Elevated atmospheric CO2 concentration alters the

Tree Physiology 26, 25–33 © 2005 Heron Publishing—Victoria, Canada

Elevated atmospheric CO2 concentration alters the effect of phosphate supply on growth of Japanese red pine (Pinus densiflora) seedlings SATOSHI KOGAWARA,1,2 MARIKO NORISADA,3 TAKESHI TANGE,1 HISAYOSHI YAGI 4 and KATSUMI KOJIMA3 1

Graduate School of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan

2

Corresponding author ([email protected])

3

Asian Natural Environmental Science Center, The University of Tokyo, Tokyo 113-8657, Japan

4

Faculty of Bioresources, Mie University, Mie 514-8507, Japan

Received August 19, 2004; accepted April 1, 2005; published online October 3, 2005

Keywords: ectomycorrhiza, ergosterol, photosynthesis, Pi requirement, root exudates.

Introduction Plants often experience inorganic phosphate (Pi ) deficiency in the field because a large amount of phosphorus (P) in soil is

unavailable, fixed in the form of metal or organic matter complexes (Dalal 1977, Harrison 1983). A deficiency in Pi decreases plant growth and alters carbon allocation (Rao and Terry 1989, Ciereszko et al. 1996). Although Pi deficiency reduces the photosynthetic rate (Brooks 1986, Rao and Terry 1989, Jacob and Lawlor 1992), growth inhibition in Pi deficient plants is primarily the result of a decreased ability to use carbohydrates rather than a decline in carbohydrate availability. This is because Pi deficiency induces starch accumulation in immature leaves (Fredeen et al. 1989) as well as other heterotrophic organs, such as roots (Paul and Stitt 1993) and stem (Qiu and Israel 1992). The inclusion of sucrose in nutrient solution enhanced biomass increment in Pi-sufficient but not Pi-deficient tobacco plants (Paul and Stitt 1993), indicating that as carbohydrate availability increases, a greater Pi supply is needed to enhance the plant’s ability to use the carbohydrates. It follows that the Pi requirement of a plant grown at an elevated CO2 concentration ([CO2]) will be higher than in ambient [CO2], because photosynthetic rate increases in response to [CO2], especially in C3 plants. Ectomycorrhizal associations of many tree species play an important role in soil Pi uptake (Cumming 1993, Casarin et al. 2004). The significance of ectomycorrhizal associations for host tree Pi uptake depends on soil Pi availability (Jones et al. 1990, Ekblad et al. 1995, Lewis and Strain 1996). The capacity of mycorrhizal mycelium to improve Pi uptake is thought to depend on its ability to extract Pi from P-metal complexes or from organic P by release of organic compounds such as organic acids (Wallander 2000, Casarin et al. 2004) or acid phosphatases (Kroehler et al. 1988). More root exudates have been observed in ectomycorrhizal pine seedlings than in nonmycorrhizal pine seedlings (Leyval and Berthelin 1993). The contribution of the ectomycorrhizal association depends on the extent to which the association develops. It has been reported that development of mycorrhizal associations is enhanced in response to low soil Pi availability (Jones et al. 1990, Koide 1991, Ekblad et al. 1995), which can be attributed to in-

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Summary We demonstrated that the inorganic phosphate (Pi ) requirement for growth of Japanese red pine (Pinus densiflora Sieb. & Zucc.) seedlings is increased by elevated CO2 concentration ([CO2]) and that responses of the ectomycorrhizal fungus Pisolithus tinctorius (Pers.) Coker & Couch to Pi supply are also altered. To investigate the growth response of non-mycorrhizal seedlings to Pi supply in elevated [CO2], non-mycorrhizal seedlings were grown for 73 days in ambient or elevated [CO2] (350 or 700 µmol mol – 1) with nutrient solutions containing one of seven phosphate concentrations (0, 0.02, 0.04, 0.06, 0.08, 0.10 and 0.20 mM). In ambient [CO2], the growth response to Pi was saturated at about 0.1 mM Pi, whereas in elevated [CO2], the growth response to Pi supply did not saturate, even at the highest Pi supply (0.2 mM), indicating that the Pi requirement is higher in elevated [CO2] than in ambient [CO2]. The increased requirement was due mainly to an altered shoot growth response to Pi supply. The enhanced Pi requirement in elevated [CO2] was not associated with a change in photosynthetic response to Pi or a change in leaf phosphorus (P) status. We investigated the effect of Pi supply (0.04, 0.08 and 0.20 mM) on the ectomycorrhizal fungus P. tinctorius in mycorrhizal seedlings grown in ambient or elevated [CO2]. Root ergosterol concentration (an indicator of fungal biomass) decreased with increasing Pi supply in ambient [CO2], but the decrease was far less in elevated [CO2]. In ambient [CO2] the ratio of extramatrical mycelium to root biomass decreased with increasing Pi supply but did not change in elevated [CO2]. We conclude that, because elevated [CO2] increased the Pi requirement for shoot growth, the significance of the ectomycorrhizal association was also increased in elevated [CO2].

26

KOGAWARA, NORISADA, TANGE, YAGI AND KOJIMA

creased allocation of photosynthate to roots (Thomson et al. 1986, Peng et al. 1993, Graham et al. 1997). We hypothesized that elevated [CO2] increases the Pi requirements of Japanese red pine (Pinus densiflora Sieb. & Zucc.) seedlings. To test this hypothesis, we examined the effects of elevated [CO2] on growth responses to increased Pi supply in non-mycorrhizal pine seedlings. In a separate experiment, we evaluated the effects of Pi supply on ectomycorrhizal biomass and organic carbon exuded from the roots of Japanese red pine seedlings inoculated with Pisolithus tinctorius (Pers.) Coker & Couch mycorrhizae and grown in ambient or elevated [CO2]. We discuss the significance of ectomycorrhizal associations under predicted elevated atmospheric [CO2] conditions. Materials and methods

Growth measurements At 73 DAT, seedlings were harvested at the end of the dark period and separated into leaves, stems and roots. Each sample was immediately dried at 80 °C for 48 h and then weighed. To assess differences in growth response to Pi supply between CO2 treatments, we compared relative values of biomass increment calculated as follows. The biomass increment that resulted from the Pi supply was determined by subtracting the mean biomass of the 0 mM Pi seedlings from the value of each seedling. This biomass increment was then converted to a value relative to the mean value of the biomass increment of seedlings at 0.2 mM Pi. The growth responses of whole plants, shoots and roots were examined by plotting the mean values of relative biomass increment against supplied Pi concentration. The following logistic regression equation was fitted to these relationships: y = bo −

Growth response of non-mycorrhizal seedlings to Pi supply

Gas exchange measurements At 72 DAT, the maximum rate of photosynthesis (Amax) under CO2- and light-saturated conditions was measured on whole leaves from each seedling at 1500 µmol mol – 1 CO2, 75% relative humidity and 25 °C, with a portable photosynthesis system (LI-6400, Li-Cor, Lincoln, NE) equipped with a conifer chamber (Li-Cor, LI-6400-05). Light was supplied by an LED light system (PLS2, Hansatech, Kings Lynn, Norfolk, U.K.) at a PPF of 1000 µmol m – 2 s – 1.

(1)

2

where y is relative biomass increment, x is the Pi concentration of the nutrient solution, and b0, b1, b2 are coefficients. Coefficient b0 provides an estimate of the asymptote, equivalent to the maximum biomass increment, b1 describes the shape of the relationship and governs the rate at which y changes with x and b2 is the Pi concentration at which the relative biomass increment is half the maximum increment, b0. Equation 1 was also used to compare Amax responses to Pi supply. Phosphorus, sugar, and starch contents of leaves The dried leaves from each seedling were ground with a mortar and pestle. For measurements of phosphorus concentration, 10 mg of ground sample were digested with a 5:3 (v/v) mixture of nitric acid and perchloric acid. The phosphorus concentration in the solution was spectrophotometrically determined by the vanadate–molybdate method (U-2000, Hitachi, Tokyo, Japan). Soluble sugars were extracted from 10 mg of ground sample with 0.6 ml of 80% (v/v) ethanol at 80 °C. The samples were centrifuged and the extraction repeated twice. The pooled supernatants (1.2 ml) were evaporated to dryness and the solids were redissolved in 600 µl of H2O. To remove protein, 100 µl of 0.3 N Ba(OH)2 and 100 µl of 5% (v/v) ZnSO4 were added to the solution. After reaction with 0.2% (v/v) anthrone in H2SO4, glucose equivalents in the solution were quantified spectrophotometrically at a wavelength of 620 nm with glucose as standard (U-2000, Hitachi). The ethanol-insoluble pellet was used for starch determinations. Starch was hydrolyzed with 0.25 ml of 8.14 N perchloric acid at 60 °C and the derived glucose was assayed as described previously. Responses of the mycorrhizal association to Pi supply Ectomycorrhizal colonization and growth conditions Each 6-month-old pine seedling infected with P. tinctorius was planted at the center of a plastic pot, then half-sib pine seeds were sown around it. Non-mycorrhizal seedlings were prepared for the comparison of organic carbon from root exudates in the same way except that the uninfected pine seedling was

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Plant materials and growth conditions Half-sib seeds of Pinus densiflora obtained from the Forest Tree Breeding Center (Ibaraki, Japan) were immersed in 0.05% benomyl hydrolyzate for 1 h, washed under running tap water for 4 h, and chilled at 4 °C for 1 week. Five seeds were sown per pot (diameter, 6 cm; height, 22 cm). Each pot was filled to 20 cm with quartz sand, which had been leached with 1 N HCl, flushed with distilled water, and sterilized in an oven at 180 °C for 30 h. Five days after germinations, seedlings were thinned to one of a uniform size per pot. Treatments were begun seven days after germination. The plants were grown in two growth chambers (Koitotron HNM-G2, Koito Industries, Tokyo, Japan; vol., 0.51 m3) at a relative humidity of 75%, a temperature of 25 °C and a photosynthetic photon flux (PPF) of 200 µmol m – 2 s – 1 during a 14-h photoperiod. Air that had been passed through soda lime to remove CO2 was injected at 3 m3 h – 1. The [CO2] in the chambers was controlled at 350 (ambient) or 700 µmol mol – 1 (elevated) by CO2 injection into the chambers. During the experiment, all pots were immersed in nutrient solutions (2 mM NH4NO3, 0.6 mM KCl, 0.35 mM CaCl2, 0.25 mM MgSO4, 10 µM FeSO4, 2 µM MnCl2, 2 µM ZnSO4, 2 µM CuSO4 and 10 µM H3BO4 with pH adjusted to 5.8 with HCl) for 5 min every 3 days. Seven concentrations of phosphate (as NaH2PO4; 0, 0.02, 0.04, 0.06, 0.08, 0.10, and 0.20 mM) were provided in the nutrient solutions. There were 11 plants per Pi treatment. Pots were rotated within each chamber every 3 days, and the chambers were switched every 6 days to eliminate chamber effects. Seedlings were grown for 73 days after treatments (DAT) began.

bo 1 + ( bx ) b 1

GROWTH RESPONSES TO P i SUPPLY IN ELEVATED [CO 2]

27

planted at the center of each pot. After 3 months of culture, each seedling was weighed and transplanted to a 50-ml plastic tube filled with sterilized quartz sand. The initial dry mass (DM) of each seedling was estimated from its initial fresh mass based on a DM conversion ratio calculated for another 10 seedlings. Seedlings were grown in two growth chambers for 93 days at one of three Pi concentrations (0.04, 0.08, and 0.20 mM) in either ambient or elevated [CO2], as in the first experiment. Relative humidity, temperature, and light regime were similar to those of the first experiment. All the tubes were watered to saturation with nutrient solution once a week until 28 DAT and then every 3 days thereafter. At the end of the experiment, each seedling was harvested and immediately lyophilized. Dry masses of lyophilized roots and shoots were measured. Three to four mycorrhizal and seven non-mycorrhizal seedlings were prepared per treatment.

tration and specific carbon loss from the roots and root biomass ratio in the second experiment. Mean values of leaf sugar and starch concentrations, root ergosterol concentration, extramatrical mycelium biomass ratio, specific carbon loss from roots and root biomass ratio were compared between Pi treatments within each CO2 treatment by the Scheffé’s test. Growth and photosynthetic responses to Pi supply were analyzed by fitting response curves with nonlinear least-squares regression models of the Statistica software (StatSoft, Version 5.1J, Tulsa, OK). Effects of Pi on leaf P concentration were assessed by linear regressions. Differences in regression coefficients between CO2 treatments were determined by t tests.

Ergosterol assay Ergosterol concentration, which is an indicator of the quantity of fungal biomass, was determined by the method of Wallander and Nylund (1991). The lyophilized root samples (about 50 mg) from mycorrhizal seedlings were ground with a mortar and pestle, and ergosterol was extracted twice in 6 ml of 95% ethanol. To assess the biomass of extramatrical mycelium in the sand, we extracted ergosterol from 30 g of sand with 50 ml of 95% ethanol at room temperature, shaking in a rotary shaker overnight. After saponification of the extracts, ergosterol content was quantified with an HPLC system (LC-10, Shimadzu, Kyoto, Japan) equipped with a reverse-phase column (Shim-pack CLC-ODS [160 × 8 mm], Shimadzu). The eluent was 100% methanol at a flow rate of 1.6 ml min – 1. The ergosterol peak was detected with an ultraviolet detector (SPD-6A, Shimadzu). The ergosterol concentra1 of P. tinctorius mycelium cultured tion, expressed as µg mg −DM on cellophane on agar, was calculated from five replicates and 1 for was used as a standard. The calculated value of 4.0 µg mg −DM this standard was used to estimate fungal biomass from the ergosterol content of extramatrical mycelium in the sand.

Growth and photosynthesis of non-mycorrhizal seedlings

Statistical analysis The experimental design was a split-plot 2 × 7 factorial for the first experiment and a split plot 2 × 3 × 2 factorial for the second experiment. Two CO2 treatments were considered the main-factor and the seven or three Pi treatments and two mycorrhizal treatments were the sub-factors. A three-way analysis of variance (ANOVA) was used to test for the CO2, Pi and mycorrhizal treatment effects on plant biomass, P concen-

Biomass increased with increasing Pi supply from 0 to 0.20 mM (Figure 1A– C). The responses to Pi supply were described satisfactorily by logistic regression (r 2 > 0.93; Figure 1A–1C, Table 1). In both CO2 treatments, Amax increased with increasing Pi supply and these responses were also satisfactorily fitted to logistic regression curves (r 2 > 0.89; Figure 1D, Tables 1 and 2). Elevated [CO2] affected the biomass responses of whole plants, shoots and roots to Pi supply. For the whole-plant growth response, the value of b2, the Pi concentration at which half the maximum biomass increment was attained, was higher in elevated [CO2] than in ambient [CO2] (0.093 versus 0.047 mM; Table 1). The value of b2 for shoot growth increased from 0.050 mM in ambient [CO2] to 0.179 mM in elevated [CO2] (Table 1), whereas elevated [CO2] increased the value of b2 for root growth only slightly, from 0.046 to 0.058 mM (Table 1). Values of b2 for Amax were unaffected by elevated [CO2] (0.028 mM in ambient [CO2] and 0.025 mM in elevated [CO2]) and were lower than those for growth of the whole plant in both CO2 treatments (Table 1). Leaf P, sugar and starch concentrations Leaf P concentration increased with increasing Pi supply (Figure 2), but the regression coefficients for responses of leaf P concentration to Pi supply did not differ significantly between CO2 treatments (t = 0.56, P = 0.58). Leaf soluble sugar concentrations were unchanged by increased Pi supply in either CO2 treatment except relative to the 0 mM Pi treatment in ambient [CO2] (Figure 3A). In both CO2 treatments, leaf starch concentration was nearly 30% of leaf DM at 0 mM Pi, but decreased with increasing Pi supply (Figure 3B). Carbon allocation to mycorrhizae in mycorrhizal pine seedlings Irrespective of Pi treatment, neither growth nor leaf P concentration was affected by mycorrhizal colonization (Table 3). Root ergosterol concentration of mycorrhizal seedlings decreased with increasing Pi supply in both CO2 treatments, but

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Quantification of total organic carbon from root exudates During the experimental period, leachate from each tube was collected at each watering and analyzed for organic carbon content with a total organic carbon analyzer (TOC-5000A, Shimadzu). At the end of the experiment, organic carbon remaining in the growth medium was extracted with pure water by shaking in a rotary shaker for 30 min and then quantified. Specific carbon loss from roots was estimated as the ratio of total exuded organic carbon to the increment of whole-plant biomass.

Results

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KOGAWARA, NORISADA, TANGE, YAGI AND KOJIMA Table 1. Values of the coefficients of the equation fitted to the growth and photosynthetic responses to inorganic phosphorus (Pi ) supply. b The equation is: ( y = b0 − b0 / (1 + ( x / b2 ) 1 ) , where y is relative biomass or maximum rate of photosynthesis (Amax ) increment and x is the Pi concentration of the nutrient solution. Parameter b0 provides an estimate of the asymptote, b1 describes the shape of the relationship and governs the rate at which y changes with x, and b2 is the Pi concentration at which the relative biomass increment is half of the maximum biomass or Amax. Abbreviation: [CO2] = carbon dioxide concentration. [CO2]

b0

b1

b2

r2

Wholeplant

Ambient Elevated

0.914 1.309

4.451 1.657

0.047 0.093

0.96 0.95

Shoot

Ambient Elevated

0.985 1.899

2.436 1.290

0.050 0.179

0.93 0.95

Root

Ambient Elevated

0.935 1.073

6.949 3.127

0.046 0.058

0.99 0.93

Amax

Ambient Elevated

1.147 1.175

2.315 3.046

0.028 0.025

0.89 0.90

Table 2. Effects of carbon dioxide and inorganic phosphorus concentrations ([CO2] and [Pi], respectively) on the maximum rate of photosynthesis (Amax ) at 72 days after germination. A two-way ANOVA was used to test each effect. Values are means (n = 11) ± standard deviations. Double asterisks (**) denote P < 0.01 (ANOVA). [CO2]

[Pi] (mM)

Amax (µmol g – 1 s – 1)

Ambient

0 0.02 0.04 0.06 0.08 0.10 0.20 0 0.02 0.04 0.06 0.08 0.10 0.20

0.51 ± 0.11 0.67 ± 0.14 0.85 ± 0.12 0.93 ± 0.14 0.90 ± 0.28 1.12 ± 0.35 0.95 ± 0.15 0.37 ± 0.09 0.52 ± 0.11 0.64 ± 0.10 0.83 ± 0.09 0.81 ± 0.11 0.75 ± 0.11 0.72 ± 0.09

Elevated

Figure 1. Growth and photosynthetic responses to inorganic phosphorus (Pi ) supply. Values are means (n = 11) with standard errors of relative whole-plant biomass increment (A), relative shoot biomass increment (B), relative root biomass increment (C) and relative maxium rate of photosynthesis (Amax) increase (D) in response to Pi treatment in ambient and elevated carbon dioxide concentrations ([CO2]). Lines represent the nonlinear regression curves fitted to the response to Pi supply in ambient and elevated [CO2].

gs (mmol g – 1 s – 1) 12 ± 3 12 ± 3 16 ± 6 23 ± 9 24 ± 10 30 ± 17 36 ± 19 11 ± 3 15 ± 4 15 ± 2 21 ± 6 28 ± 7 30 ± 19 39 ± 14

ANOVA results

CO2 Pi CO2 × Pi

df

F value

F value

1 6 6

25.45** 26.95** 2.04

0.49 16.42** 0.32



elevated [CO2] greatly reduced the extent of the decrease (Figure 4A). Root ergosterol concentration at the highest Pi supply (0.20 mM) was reduced to 12% of the value at 0.04 mM Pi in ambient [CO2], but was reduced to only 55% of the value at 0.04 mM Pi in elevated [CO2] (Figure 4A). The ratio of extramatrical mycelium to root biomass, which was calculated


29

Root exudates The total organic carbon exuded from roots was unaffected by Pi supply, mycorrhizal association, or elevated [CO2] (data not shown). Specific carbon loss (expressed as the amount of exudation per plant biomass increment) decreased with increasing Pi supply (P < 0.001), and increased with ectomycorrhizal association (P < 0.001; Table 4), but elevated [CO2] had no significant effect on specific carbon loss (P = 0.07; Table 4). The root biomass to whole plant biomass ratio decreased with increasing Pi supply (P < 0.001; Table 4).

Discussion CO2 enrichment alters growth response to Pi supply

from the ergosterol concentration in the medium, decreased with increasing Pi supply in ambient [CO2] but not in elevated [CO2] (Figure 4B).

Table 3. Effects of carbon dioxide concentration ([CO2]), inorganic phosphorus concentration ([Pi]) and mycorrhizal infection on whole-plant biomass and leaf and root phosphorus (P) concentrations. Three-way ANOVA was used to test each effect. Values are means ± standard deviations of non-mycorrhizal (n = 7) and mycorrhizal (n = 3 to 4) seedlings. Abbreviations: DM = dry mass; M = mycorrhizal seedlings; NM = non-mycorrhizal seedlings. Asterisks indicate significance level: * = P < 0.05; and *** = P < 0.001 (ANOVA). [CO2]

Ambient

Mycorrhizal status

[Pi] (mM)

NM

M

Elevated

NM

M

−1 P concentration (mg g DM )

Biomass −1 ( mg DM )

Leaf

Root

0.04 0.08 0.20 0.04 0.08 0.20

1.16 ± 0.30 1.43 ± 0.23 1.83 ± 0.18 1.02 ± 0.15 1.11 ± 0.27 1.73 ± 0.20

1.10 ± 0.10 1.44 ± 0.09 2.58 ± 0.39 1.23 ± 0.08 1.47 ± 0.18 2.68 ± 0.25

466 ± 83 667 ± 102 836 ± 56 484 ± 53 744 ± 81 771 ± 132

0.04 0.08 0.20 0.04 0.08 0.20

0.77 ± 0.16 0.99 ± 0.21 1.47 ± 0.05 1.03 ± 0.22 1.04 ± 0.15 1.46 ± 0.13

1.09 ± 0.10 1.17 ± 0.14 2.27 ± 0.38 1.16 ± 0.04 1.43 ± 0.14 2.40 ± 0.40

530 ± 45 869 ± 158 1040 ± 180 515 ± 41 732 ± 118 978 ± 136

df

F value

F value

F value

1 2 1 1 2 2 2

11.70* 48.80*** 0.63 7.16* 0.48 1.10 0.88

6.83* 171.09*** 3.67 0.31 1.37 0.05 0.48

8.02* 60.00*** 1.03 1.79 2.35 0.38 1.22

ANOVA results

CO2 Pi Mycorrhizal CO2 × Mycorrhizal CO2 × Pi Mycorrhizal × Pi CO2 × Pi × Mycorrhizal



Figure 2. Effects of carbon dioxide (CO2) treatment on leaf phosphorus (P) concentration. Values are means (n = 11) with standard deviations in ambient and elevated [CO2]. Linear regressions were performed on the responses of leaf P concentrations to inorganic phosphorus (Pi ) supply in ambient and elevated [CO2]. Triple asterisks (***) indicate the regressions differ significantly at P < 0.001.

Elevated [CO2] increased the Pi requirement for growth of Japanese red pine seedlings (Figure 1A, Table 1), indicating that an adequate Pi supply for growth in ambient [CO2] can limit growth in elevated [CO2]. The effects of elevated [CO2] on the Pi growth response curve can be divided into three phases. In the first phase, represented by the treatment lacking Pi (deficient supply), growth was limited in both ambient and elevated [CO2]. At 0.04 mM Pi, growth was not limited in ambient [CO2] but it was inhibited in elevated [CO2] (transiently sufficient scenario). A third phase (sufficient supply) occurred when the Pi supply was saturating for growth in both ambient and elevated [CO2]. Under conditions of a transiently suffi-

30


Figure 4. Effects of inorganic phosphorus (Pi ) treatment on root ergosterol concentration (A) and ratio of ectomycorrhizal mycelium biomass to root biomass (B) in ambient (䉭) and elevated (䊉) carbon dioxide concentrations ([CO2]). Mean values (n = 3 or 4) with standard deviations are shown. Different letters indicate statistically significant differences between Pi treatments in ambient (lowercase) and elevated (uppercase) [CO2] at P = 0.05 according to Scheffé’s test. The ectomycorrhizal mycelium biomass to root biomass ratios in elevated [CO2] (B) were not significantly different between treatments.

cient supply (Phase 2), trees growing in elevated [CO2] exhibited symptoms of Pi deficiency even though growth was enhanced compared with ambient CO2 conditions. Examination of nutrient growth response curves in elevated [CO2] can also be used to identify when nutrients other than Pi reach the threshold of transiently sufficient supply. At a transiently sufficient supply, seedling growth could become more dependent on the nutrient characteristic when the atmospheric [CO2] is elevated. Species differ in their ability to overcome or ameliorate nutrient deficiency, and the extent to which nutrient requirements change in response to elevated [CO2] could also differ among species and developmental stages. Thus, elevated [CO2] has the potential to alter site–species relationships.

Induction of Pi deficiency by elevated [CO2] was more pronounced in shoots than in roots (Figures 1B and 1C). The b2 value for root growth, which represents the Pi concentration required to attain half the maximum biomass increment, changed only slightly in response to elevated [CO2] (Figure 1C, Table 1), whereas b2 for shoot growth was affected by elevated [CO2] (Figure 1B, Table 1), indicating that the change in Pi requirements for whole-plant growth in response to elevated [CO2] depended mainly on the change in shoot growth response. Although Pi deficiency reduces plant growth rate (Rao and Terry 1989, Ciereszko et al. 1996) and leaf expansion rate (Radin and Eidenbock 1986, Fredeen et al. 1989), root growth is often less affected (Khamis et al. 1990, Benbrahim et al.



Figure 3. Effects of inorganic phosphorus (Pi ) treatment on leaf soluble sugar concentration (A) and starch concentration (B) in ambient (䉭) and elevated (䊉) carbon dioxide concentrations ([CO2]). Mean values (n = 11) with standard deviations are shown. Different letters indicate statistically significant differences between Pi treatments in ambient (lowercase) and elevated (uppercase) [CO2] at P = 0.05 according to Scheffé’s test. Sugar concentrations in elevated [CO2] (A) were not significantly different between Pi treatments.


31

Table 4. Effects of carbon dioxide concentration ([CO2]), inorganic phosphorus concentration ([Pi]) and mycorrhizal infection on specific carbon loss from roots and root biomass ratio. Values are means and standard deviations of non-mycorrhizal (n = 7) and mycorrhizal (n = 3 to 4) seedlings. Abbreviations: DM = dry mass; M = mycorrhizal seedlings; and NM = non-mycorrhizal seedlings. Means within a column followed by the same letter are not significantly different by the Scheffé’s test at P = 0.05. Three-way ANOVA was used to test each effect, with ** = P < 0.01, and *** = P < 0.001. Mycorrhizal status

[Pi] (mM)

Specific carbon loss (carbon % DM plant increment)

Root biomass ratio (% DM whole plant biomass)

Ambient

NM

0.04 0.08 0.20 0.04 0.08 0.20

2.3 ± 0.5 abc 1.6 ± 0.4 abc 1.5 ± 0.5 b 3.4 ± 0.6 a 2.2 ± 0.9 abc 1.6 ± 0.5 abc

42 ± 5 a 33 ± 5 abc 26 ± 3 c 38 ± 1 abc 32 ± 6 abc 27 ± 1 c

0.04 0.08 0.20 0.04 0.08 0.20

3.0 ± 1.0 ab 1.7 ± 0.3 abc 1.4 ± 0.3 c 3.6 ± 0.9 a 2.6 ± 0.9 abc 2.2 ± 1.3 abc

41 ± 4 ab 36 ± 5 abc 29 ± 5 bc 41 ± 3 abc 39 ± 4 abc 31 ± 7 abc

df

F value

F value

1 2 1 1 2 2 2

5.23 23.89 *** 16.36 *** 0.17 0.04 0.18 0.97

6.25 34.58 ** 0.00 1.42 1.73 0.80 0.12

M

Elevated

NM

M

ANOVA results

CO2 Pi Mycorrhizal CO2 × Mycorrhizal CO2 × Pi Mycorrhizal × Pi CO2 × Pi × Mycorrhizal

1996). These reports support our finding that shoots are more sensitive to Pi deficiency than roots. Although the mechanism responsible for this phenomenon has not been clarified, increased carbon allocation to roots under Pi-deficient conditions (Sicher and Kremer 1988, Ciereszko et al. 1996) could account for the negligible change in root Pi requirements in response to elevated [CO2] (Figure 1C, Table 1). The effect of elevated [CO2] on shoot growth response to Pi supply cannot be attributed to a difference in internal P status, because leaf P concentrations were unaffected by CO2 treatment (Figure 2). This result contrasts with the result of the experiment on mycorrhizal association (Table 3) and with other studies showing that a decrease in leaf P concentration occurs in response to elevated [CO2] (Porter and Grodzinski 1984, Kuehny et al. 1991, Duchein et al. 1993). It has been observed that elevated [CO2] decreases the ratio of orthophosphate (free Pi ) to esterified phosphate in leaves (Morin et al. 1992). If a similar response occurred in our Japanese red pine leaves, elevated [CO2] could have affected Pi requirements through a decrease in free Pi content. Photosynthetic response to Pi supply in elevated CO2 Photosynthesis is generally limited by Rubisco activity or the capacity to regenerate ribulose 1,5-bisphosphate (RuBP, Farquhar et al. 1980). Compared with Pi-sufficient plants, it has

been reported that Rubisco activity is less repressed (Brooks 1986, Rao and Terry 1989, Nielsen et al. 1998) and photosynthesis is not down-regulated in Pi-deficient plants growing in elevated [CO2], which contrasts with the response of nitrogen-deficient plants to elevated [CO2] (Riviere-Rolland et al. 1996). However, the capacity for RuBP regeneration is reported to be repressed in Pi-deficient plants (Sharkey 1985, Lewis et al. 1994), manifested as a decrease in Amax (Sharkey 1985). In our study, however, Amax responses to Pi supply were unaffected by elevated [CO2] (Figure 1D, Tables 1 and 2), indicating that the limitation on the capacity for RuBP regeneration by Pi deficiency was unaffected by elevated [CO2]. Because b2 for Amax was unchanged by elevated [CO2] and it was much lower than the b2 value for whole-plant growth in both ambient and elevated [CO2] (Table 1), the change in Pi requirement for growth caused by elevated [CO2] could not have been mediated by the changes in the Pi requirement for photosynthesis. Carbon allocation to ectomycorrhizae In ambient [CO2], ectomycorrhizal colonization was decreased by a Pi supply > 0.04 mM (Figure 4A), a finding that is consistent with other studies (Jones et al. 1990, Koide 1991). In elevated [CO2], however, some ectomycorrhizal colonization was observed even at the highest Pi supply (0.20 mM; Fig-



[CO2]

32


Carbon loss from roots under elevated CO2 Studies with 14C-labeled CO2 have shown that increased allocation of photosynthate to fine root biomass and the mycorrhizal symbiont (Norby et al. 1987), or to root exudates and root respiration (Rouhier et al. 1996) in response to elevated [CO2], result in increased total carbon input to soils. However, such effects have not been consistently observed every sampling time (Norby et al. 1987, Rouhier et al. 1996), raising the possibility of transient effects of elevated [CO2] on root exudates. Most analyses on allocation of 14C-labeled CO2 have focused on allocation of recently formed photosynthate. From the viewpoint of carbon loss, we evaluated the ratio of cumulative root exudates to plant biomass increment throughout the experiment. We found elevated [CO2] had no effect on specific carbon loss from roots (Table 4), indicating that the carbon cost imposed by root exudation is unaffected by long-term exposure to elevated [CO2]. Increasing the Pi supply reduced spe-

cific carbon loss (Table 4), implying a decrease in the root biomass to whole-plant biomass ratio with increasing Pi supply (Table 4). Conclusions Elevated [CO2] increased the Pi requirement for maximum growth of Japanese red pine seedlings, especially in shoots, indicating that a Pi supply that is adequate for pine seedlings under the current atmospheric [CO2] will not necessarily permit this species to realize its full potential shoot growth in elevated [CO2] conditions. Because Pi availability varies widely in the field, an increased Pi requirement in elevated [CO2] must be taken into account when predicting the responses of forest trees to elevated [CO2]. Much soil P exists in fixed forms as organic or inorganic compounds, indicating that the observed enhanced development of ectomycorrhizal associations in response to elevated [CO2] may play an increasingly important role in increasing Pi acquisition from such Pi compounds to support Japanese red pine growth under conditions of elevated [CO2]. Acknowledgments We thank Prof. K. Suzuki (University of Tokyo) for providing the P. tinctorius isolate and the Forest Tree Breeding Center for providing the P. densiflora seeds. This work was supported in part by a grant from the Program for Bio-oriented Technology Research Advancement Institution. References Benbrahim, M., D. Loustau, J.P. Gaudillère and E. Saur. 1996. Effects of phosphate deficiency on photosynthesis and accumulation of starch and soluble sugars in 1-year-old seedlings of maritime pine (Pinus pinaster Ait.). Ann. Sci. For. 53:801–810. Brooks, A. 1986. Effects of phosphorus nutrition on ribulose-1,5bisphosphate carboxylase activation, photosynthetic quantum yield and amounts of some Calvin-cycle metabolites in spinach leaves. Aust. J. Plant Physiol. 13:221–237. Buwalda, J.G. and K.M. Goh. 1982. Host-fungus competition for carbon as a cause of growth depressions in vesicular-arbuscular mycorrhizal ryegrass. Soil Biol. Biochm. 14:103–106. Casarin, V., C. Plassard, P. Hinsinger and J.C. Arvieu. 2004. Quantification of ectomycorrhizal fungal effects on the bioavailability and mobilization of soil Pi in the rhizosphere of Pinus pinaster. New Phytol. 163:177–185. Ciereszko, I., A. Gniazdowska, M. Mikulska and A.M. Rychter. 1996. Assimilate translocation in bean plants (Phaseolus vulgaris L.) during phosphate deficiency. J. Plant Physiol. 149:343–348. Cumming, J.R. 1993. Growth and nutrition of nonmycorrhizal and mycorrhizal pitch pine (Pinus rigida) seedlings under phosphorus limitation. Tree Physiol. 13:173–187. Dalal, R.C. 1977. Soil organic phosphorus. Adv. Agron. 29:83–117. Duchein, M-C., A. Bonicel and T. Betscher. 1993. Photosynthetic net CO2 uptake and leaf phosphate concentrations in CO2 enriched clover (Trifolium subterraneum L.) at three levels of phosphate nutrition. J. Exp. Bot. 44:17–22. Ekblad, A., H. Wallander, R. Carlsson and K. Huss-Danell. 1995. Fungal biomass in roots and extramatrical mycelium in relation to macronutrients and plant biomass of ectomycorrhizal Pinus sylvestris and Alnus incana. New Phytol. 131:443–451.



ure 4A). Ectomycorrhizal colonization at 0.2 mM Pi in elevated [CO2] might be associated with the Pi deficiency induced by elevated [CO2], because several studies have shown elevated [CO2] has a positive effect on ectomycorrhizal colonization when soil nutrient availability is low (Norby et al. 1986, O’Neill et al. 1987). Carbon allocation to extramatrical mycelium declined with increasing Pi supply and this effect was more pronounced in roots of plants grown in ambient [CO2] than in elevated [CO2] (Figure 4B). Reduced growth as a result of arbuscular mycorrhizal colonization at high P supply has been observed (Buwalda and Goh 1982, Peng et al. 1993, Graham and Eissenstat 1998) and explained on the basis of the high carbon cost of mycorrhizae formation in the absence of the positive effects of increased P uptake on photosynthesis and growth (Jifon et al. 2002). In contrast, ectomycorrhizal association did not enhance Pi uptake or depress growth in seedlings in any of our Pi treatments (Table 3). Wright et al. (1998) reported that increased carbon allocation to roots colonized by arbuscular mycorrhiza is associated with stimulation of sucrolytic enzyme activity; however, in other studies, this enzyme activity did not increase in roots colonized by ectomycorrhiza (Salzer and Hager 1993, Schaeffer et al. 1995). Wright et al. (2000) speculated that the difference in the effects of arbuscular and ectomycorrhizal colonization on sucrolytic enzyme activity was correlated with a difference in sink strength. Our finding that mycorrhizal association did not depress growth might be associated with the low carbon cost of P. tinctorius, whereas the absence of a stimulatory effect of mycorrhizal colonization on Pi uptake can be explained by the application of inorganic soluble Pi as the Pi source. It is known that ectomycorrhizal fungi can take up Pi from insoluble organic or inorganic P-compounds (Kroehler et al. 1988, Wallander 2000, Casarin et al. 2004). Retention of the ectomycorrhizal association in the 0.2 mM Pi treatment in elevated [CO2] (Figure 4) indicates that growth in elevated [CO2] could be affected by increased ectomycorrhizal colonization in the field where a large amount of P is fixed in forms unavailable to host plants.


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