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

Phosphorus (P) nutrition is always a key issue regarding plants responses to elevated CO(2). Yet it is unclear of how elevated CO(2) affects P uptake under different nitrogen (N) forms. This study investigated the influence of elevated CO(2) (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 (NO(3)(-)) or ammonium (NH(4)(+)). After 7 d treatment, elevated CO(2) enhanced the biomass production of both NO(3)(-)- and NH(4) (+)-fed plants but decreased the P amount absorbed per weight of roots and the P concentration in the shoots of plants supplied with NH(4)(+). In comparison, elevated CO(2) 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 NO(3)(-). Elevated CO(2) 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 CO(2) increased the nitric oxide (NO) level in roots of NO(3)(-)-fed plants but decreased it in NH(4)(+)-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 CO(2). Considering all of these findings, this study concluded that a combination of elevated CO(2) and NO(3)(-) 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 n class="Chemical">CO2 have risenpan> from about 270 µl l–1 inpan> pre-inpan>dustrial times to over 380 µl l–1 at presenpan>t, anpan>d are predicted to reach betweenpan> 530 anpan>d 970 µl l–1 by the enpan>d of this cenpan>tury (IPCC, 2007). It has beenpan> demonpan>strated that inpan>creasinpan>g atmospheric pan> class="Chemical">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 ; Teng ; Jin ; Niu ). 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 ; Drake ). 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.

n class="Chemical">Phosphorus is anpan> essenpan>tial elemenpan>t for planpan>t growth anpan>d developmenpan>t. It plays importanpan>t roles inpan> enpan>ergy tranpan>sfer, signpan>al tranpan>sductionpan>, photosynthesis, enpan>zyme activationpan>/inpan>activationpan>, membranpan>e synthesis anpan>d stability, anpan>d respirationpan>, anpan>d is a structural componpan>enpan>t of nucleic acids anpan>d pan> class="Chemical">phospholipids (Abel ; Vance ; Chiou and Lin, 2011; Péret ). 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 ; 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 ). Hence, low P availability is one of the major growth-limiting factors for plants in many natural and agricultural ecosystems (Barber ; 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 ). 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 ). 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 ; Abel ; Rubio ; Ramaekers ; Péret ; Jin ; Veneklaas ). However, it is unclear how plants sense and respond to P deficiency under conditions of elevated CO2.

n class="Chemical">Nitrogen (N) has beenpan> the cenpan>tre of attenpan>tionpan> not onpan>ly because it is the primary limitinpan>g nutritionpan> inpan> most temperate ecosystems, but also because the form of N supply canpan> modify the responpan>se of planpan>t growth to nutritionpan> acquisitionpan> anpan>d elevated pan> class="Chemical">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 ). 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 , 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 ), but was inhibited by ammonium, glutamine, or related downstream products of nitrate assimilation (Gojon ). Recently, Carlisle 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 n class="Species">Arabidopsis thaliana wild-type ecotype anpan>d mutanpan>ts pan> class="Gene">nr, AT1G77760 and AT1G37130 locus (Wang ) and atnos1, AT3G47450 locus (Guo ), were surface-sterilized, and germinated on Petri plates containing 0.8% agar medium supplemented with one-fifth-strength nutrition solution (Niu ). 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 n class="Chemical">CO2 conpan>cenpan>trationpan> of either 350±50 (ambienpan>t) or 800±50 µl l–1 (elevated pan> class="Chemical">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 n class="Chemical">CO2; (ii) elevated pan> class="Chemical">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 treatmenpan>ts were harvested, washed thoroughly with deionpan>ized 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, Minpan>olta, Japan) was used to measure chlorophyll content (Jin ). 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 n class="Chemical">anthocyanins inpan> leaves were extracted anpan>d determinpan>ed as described by Hodges with minpan>or modificationpan>s. Approximately 0.1g of fresh leaves was homogenpan>ized inpan> 3ml of pan> class="Chemical">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 ).

Root morphology

After 7 d treatment, the root parameters of individual plants were measured using a root analysis instrument (Win class="Gene">nRHIZO, Regenpan>t, Canada). The number and lenpan>gth of root hairs inpan> each segmenpan>t were measured usinpan>g light microscopy with differenpan>tial inpan>terferenpan>ce conpan>trast optics. Micrographs were recorded onpan> a CCD camera (Nikonpan> 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 with minor modifications. After 7 d in low-P treatment, the seedlings were washed thoroughly with deionized n class="Chemical">water, divided inpan>to shoots anpan>d roots, weighed, anpan>d thenpan> chilled immediately onpan> ice. About 0.1–0.2g of fresh samples was frozenpan> with liquid pan> class="Chemical">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 n class="Chemical">diaminofluorescein-FM diacetate (pan> class="Chemical">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 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 n class="Chemical">nitrogen anpan>d grounpan>d to a finpan>e powder. The powder was homogenpan>ized inpan> 1ml of extractionpan> buffer conpan>tainpan>inpan>g 100mM pan> class="Chemical">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 determinpan>ed as described by Tian with some modificationpan>s. Briefly, a total of 0.2ml of clear supernpan>atant prepared as described above was added inpan>to 0.4ml of pre-warmed assay buffer conpan>tainpan>inpan>g 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 ). The mRNA levels of the genes n class="Chemical">PHOSPHATE STARVATION RESPONSE 1 (pan> class="Gene">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 . A pair of primers for the housekeeping gene n class="Gene">UBQ10 (forward: 5’-GGTTCGTACCTTTGTCCAAGCA-3’, reverse: 5’-CCTTCGTTA AACCAAGCTCAGTATC-3’) was used for a conpan>trol referenpan>ce genpan>e for the PCR (Ganpan> ). Meltinpan>g-curve anpan>alysis anpan>d gel electrophoresis of the PCR products were used to conpan>firm the absenpan>ce of nonpan>-specific amplificationpan> products. Relative expressionpan> levels were calculated by subtractinpan>g the threshold cycle (C t) values for pan> class="Gene">UBQ10 from those of the target gene (to give ΔC t) and then calculating 2−Δ, 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 n class="Chemical">CO2 anpan>d pan> class="Disease">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.

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 n class="Species">Arabidopsis grownpan> for 7 d inpan> pan> class="Disease">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 n class="Chemical">CO2 enpan>hanpan>ced shoot growth exclusively unpan>der NH4 + nutritionpan> (Fig. 1b), whereas it enpan>hanpan>ced root growth unpan>der pan> class="Gene">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 treatmenpan>t did not affect the conpan>cenpan>trationpan> 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 enpan>hanced the number of lateral roots inpan> 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).

Root morphology (a), and root length, number of root tips, root surface area, and root/shoot ratio (b) of 5-week-old wild-type n class="Species">Arabidopsis grownpan> for 7 d inpan> pan> class="Disease">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 n class="Chemical">CO2 treatmenpan>ts (Fig. 3a, b). However, elevated pan> class="Chemical">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.

APase activity in shoots (a) and roots (b) of 5-week-old wild-type n class="Species">Arabidopsis grownpan> for 7 d inpan> pan> class="Disease">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 ; Vance ; Chiou and Lin, 2011). Elevated n class="Chemical">CO2 enpan>hanpan>ced the expressionpan> of the P-uptake genpan>e pan> class="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 n class="Gene">AtPHO1 anpan>d pan> class="Gene">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 n class="Disease">P deficiency (Wanpan>g ). We observed that elevated pan> class="Chemical">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).

NO production of 5-week-old wild-type n class="Species">Arabidopsis grownpan> for 7 d inpan> pan> class="Disease">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.

Effect of SNP under ambient n class="Chemical">CO2 (350±50 µl l–1) (a) anpan>d cPTIO unpan>der elevated pan> class="Chemical">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 onpan> 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).

NR activity inpan> shoots (a) and roots (b) and NOS activity inpan> 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 n class="Chemical">CO2-inpan>duced uptake of P durinpan>g pan> class="Disease">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).

P concentration in shoots (a, c) and roots (b, d) of n class="Species">Arabidopsis pan> class="Gene">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-depenpan>denpan>t stimulationpan> of growth is likely to enpan>hance the requiremenpan>ts of plants for macronpan>utrienpan>ts such as N and P (Baxter ; Kogawara ; Lagomarsinpan>o ). This study demonpan>strated that a combinpan>ationpan> 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 n class="Chemical">CO2 would be associated with inpan>creased requiremenpan>ts for minpan>eral nutritionpan> to optimize the nutritionpan> balanpan>ce inpan> planpan>ts. This was the case inpan> a 6-year Free Air pan> class="Chemical">CO2 Enrichment (FACE) experiment in which photosynthetic stimulation by elevated CO2 was largely regulated by N and P availability in the soil (Lagomarsino ). 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 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 n class="Gene">NO3 −-fed pan> class="Disease">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 ; Shenoy and Kalagudi, 2005; Shulaev ; Wang ). 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 n class="Gene">NO3 −-fed planpan>ts had more lateral roots but a shorter taproot unpan>der elevated compared with ambienpan>t pan> class="Chemical">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 ). 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 ; 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 ; Tran ). 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 ). Similarly, the physiological function of root APases has also been demonstrated by Xiao and Wang where transgenic plants overexpressing APase genes exhibited greater P acquisition from an organic P resource than wild-type plants. Our results showed that elevated n class="Chemical">CO2 inpan>creased APase activity inpan> both the shoots anpan>d roots of pan> class="Species">Arabidopsis plants supplied with NO3 −, which is consistent with results reported in the literature (Lagomarsino ). 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. n class="Gene">PHR1 is a key tranpan>scriptionpan>al activator inpan> conpan>trollinpan>g P uptake, allocationpan>, anpan>d pan> class="Chemical">anthyocyanin accumulation (Rubio ; Nilsson ). 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 ; Jain ). 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 n class="Disease">P homeostasis withinpan> planpan>ts. pan> class="Gene">AtPHO1 was first identified in Arabidopsis as a gene playing an important role in P transport from the roots to the shoots (Poirier ), 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 ; Hamburger ; Stefanovic ). 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 ). 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 n class="Disease">P deficiency signpan>ificanpan>tly inpan>creases the enpan>dogenpan>ous NO conpan>cenpan>trationpan> inpan> roots of white lupinpan> (Wanpan>g ). In this study, we observed that elevated pan> class="Chemical">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 ). These results suggest that NO is partly responsible for NO3 −-facilitated P accumulation in plants.

It is unclear how elevated n class="Chemical">CO2 chanpan>ges the NO levels inpan> roots unpan>der pan> class="Disease">P deficiency. It is recognized that NR and NOS are two potential enzymatic sources of NO in plants (Neill ). Although NOS genes have not yet been identified in plants (Neill ), several studies support the presence of NOS activity in plants (Corpas ; Valderrama ). 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 ; 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 ). 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 ). 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 inpan>creased 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 ; 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 ; Stitt and Krapp, 1999), which supports our findings.

In n class="Species">Arabidopsis, two mutanpan>ts defective inpan> NO productionpan> displayed altered phenpan>otypes. The pan> class="Gene">nr mutant had a deletion of the major NR gene, NIA2, and an insertion in the NIA1 NR gene (Wang ), and the atnos1 mutant has a mutation in an NOS structural gene NOS1 (Guo ). 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 and Niu 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 n class="Species">Arabidopsis planpan>ts, we demonpan>strated that pan> class="Gene">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 n class="Chemical">CO2 and N form onpan> shoot and root fresh biomass productionpan>, P conpan>cenpan>trationpan> inpan> whole plants, and amount of P absorbed per weight of roots.

Supplementary Fig. S2. Effect of elevated CO2 and N form onpan> 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 onpan> 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 +.