff of P (Abel et al., 2002). Hence, low P availability is one of the major growth-limiting factors for plants in many natural and agricultural ecosystems (Barber et al., 1963; Raghothama, 1999).

When soil phosphorus availability is low, phosphorus uptake is increased by elevated CO₂; however, higher foliar phosphorus concentrations are also required to realize the maximum growth potential at elevated CO₂ (Conroy et al., 1990). Although the absolute increase in growth is greatest at high nutrient availability, relatively large gains in productivity can also be achieved at lower nutrient availability (Conroy et al., 1992). Notably, P deficiency reduces the assimilation of NO₃ ⁻ into the proteins, which might cause negative feedback on NO₃ ⁻ influx and/or stimulate NO₃ ⁻ efflux (Schjørring, 1986). Therefore, it seems that, under conditions of nutrition deficiency and elevated CO₂, plants sense changes in nutrition availability and trigger a set of plant adaptive responses to increase nutrition uptake and recycling. Numerous studies indicate that plants enhance P acquisition and mobilization to cope with low P availability by inducing a series of biochemical, physiological, and molecular adaptation strategies (Raghothama, 1999; Mukatira et al., 2001; Abel et al., 2002; Rubio et al., 2009; Ramaekers et al., 2010; Péret et al., 2011; Jin et al., 2012; Veneklaas et al., 2012). However, it is unclear how plants sense and respond to P deficiency under conditions of elevated CO₂.

Nitrogen (N) has been the centre of attention not only because it is the primary limiting nutrition in most temperate ecosystems, but also because the form of N supply can modify the response of plant growth to nutrition acquisition and elevated CO₂. It has been reported that N forms (NO₃ ⁻ versus NH₄ ⁺) can lead to contrasting responses of plant growth and metabolism to elevated CO₂. For example, elevated CO₂ increased NO₃ ⁻ uptake and nitrate reductase activity when tobacco was fed with NO₃ ⁻ but increased NH₄ ⁺ uptake and inhibited nitrate reductase activity with an NH₄ ⁺ supply (Matt et al., 2001). Likewise, under elevated CO₂, NH₄ ⁺-fed wheat showed greater increases in leaf area and smaller decreases in shoot protein concentration than NO₃ ⁻-fed plants (Bloom et al., 2002, 2010). Pharmacological experiments and studies of mutants with low activity of NIA (encoding nitrate reductase) showed that nitric oxide (NO) production was induced by NO₃ ⁻ and elevated CO₂ (Gojon et al., 1998), but was inhibited by ammonium, glutamine, or related downstream products of nitrate assimilation (Gojon et al., 1998). Recently, Carlisle et al. (2012) reported that both CO₂ concentration and N form strongly affected biomass production in hydroponically grown wheat, as well as nutrient concentrations in above- and below-ground tissues. Consequently, these differences between NO₃ ⁻ and NH₄ ⁺ nutrition led us to investigate how N form regulates the responses of P homeostasis of plants grown under elevated CO₂. Therefore, the aim of this study was to investigate the interactive effects of N form and elevated CO₂ on plant P uptake and utilization in a P-deficient medium. Arabidopsis was used as the test plant.

Materials and methods

Plant material and growth conditions

Seeds of Arabidopsis thaliana wild-type ecotype and mutants nr, AT1G77760 and AT1G37130 locus (Wang et al., 2004) and atnos1, AT3G47450 locus (Guo et al., 2003), were surface-sterilized, and germinated on Petri plates containing 0.8% agar medium supplemented with one-fifth-strength nutrition solution (Niu et al., 2011a ). After 7 d, seedlings of uniform size were transferred to 1.5ml Eppendorf tubes whose bottoms were cut about 0.7cm from the bottom to allow root growth into the nutrition solution. The tubes were inserted in holes of plastic boxes containing 200ml of a half-strength nutrition solution, and plants were grown for 14 d. Then, each seedling was transferred to each pot containing 300ml of nutrition solution. The composition (µM) of the nutrition solution was as follows: 1500 KNO₃, 500 MgSO₄, 1000 CaCl₂, 500 NaH₂PO₄, 250 (NH₄)₂SO₄, 10 H₃BO₃, 0.5 MnSO₄, 0.5 ZnSO₄, 0.1 CuSO₄, 0.1 (NH₄)₆Mo₇O₂₄, and 25 Fe-EDTA (Hoagland and Arnon, 1938). The nutrition solution was renewed every 3 d and its pH was adjusted daily to 6.0 using 1M NaOH. All plants were grown in controlled-environment growth chambers (Conviron E7/2, Winnipeg, Manitoba, Canada) with a humidity of 80%, a daily cycle of 22 °C/10h day and 20 °C/14h night. The daytime light intensity was 120 µmol photons m⁻² s⁻¹. At least eight independent replicates were used for each treatment.

After plants were further grown in pots for 14 d, treatments commenced. Plants were grown in chambers with a CO₂ concentration of either 350±50 (ambient) or 800±50 µl l^–1 (elevated CO₂). Meanwhile, N and P treatments were initiated. One set of plants were fully supplied with 2mM NO₃ ⁻ as KNO₃, while the other was fully supplied with 2mM NH₄ ⁺ in each CO₂ treatment. For the treatment of NH₄ ⁺ as the sole N source, 1mM (NH₄)₂SO₄ and 1mM K₂SO₄ were added. The low-P treatment had 0.5 µM NaH₂PO₄. The solution pH was buffered with 2mM HEPES at pH 6.8 to minimize pH change, and was adjusted daily to 6.8 using 1M NaOH. The treatment solutions were renewed every 2 d.

The NO-related treatments on wild-type plants were also initiated as follows: (i) ambient treatment: plants were grown continuously under ambient CO₂; (ii) elevated CO₂ treatment: plants were transferred to a chamber with a CO₂ concentration of 800±50 µl l^–1; (iii) ambient+sodium nitroprusside (SNP) treatment: plants were transferred to the nutrition solution containing 100 µM SNP under ambient CO₂; and (iv) elevated CO₂+2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO) treatment: plants were transferred to the nutrition solution containing 100 µM cPTIO under elevated CO₂.

Analysis of total P in plant tissue

After 7 d treatment with ambient or elevated CO₂, plants from various treatments were harvested, washed thoroughly with deionized water, divided into shoots and roots, and dried in an oven at 75 °C for 1 d. The samples were then weighed, digested in sulfuric acid/hydrogen peroxide, and analysed for total P concentration using the vanadium-molybdenum blue photometric method. The ‘P absorbed per unit weight of roots’ was calculated as the total amount of P per plant/root dry weight.

Chlorophyll concentration in leaves

After 7 d treatment, a portable chlorophyll meter (SPAD-502, Minolta, Japan) was used to measure chlorophyll content (Jin et al., 2009). Three leaves were selected randomly from each treatment and two SPAD readings were recorded for each leaf, avoiding the main veins during measurement.

Anthocyanins

Total anthocyanins in leaves were extracted and determined as described by Hodges et al. (1999) with minor modifications. Approximately 0.1g of fresh leaves was homogenized in 3ml of methanol-1% HCl. Samples were centrifuged and 1ml of the supernatant appropriately diluted in methanol-1% HCl was used for analysis. The difference in absorbance at 536 and 600nm was used to calculate total anthocyanin content. Results were expressed as cyanidin 3-glucoside equivalents (mmol g⁻¹ of fresh weight) using an extinction coefficient of 0.449mol l⁻¹ at 536nm (Fiorani et al., 2005).

Root morphology

After 7 d treatment, the root parameters of individual plants were measured using a root analysis instrument (WinRHIZO, Regent, Canada). The number and length of root hairs in each segment were measured using light microscopy with differential interference contrast optics. Micrographs were recorded on a CCD camera (Nikon Eclipse E600, Melville, NY, USA).

Activity of acid phosphatases (APases)

The activity of APases in shoots and roots was determined according to the method of McLachlan et al. (1987) with minor modifications. After 7 d in low-P treatment, the seedlings were washed thoroughly with deionized water, divided into shoots and roots, weighed, and then chilled immediately on ice. About 0.1–0.2g of fresh samples was frozen with liquid nitrogen and homogenized in a cold mortar, and the powder was macerated with 5ml of extraction buffer (0.2M glacial acetic acid-sodium acetate buffer, pH 5.8). The extract was centrifuged at 27 000g at 4 °C for 10min, and the supernatant was used for an assay of APase activity. APase activity was determined by measuring enzyme activity in extraction buffer. Briefly, the APase reaction mixture contained 0.05ml of enzyme extract, 0.45ml of buffer solution and 4.5ml of 5M p-nitrophenyl phosphate. All APase reaction mixtures were incubated at 30 °C for 30min in the dark. The reaction mixtures were stopped by the addition of 2ml of 2M NaOH. After this, the concentration of p-nitrophenol in the APase reaction mixtures was determined in a spectrophotometer at 405nm. The APase activity was expressed as mol substrate hydrolysed g^–1 of fresh weight min^–1.

In situ measurement of NO in the root

NO was imaged using diaminofluorescein-FM diacetate (DAF-FM DA) and epifluorescence microscopy. DAF-FM DA has been used successfully to detect NO production in both plants and animals. Roots were loaded with 5 µM DAF-FM DA in 20mM HEPES-NaOH buffer (pH 7.4) for 30min, washed three times in fresh buffer and observed under a microscope (Nikon Eclipse E600, Nikon; excitation 488nm, emission 495–575nm). A 100W high-pressure mercury-vapour lamp was used as a light source (HB-10103AF-Hg, Nikon). Fifteen roots of each treatment were measured each time. The signal intensities of green fluorescence in the images of the root tip and young root-hair zone were quantified using Image J.

Determination of nitric oxide synthase (NOS) activity

The activity of NOS was determined as described by Tian et al. (2007) with some modifications. The activity of NOS was determined with an NOS assay kit (Beyotime, Haimen, China). Briefly, 0.1g of roots was frozen in liquid nitrogen and ground to a fine powder. The powder was homogenized in 1ml of extraction buffer containing 100mM HEPES-KOH (pH 7.5), 1mM EDTA, 10% glycerol, 5mM dithiothreitol, 0.1% Triton X-100, 0.5mM phenylmethylsulfonyl, 20 µM flavin adenine dinucleotide, 25 µM leupeptin, 5 µM Na₂MoO₄, and 1% polyvinylpyrrolidone. The contents were centrifuged at 13 000g for 20min at 4 °C and 0.2ml of clear supernatant was then added to 0.1ml of assay mixture (containing NADPH, L-arginine, NOS assay buffer, and DAF-FM DA) and reacted at 37 °C in the dark for 1h. The NO content was detected under a microscope as described above. The fluorescence intensity was expressed as colour level on a scale ranging from 0 to 255, and the fluorescence intensity was quantified by using Image J. Data are presented as the mean of fluorescence intensity relative to the control treatment. The control treatment was the plant grown in ambient CO₂.

Determination of maximum nitrate reductase (NR) activity

The activity of maximum NR was determined as described by Tian et al. (2007) with some modifications. Briefly, a total of 0.2ml of clear supernatant prepared as described above was added into 0.4ml of pre-warmed assay buffer containing 100mM HEPES-KOH (pH 7.5), 5mM KNO₃, and 0.25mM NADPH. The mixed solution was reacted at 30 °C for 1h, and the reaction was stopped by adding zinc acetate. The nitrite produced was measured colorimetrically at 540nm by adding 1ml of 1% sulfanilamide in 3M HCl plus 1ml of 0.2% N-(1-naphthyl)ethylenediamine.

Quantitative PCR analysis

Total RNA was extracted using RNAiso Plus (Takara, Otsu, Shiga, Japan) from about 70mg of fresh root tissues. At least five plants were used for extraction. All RNA samples were checked for DNA contamination before cDNA synthesis. cDNA was synthesized, and possible residual genomic DNA contamination was verified as described by us previously (Niu et al., 2011a ). The mRNA levels of the genes PHOSPHATE STARVATION RESPONSE 1 (AtPHR1), PHOSPHATE TRANSPORTER 1 (AtPHT1), PHOSPHATE 1 (AtPHO1), PHOSPHATE 2 (AtPHO2), and PURPLE ACID PHOSPHATASE 2 (AtPAP2) were detected using a SYBR Green RT-PCR kit (Takara) with the following pairs of gene-specific primers: AtPHR1, forward: 5’-ACAGCAATAACGGAACGGGCAAG-3’ and reverse: 5’-GCTCTTTCACTACCGCCAAGACTG-3’; AtPHT1, forward: 5’-GCCAAGGTAGACGCAGGATA-3’ and reverse: 5’-AACCTCAG CCTCACCAGAGA-3’; AtPHO1, forward: 5’-TACGCGAGAGAAA ACAACGA-3’ and reverse: 5’-TTCCGGAGAACCAAATTGTC-3’; AtPHO2, forward: 5’- TTTTACACAAGCCACCAAAGC-3’ and reverse: 5’-TCACGAGCATGTCCAACAA-3’; AtPAP2, forward: 5’-CATCAAGTTCCTTTGAGAGC-3’ and reverse: 5’-TTGGACCGG TGTTGTAGGAG-3’.

Real-time PCR was carried out similar to as described by Niu et al. (2011a). A pair of primers for the housekeeping gene UBQ10 (forward: 5’-GGTTCGTACCTTTGTCCAAGCA-3’, reverse: 5’-CCTTCGTTA AACCAAGCTCAGTATC-3’) was used for a control reference gene for the PCR (Gan et al., 2006). Melting-curve analysis and gel electrophoresis of the PCR products were used to confirm the absence of non-specific amplification products. Relative expression levels were calculated by subtracting the threshold cycle (C _t) values for UBQ10 from those of the target gene (to give ΔC _t) and then calculating 2^−ΔCt, where C _t was the cycle number at which the fluorescence rose above the set threshold of the quantitative PCR.

Statistical analyses

All statistical analyses were conducted with DPS software (Stirling Technologies Inc., China). Means were compared using Student’s t-test or Fisher’s least significant difference test at P=0.05 in all cases.

Results

Plant growth and P uptake in low-P culture

After 7 d growth in ambient CO₂ and P-deficient medium containing NO₃ ⁻ supplement as the sole N source, Arabidopsis leaves were dark green with a SPAD reading of 33. By comparison, the leaves of plants grown under the same nutrition conditions but with elevated CO₂ were a normal green colour (Fig. 1a) with SPAD readings of 26 (see Supplementary Fig. S2a at JXB online). Elevated CO₂ decreased the concentration of anthocyanins in leaves of NO₃ ⁻-supplied plants (see Supplementary Fig. S3a at JXB online), indicating that elevated CO₂ alleviated the P-deficiency-induced symptoms of the NO₃ ⁻-supplied plants. However, with a supply of NH₄ ⁺ as the sole N source, plants grown in the low-P solution had a similar appearance under both ambient and elevated CO₂ treatments (Fig. 1a). Elevated CO₂ did not change the concentrations of chlorophyll and anthocyanins (Supplementary Figs S2a and S3a). Furthermore, ammonium toxicity was not evident in the NH₄ ⁺-fed plants.

Fig. 1.

Images (a), fresh biomass production of shoots (b) and roots (c), concentration of P in shoots (d) and roots (e), total P content (f), and amount of P absorbed per weight of roots (g) of 5-week-old wild-type Arabidopsis grown for 7 d in P-deficient nutrition solutions with NO₃ ⁻ or NH₄ ⁺ exposed to ambient CO₂ (350±50 µl l^–1) or elevated CO₂ (800±50 µl l^–1). Data are means ±SD (n=5). Asterisks indicate that the mean values are significantly different between the ambient and elevated CO₂ treatments (P <0.05). DW, dry weight.

Elevated CO₂ enhanced shoot growth exclusively under NH₄ ⁺ nutrition (Fig. 1b), whereas it enhanced root growth under NO₃ ^– nutrition (Fig. 1c). Elevated CO₂ decreased the P concentration in shoots by 27% but did not alter it in the roots of NH₄ ⁺-supplied plants. However, elevated CO₂ increased the P concentration by 32 and 21%, respectively, in the shoots and roots of NO₃ ⁻-fed plants (Fig. 1d, e). Meanwhile, elevated CO₂ increased the amount of P absorbed per unit weight of roots of NO₃ ^–-fed plants by 56% but decreased that of NH₄ ⁺-fed plants by 33% (Fig. 1g), indicating that NO₃ ⁻ nutrition facilitates P uptake in roots.

After 7 d growth in P-adequate medium with either N source, the leaves were a normal green colour (data not shown). CO₂ treatment did not affect the concentration of anthocyanins in leaves, but NH₄ ⁺ supply significantly increased SPAD readings (Supplementary Figs S2b and S3b). Elevated CO₂ increased biomass production (Supplementary Fig. S1a, b), but decreased the P concentration and the amount of P absorbed per weight of roots, with the decrease being greater in the NO₃ ⁻ than in the NH₄ ⁺ treatment (Fig. S1c, d).

Physiological and morphological responses to P deficiency

Elevated CO₂ greatly enhanced the number of lateral roots in the NO₃ ⁻-supplied plants but restrained it in the NH₄ ⁺-supplied plants (Fig. 2a). The root/shoot ratio of NO₃ ⁻-fed plants was greater and that of NH₄ ⁺-fed plants was lower under elevated compared with ambient CO₂ (Fig. 2b). Furthermore, compared with ambient CO₂, elevated CO₂ increased the total root surface area and root/shoot ratio of the NO₃ ⁻-supplied plants but not that of the NH₄ ⁺-supplied plants (Fig. 2b). Elevated CO₂ did not affect the length or density of root hairs of plants grown in either N treatment (data not shown).

Fig. 2.

Root morphology (a), and root length, number of root tips, root surface area, and root/shoot ratio (b) of 5-week-old wild-type Arabidopsis grown for 7 d in P-deficient nutrition solutions containing NO₃ ⁻ or NH₄ ⁺ exposed to ambient CO₂ (350±50 µl l^–1) or elevated CO₂ (800±50 µl l^–1). Data are means ±SD (n=8) of two independent experiments. Asterisks indicate that the mean values are significantly different between the ambient and elevated CO₂ treatments (P <0.05).

Another general response of plants to P starvation is the enhanced activity of APases. APase activity in both the shoots and roots of NH₄ ⁺-supplied plants was similar between the ambient and elevated CO₂ treatments (Fig. 3a, b). However, elevated CO₂ strongly increased APase activity in the shoots and roots of NO₃ ⁻-fed plants (Fig. 3a, b).

Fig. 3.

APase activity in shoots (a) and roots (b) of 5-week-old wild-type Arabidopsis grown for 7 d in P-deficient nutrition solutions containing NO₃ ⁻ or NH₄ ⁺ exposed to ambient CO₂ (350±50 µl l^–1) or elevated CO₂ (800±50 µl l^–1). Data are means ±SD (n=5). Asterisks indicate that the mean values are significantly different between the ambient and elevated CO₂ treatments (P <0.05). FW, fresh weight.

Expressions of genes involved in P uptake, translocation, and allocation in P-deficient plants

Increased expression of genes involved in P uptake, transport, allocation, and remobilization is a significant adaptive response of plants to facilitate external P acquisition and internal P utilization under P-limited conditions (Rubio et al., 2001; Vance et al., 2003; Chiou and Lin, 2011). Elevated CO₂ enhanced the expression of the P-uptake gene AtPHR1 and the P-transport gene AtPHT1; their expression in shoots were increased by 8-fold and 3.7-fold, respectively (Fig. 4a–d). By contrast, elevated CO₂ did not change the expression of AtPHR1 and AtPHT1 in the roots of NH₄ ⁺-fed plants, and decreased their expression in the shoots (Fig. 4a–d).

Fig. 4.

Relative expression levels of the genes AtPHR1, AtPHT1, AtPHO1, AtPHO2, and AtPAP2 in the shoots and roots of 5-week-old wild-type Arabidopsis grown for 7 d in P-deficient nutrition solutions with NO₃ ⁻ or NH₄ ⁺ exposed to ambient CO₂ (350±50 µl l^–1) or elevated CO₂ (800±50 µl l^–1). Relative expression levels were calculated and normalized with respect to expression of UBQ10 mRNA. Data are means ±SD (n=5). Asterisks indicate that the mean values are significantly different between the ambient and elevated CO₂ treatments (P <0.05).

In order to observe the allocation and retranslocation of internal P between shoots and roots, the expression of AtPHO1 and AtPHO2 was analysed. Elevated CO₂ increased AtPHO1 expression in the shoots and roots of NO₃ ⁻-supplied plants by 2.6-fold and 32%, respectively. In comparison, elevated CO₂ increased AtPHO1 expression in the shoots by 4.7-fold but decreased it by 34% in the roots of NH₄ ⁺-fed plants (Fig. 4e, f). Elevated CO₂ increased the expression of AtPHO2 by 8.8-fold in the shoots but decreased it by 76% in the roots of NO₃ ⁻-supplied plants, whereas it did not significantly change its expression in the shoots of NH₄ ⁺-fed plants (Fig. 4g, h).

Possible role of NO in P nutrition under P deficiency and elevated CO₂

NO was demonstrated recently to be a signal molecule involved in the regulation of root growth during P deficiency (Wang et al., 2010). We observed that elevated CO₂ resulted in stronger fluorescence intensity in the root tip and root-hair region in NO₃ ⁻-fed plants but decreased it in NH₄ ⁺-fed plants, compared with results under ambient CO₂ (Fig. 5a). To confirm the observation, the fluorescence of 15 roots from each treatment was measured. As shown in Fig. 5b, elevated CO₂ significantly increased the NO level in the roots of NO₃ ⁻-supplied plants but decreased it in NH₄ ⁺-supplied plants. A correlation was found between endogenous NO level and P concentration in the roots. Whereas the application of SNP (a NO donor) to the plants under ambient CO₂ tended to promote the P concentration to levels below those measured under elevated CO₂ (Fig. 6a), the addition of the NO scavenger cPTIO under elevated CO₂ reduced the amount of P absorbed per unit weight of roots in both NO₃ ⁻- and NH₄ ⁺-supplied plants and inhibited it to levels significantly below those measured under ambient CO₂ (Fig. 6c), suggesting some involvement of NO in modulating P uptake in roots. The treatment effect of total P uptake followed the same pattern (data not shown). These results implied that an appropriate concentration of endogenous NO in roots is critical for the regulation of elevated CO₂-induced P uptake in both NO₃ ⁻ and NH₄ ⁺ medium.

Fig. 5.

NO production of 5-week-old wild-type Arabidopsis grown for 7 d in P-deficient nutrition solutions containing NO₃ ⁻ or NH₄ ⁺ exposed to ambient CO₂ (350±50 µl l^–1) or elevated CO₂ (800±50 µl l^–1). (a) Photographs of NO production shown as green fluorescence in the root tips and root-hair region of representative roots of plants supplied with NO₃ ⁻ and exposed to ambient or elevated CO₂, and with NH₄ ⁺ exposed to ambient or elevated CO₂. Bar, 500 µm. (b) Effects of N form and CO₂ treatment on NO production expressed as relative fluorescence. NO production was visualized with DAF-FM DA dye. Data are means ±SD (n=15). Means followed by the same letter within a root segment are not significantly different at P <0.05.

Fig. 6.

Effect of SNP under ambient CO₂ (350±50 µl l^–1) (a) and cPTIO under elevated CO₂ (800±50 µl l^–1) (b) on P concentration per plant, and the effect of cPTIO under elevated CO₂ (800±50 µl l^–1) (c) on the amount of P absorbed per weight of roots of 5-week-old wild-type Arabidopsis treated with P-deficient nutrition solutions containing NO₃ ⁻ or NH₄ ⁺. Data are means ±SD (n=5). Asterisks indicate that the mean values are significantly different between the ambient and elevated CO₂ treatments (P <0.05).

To confirm the above findings further, the effects of elevated CO₂ on the activity of NR and NOS were studied (Fig. 7). In the NO₃ ⁻ treatment, elevated CO₂ increased NR activity in the shoots but decreased it in the roots (Fig. 7a, b). Elevated CO₂ also increased NOS activity in both the shoots and roots of the NO₃ ⁻-fed plants (Fig. 7c, d). In the NH₄ ⁺ treatment, elevated CO₂ did not affect the NOS and NR activity in shoots but inhibited their activity in roots (Fig. 7).

Fig. 7.

NR activity in shoots (a) and roots (b) and NOS activity in shoots (c) and roots (d) of 5-week-old wild-type Arabidopsis treated with P-deficient nutrition solutions containing NO₃ ⁻ or NH₄ ⁺ exposed to ambient CO₂ (350±50 µl l^–1) or elevated CO₂ (800±50 µl l^–1) for 7 d. Data are means ±SD (n=5 for NR; n=10 for NOS). Asterisks indicate that the mean values are significantly different between the ambient and elevated CO₂ treatments (P <0.05).

To further verify the role of NO in mediating elevated CO₂-induced uptake of P during P deficiency, mutants nr (an NR-reduced mutant) and atnos1 (an NOS-reduced mutant) were used to observe their capacity of P uptake compared with wild-type Arabidopsis. Overall, the P concentrations of mutants nr and atnos1 grown in both NO₃ ⁻- and NH₄ ⁺-supplied media were similar under ambient and elevated CO₂ (Fig. 8a, b). Specially, elevated CO₂ increased the P concentration in shoots of the nr mutant supplied with NO₃ ⁻. These results demonstrated that NO is the signal involved in the regulation of elevated CO₂-induced uptake of P during P deficiency.

Fig. 8.

P concentration in shoots (a, c) and roots (b, d) of Arabidopsis NR-reduced mutant nr and nitric oxide synthase-reduced mutant atnos1 treated with P-deficient nutrition solutions containing NO₃ ⁻ or NH₄ ⁺ exposed to ambient CO₂ (350±50 µl l^–1) or elevated CO₂ (800±50 µl l^–1) for 7 d. Data are means ±SD (n=5). Asterisks indicate that the mean values are significantly different between the ambient and elevated CO₂ treatments (P <0.05).

Discussion

Previous studies have indicated that CO₂-dependent stimulation of growth is likely to enhance the requirements of plants for macronutrients such as N and P (Baxter et al., 1994; Kogawara et al., 2006; Lagomarsino et al., 2008). This study demonstrated that a combination of elevated CO₂ and nitrate induced a set of morphological, physiological, and molecular responses that enabled the plants to better access and utilize P and to alleviate P-deficiency symptoms.

Elevated CO₂ improves P nutrition under a nitrate supply

The promotion of plant growth under elevated CO₂ would be associated with increased requirements for mineral nutrition to optimize the nutrition balance in plants. This was the case in a 6-year Free Air CO₂ Enrichment (FACE) experiment in which photosynthetic stimulation by elevated CO₂ was largely regulated by N and P availability in the soil (Lagomarsino et al., 2008). In the present study, elevated CO₂ enhanced biomass production in both NO₃ ⁻- and NH₄ ⁺-fed Arabidopsis plants during P deficiency (Fig. 1). However, elevated CO₂ preferentially stimulated specific P (pool) concentration as well as total P concentration and reduced the accumulation of anthocyanins in NO₃ ⁻-fed plants. This result is consistent with the findings of Carlisle et al. (2012) that NH₄ ⁺-supplied plants tended to have decreased nutrient concentrations with increasing CO₂ concentration, while NO₃ ⁻-supplied plants varied widely across CO₂ treatments. The present study concluded that NO₃ ⁻ nutrition facilitates the P uptake of roots under elevated CO₂.

To test whether the improved P uptake occurred only in NO₃ ⁻-fed P-deficient plants under elevated CO₂ conditions, we further examined the influence of elevated CO₂ on P nutrition of P-adequate Arabidopsis supplied with different N forms. Interestingly, elevated CO₂ only increased the shoot biomass, but decreased specific P uptake with the decrease being greater in the NO₃ ⁻ than the NH₄ ⁺ treatment (Fig. S1). Numerous studies have elucidated that plants adapt to P deficiency through modifications of root architecture, carbon metabolism and membrane structure; exudation of carboxylates, protons and enzymes; and enhanced expressions of genes (Raghothama, 1999; Vance et al., 2003; Shenoy and Kalagudi, 2005; Shulaev et al., 2008; Wang et al., 2010). Therefore, the increase in P concentration in NO₃ ⁻-fed Arabidopsis plants by elevated CO₂ cannot be attributed to increased photosynthesis alone but also to the improved P nutrition of the plants through morphological, physiological, and molecular responses to P deficiency.

This study showed that NO₃ ⁻-fed plants had more lateral roots but a shorter taproot under elevated compared with ambient CO₂, whereas NH₄ ⁺-fed plants had longer roots with fewer lateral roots under elevated CO₂ (Fig. 2). In the field, particularly with no-till systems, P concentration is great in topsoil and declines substantially with depth (Lynch and Brown, 2001; Ao et al., 2010). The reduced primary root and increased lateral roots, resulting in a shallower root system, is a positive adaptive response to low-P availability by reducing inter-root competition within the same plant (Miller et al., 2003; Lynch, 2007). In addition, the greater root/shoot ratios seen in NO₃ ⁻- compared with NH₄ ⁺-supplied plants during P deficiency under high CO₂ concentrations (Fig. 2) also favour P acquisition. Nevertheless, favourable root architecture may only play a minor role in increasing P uptake of plants under the combined conditions of P limitation and elevated CO₂ in the hydroponic culture supplied with H₂PO₄ ⁻.

Another general response of plants to P starvation is the enhanced activity of APases (Richardson et al., 2009; Tran et al., 2010). In bean plants, a high activity of APases contributes to the increased P-utilization efficiency through remobilization of P from old leaves to young tissues (Kouas et al., 2009). Similarly, the physiological function of root APases has also been demonstrated by Xiao et al. (2006) and Wang et al. (2009) where transgenic plants overexpressing APase genes exhibited greater P acquisition from an organic P resource than wild-type plants. Our results showed that elevated CO₂ increased APase activity in both the shoots and roots of Arabidopsis plants supplied with NO₃ ⁻, which is consistent with results reported in the literature (Lagomarsino et al., 2008). The enhanced APase activity, especially in the root of NO₃ ⁻-fed plants, under elevated CO₂ conditions (Fig. 3) would be expected to facilitate hydrolysis of organic P and thus plant P uptake in the field.

Biochemical and molecular studies have identified a number of genes that are involved in plant alteration in response to P starvation. PHR1 is a key transcriptional activator in controlling P uptake, allocation, and anthyocyanin accumulation (Rubio et al., 2001; Nilsson et al., 2007). The enhanced expression of AtPHR1 under elevated CO₂ could activate numerous P-deficiency-response genes, which in turn change molecular, cellular, and physiological processes to cope with P deficiency. P assimilation is initiated by the transport of P into the roots and the number of P transporters present in the roots reflects the complexity and significance of the process. In Arabidopsis, the reduced P uptake activity is caused by a defect in targeting the PHT1 transporter to the plasma membrane (González et al., 2005; Jain et al., 2007). Our present study showed that elevated CO₂ enhanced the expression of AtPHT1 genes in the shoots and roots of NO₃ ⁻-fed plants with expression being much greater in the roots (Fig. 4), indicting that P transport was stimulated by elevated CO₂ in the NO₃ ⁻ medium.

Partitioning and allocation of internal P is also crucial for maintenance of P homeostasis within plants. AtPHO1 was first identified in Arabidopsis as a gene playing an important role in P transport from the roots to the shoots (Poirier et al., 1991), while AtPHO2 is responsible for P translocation from the shoots to the roots. It was reported that a pho1 mutant plant accumulated adequate P in the roots but less P in the shoots (Poirier et al., 1991; Hamburger et al., 2002; Stefanovic et al., 2007). The downregulated expression of AtPHO1 in the roots and the similar expression of AtPHO2 in the shoots of NH₄ ⁺-supplied plants in the present study (Fig. 4) probably form a negative-feedback loop against internal P recycling between shoots and roots. In contrast, the PHO2 gene, which regulates P uptake, allocation, and remobilization, is a target gene of miR399s pho2, which displays P toxicity with excessive P accumulation in the shoots of Arabidopsis (Aung et al., 2006). Accordingly, the high expression of AtPHO1 in the roots and AtPHO2 in the shoots of the NO₃ ⁻-supplied plants under elevated CO₂ (Fig. 4) indicated that internal P recycling between shoots and roots was stimulated by elevated CO₂ during the onset of P starvation. Furthermore, the enhanced AtPAP2 expression in shoots under elevated CO₂ (Fig. 4) indicated that elevated CO₂ could excite intracellular P remobilization during P deficiency.

A possible role of NO

It has been shown that P deficiency significantly increases the endogenous NO concentration in roots of white lupin (Wang et al., 2010). In this study, we observed that elevated CO₂ increased the NO level in roots of NO₃ ⁻-supplied plants, while the NO scavenger cPTIO inhibited specific P uptake by the root. In contrast, the NH₄ ⁺ supply was associated with a rapid decrease in NO production under elevated CO₂, while the NO scavenger cPTIO inhibited specific P uptake (Figs 5 and 6). These results are supported by recent observations that NO₃ ⁻ but not NH₄ ⁺ increased the NO level in the roots of tomato (Solanum lycopersicum) in both cadmium-free and cadmium-supplemented growth solutions (Luo et al., 2012). These results suggest that NO is partly responsible for NO₃ ⁻-facilitated P accumulation in plants.

It is unclear how elevated CO₂ changes the NO levels in roots under P deficiency. It is recognized that NR and NOS are two potential enzymatic sources of NO in plants (Neill et al., 2008). Although NOS genes have not yet been identified in plants (Neill et al., 2008), several studies support the presence of NOS activity in plants (Corpas et al., 2006; Valderrama et al., 2007). As shown in Fig. 7, NR activity in the shoots of NO₃ ⁻-fed plants was greater under elevated compared with ambient CO₂, while NR activity in the roots was lower under elevated CO₂, irrespective of N form. These results are in accordance with the findings of other studies (Buchanan et al., 2000; Poonnachit and Darnell, 2004). Nitrate supply has been shown to enhance NR activity (Shaner and Boyer, 1976), whereas NH₄ ⁺ is an inhibitor of NR (Jin et al., 2011). On the one hand, increasing NO₃ ⁻ availability should enhance the NR-dependent NO production. Accordingly, a higher NO level in the roots of the NO₃ ⁻-fed plants and a lower NO level in the roots of NH₄ ⁺-fed plants under elevated CO₂ is partly due to the change in NR activity. On the other hand, elevated CO₂ enhanced the root uptake capacity for NO₃ ⁻, but not for NH₄ ⁺, in field-grown loblolly pine saplings (Bassirirad et al., 1996). Therefore, it is reasonable to propose that stimulated NO₃ ⁻ uptake under elevated CO₂ may facilitate NO production in roots, form a positive feedback loop, and upregulate P uptake.

Elevated CO₂ also increased the NOS activity of Arabidopsis plants supplied with NO₃ ⁻ but decreased it in NH₄ ⁺-fed plants (Fig. 7). It is well established that elevated CO₂ represses NIA in NH₄ ⁺-fed plants and increases NH₄ ⁺ uptake and assimilation, and the synthesis of glutamine (Gojon et al., 1998; Stitt and Krapp, 1999). Thus, increased NH₄ ⁺ uptake and assimilation under elevated CO₂ may restrain the activity of NR, and possibly of NOS. Furthermore, in mutants with low NIA activity, NO production was suppressed by NH₄ ⁺ (Gojon et al., 1998; Stitt and Krapp, 1999), which supports our findings.

In Arabidopsis, two mutants defective in NO production displayed altered phenotypes. The nr mutant had a deletion of the major NR gene, NIA2, and an insertion in the NIA1 NR gene (Wang et al., 2004), and the atnos1 mutant has a mutation in an NOS structural gene NOS1 (Guo et al., 2003). In this study, the P concentrations of both mutants, nr and nos, were similar under elevated and ambient CO₂ (Fig. 8), suggesting that regulation of P nutrition under elevated CO₂ is NO-signalling dependent. However, relative to the ambient CO₂ condition, elevated CO₂ tended to increase the P concentration in both the shoots and roots of NO₃ ⁻-fed plants. These results are consistent with a major or general role of NO in the regulation of plant development and metabolism. The results also support our hypothesis that elevated CO₂ increases P uptake in NO₃ ⁻-fed plants but suppresses it in NH₄ ⁺-fed plants through the effect on the activity of NR and/or NOS, leading to changes in NO production and physiology. However, the details about how NO exerts its effects on P nutrition remain unclear. The studies by Neill et al. (2003) and Niu et al. (2011b) on interrelations between NO, Ca²⁺, cyclic ADP ribose, cyclic GMP, salicylic acid, reactive oxygen species, hydrogen peroxide signalling, and protein kinases have provided an important focal point for future research.

Overall, although previous reports have provided other evidence concerning NH₄ ⁺ nutrition facilitating P uptake in the roots of different plant species. Here, using wild-type Arabidopsis plants, we demonstrated that NO₃ ⁻ nutrition facilitates the P uptake in roots from P-deficient soluble sources under elevated CO₂ by upregulating plant adaptive strategies. The increase in NO production may be a signalling pathway controlling the above process. To our knowledge, this finding uncovers a mechanism that has not been described previously and provides a new insight into N forms regulating P nutrition under elevated CO₂, an important part of climate change in the 21st century. This study also helps to determine whether the carbon costs of P efficiency under elevated CO₂ should be prioritized in crop improvement programmes.

Supplementary data

Supplementary data are available at JXB online.

Supplementary Fig. S1. Effect of elevated CO₂ and N form on shoot and root fresh biomass production, P concentration in whole plants, and amount of P absorbed per weight of roots.

Supplementary Fig. S2. Effect of elevated CO₂ and N form on chlorophyll content (SPAD readings) of wild-type Arabidopsis grown in P-deficient (0.5 µM) (a) and P-adequate (0.5mM) (b) nutrition solutions containing NO₃ ⁻ or NH₄ ⁺.

Supplementary Fig. S3. Effect of elevated CO₂ and N form on anthocyanin content of wild-type Arabidopsis grown in P-deficient (0.5 µM) (a) and P-adequate (0.5mM) (b) nutrition solutions containing NO₃ ⁻ or NH₄ ⁺.

Glossary

Abbreviations:

APase

acid phosphatase

cPTIO

2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide

DAF-FM DA

diaminofluorescein-FM diacetate

N

nitrogen

NO

nitric oxide

NOS

nitric oxide synthase

NR

nitrate reductase

P

phosphorus

SNP

sodium nitroprusside

AtPHR1

PHOSPHATE STARVATION RESPONSE 1

AtPHT1

PHOSPHATE TRANSPORTER 1

AtPHO1

PHOSPHATE 1

AtPHO2

PHOSPHATE 2. AtPAP2, PURPLE ACID PHOSPHATASE 2