Acta Physiologiae Plantarum

, 31:163

Growth and physiological responses of Picea asperata seedlings to elevated temperature and to nitrogen fertilization

Authors

  • Chunzhang Zhao
    • Ecological Restoration and Rehabilitation Center, Chengdu Institute of BiologyChinese Academy of Sciences
    • Graduate University of the Chinese Academy of Sciences
    • Ecological Restoration and Rehabilitation Center, Chengdu Institute of BiologyChinese Academy of Sciences
Original Paper

DOI: 10.1007/s11738-008-0217-8

Cite this article as:
Zhao, C. & Liu, Q. Acta Physiol Plant (2009) 31: 163. doi:10.1007/s11738-008-0217-8

Abstract

Picea asperata is a dominant species in the subalpine coniferous forests distributed in eastern edges of Tibetan Plateau and upper reaches of the Yangtze River. The paper mainly identified the short-term influences of experimental warming, nitrogen fertilization, and their combination on growth and physiological performances of Picea asperata seedlings. These seedlings were subjected to two levels of temperature (ambient; infrared heater warming) and two nitrogen levels (0; 25 g m−2 a−1 N) for 6 months. We used a free air temperature increase of overhead infrared heater to raise both air and soil temperature by 2.1 and 2.6°C, respectively. The temperature increment induced an obvious enhancement in biomass accumulation and the maximum net photosynthetic rate, and decreased AOS and MDA level under ambient nitrogen conditions. Whereas, negative effects of experimental warming on growth and physiology was observed under nitrogen fertilization condition. On the other hand, nitrogen fertilization significantly improved plant growth in unwarmed plots, by stimulating total biomass, maximum net photosynthetic rate (Amax), antioxidant compounds, as well as reducing the content of AOS and MDA. However, in warmed plots, nitrogen addition clearly decreased Amax, antioxidant compounds, and induced higher accumulation of AOS and MDA. Obviously, the beneficial effects of sole nitrogen on growth and physiology of Picea asperata seedlings could not be magnified by artificial warming.

Keywords

Picea asperataWarmingNitrogenGrowthPhysiological performance

Abbreviations

AOS

Active oxygen species

APX

Ascorbate peroxidase

ASA

Ascorbic acid

CAT

Catalase

H2O2

Hydrogen peroxide

LMR

Leaf mass ratio

MDA

Malondialdehyde

MSI

Membrane stability index

O2

Superoxide radical

POD

Peroxidase

RMR

Root mass ratio

SMR

Stem mass ratio

SOD

Superoxide dismutase

UF

Unwarmed fertilized

UU

Unwarmed unfertilized

WF

Warmed fertilized

WU

Warmed unfertilized

Introduction

The mean global surface temperature has increased by about 0.74°C over the past century and is predicted to rise by as much as 6.4°C during this century, because of continued increment in atmospheric concentration of greenhouse gases (IPCC 2007). Thus such projected warming will alter the establishment, survival and reproduction of terrestrial vegetation, resulting in dramatic effects on ecosystem (Loik et al. 2004). And the temperature increment also exercises a great influence on plant growth, on account of its great impact on the regulation of biochemical reaction rates, plant physiological processes and biomass allocation (Starr et al. 2000; Saxe et al. 2001).Besides global warming, nitrogen deposition is another important environment problem due to human activity. The NEGTAP (2001) report shows that atmospheric nitrogen deposition has slightly changed since its peak in 1986, and widespread excess of critical nitrogen load are still affecting the biotic and abiotic environment. As the largest developing country in the world, China consumes large amounts of N fertilizer, animal products and fossil fuels, causing large emissions of N into atmosphere. During the development of intensive agriculture, over-fertilization of agricultural crops has become very common in China, and more than 24 Tg year−1 fertilizer N have been consumed in recent years (China Agricultural Yearbook 2003), accounting for 30% of total fertilizer N used in the world (IFA 2005). The total N deposition at the five IMPACT (Integrated Monitoring Program on Acidification of Chinese Terrestrial Systems) sites in south China ranges from 6 to 44 kg N ha−1 year−1 in 2003, which is in the same range as that observed in Europe and North America (Larssen et al. 2006).

Nitrogen is the mineral nutrient needed in the largest amounts by plants and it is usually also a major limiting factor to plant growth in some temperate coniferous forests (Vitousek and Howarth 1991), so the atmospheric nitrogen deposition usually stimulates plant growth to some extent. According to previous studies, additional nitrogen can also induce a great deal of physiological alterations, such as reducing accumulation of active oxygen species (AOS) and MDA (malondialdehyde), increasing foliar proline content, and affecting photosynthesis (Ramalho et al. 1998; Nakaji et al. 2001; Yao and Liu 2006). Similar to nitrogen, temperature also is one of the most important factors controlling the physiological activity and growth of plants. It is reported that the elevated temperature can promote growth and photosynthesis in all plants as long as their optimal temperatures had not been exceeded (Warren Wilson 1966). Up to now, substantial efforts have been made in order to study the potential impacts of climate changes on terrestrial ecosystems. Previous studies were concerned about the interaction between warming and N-fertilization on soil microarthropod abundances (Sjursen et al. 2005), carbon sequestration (Mäkipää et al. 1999), and root growth (Majdi and Öhrvik 2004). However, the interactive effects of warming and nitrogen on growth and physiology of plants have not been well documented. Substantial amounts of AOS are produced by organisms, and this highly reactive form of oxygen is capable of oxidizing electron-rich substrates of biological importance, such as lipids and proteins to give peroxides or other oxidized products ultimately resulting in serious damage to the tissue. Whereas plants have evolved oxygen-scavenging system consisting of non-enzyme antioxidant metabolites, such as ascorbic acid (ASA) and proline, and antioxidant enzymes (SOD, CAT, POD, and APX) to combat the deleterious effects of AOS (Bowler et al. 1992; Jebara et al. 2005); it is important to understand the possible antioxidant defense responses of plant to future climate warming combined with high N-deposition. On the basis of previous study, we hypothesized that the interaction of artificial warming and nitrogen addition would further improve physiology and growth of plant.

The subalpine coniferous forests distributed in eastern edges of Tibetan Plateau and upper reaches of the Yangtze River, comprise the second largest biome in China, and Picea asperata is a key species in those regions (Liu 2002). The potential influences of climate warming and nitrogen deposition on growth and physiology of P asperata seedlings may be crucial to understand the regeneration behavior of subalpine coniferous forests under future climate changes. The beneficial effects of experimental warming or additional nitrogen on growth and physiology of P asperata seedlings would be magnified by the interaction of both warming and nitrogen.

The specific objectives in this study were to identify the short-term effects of artificial warming, nitrogen fertilization, and their possible interaction on growth and physiological alterations of P. asperata seedlings, such as biomass accumulation and allocation, photosynthesis, and antioxidant responses.

Materials and methods

Plant material and experiment design

The experiment was conducted in open semi-field condition during the growing season from April to October, 2007 in Maoxian Ecological Station of Chinese Academy of Sciences, Sichuan province, China (31° 41 N, 103° 53′E; 1,820 m a.s.l.). Experiment design used a blocked split-plot design with warming as the main factor and nitrogen fertilization nested within temperature manipulations according to Wan’s research (Wan et al. 2002). There were four blocks, and each block contained a pair of 2 × 2 m plots (a warmed plot and a control plot). The warmed plot were continuously heated by a 165 × 15 cm overhead infrared heater (Kalglo Electronics Inc., Bethlehem, PA, USA) suspended 1.5 m above the middle of the plots. The infrared heater had a radiation output of approximately 100 w m−2, and it’s warming effect on soil temperature was spatially uniform in plots according to previous similar study (Wan et al. 2002). The control plot of each pair had a ‘dummy’ heater of the same shape and size as the infrared heater suspended 1.5 m high in order to simulate the shading effects of the heater. The distance between the control plot and the warmed plot was 6 m in order to avoid heating the control plot. Each 2 × 2-m plot was divided into four 1 × 1-m subplots, and indigenous soil of the plots until the depth of 50 cm was replaced by the sieved topsoil from a forest. Ten PVC pipes (20 × 50 cm) were buried vertically in each subplot ground for planting experimental seedlings. In the preliminary experiment, the PVC pipes and the soil depth did not affect growth of seedling root during a 2-year growth period.

Three-year-old P. asperata seedlings were selected from a local tree nursery with uniform plant height, basal diameter and fresh weight. The average plant height, basal diameter and whole-plant fresh weight were 10.45 ± 0.57 cm, 2.94 ± 0.41 mm, 4.82 ± 0.65 g for these seedlings, respectively. In March 2007, healthy seedlings were randomly selected and transplanted into PVC pipes. The seedlings grown in two diagonal subplots in each plot were weekly watered with 200 ml 2.7 mM ammonium nitrate solution (for a total equivalent to 25 g N m−2 a−1) to the soil surface of the PVC pipes, and the seedlings in the other two subplots were watered with the equivalent water. Nitrogen amount was based on the similar studies (Nakaji et al. 2001; Li et al. 2003). Artificial warming and nitrogen addition was conducted from 15 April 2007 to 15 October 2007. During the experimental period, infrared heaters were continually powered on for 24 h a day, and seedlings were watered frequently as needed. Four treatments in this study were: (1) unwarmed unfertilized (UU); (2) unwarmed fertilized (UF); (3) warmed unfertilized (WU); (4) warmed fertilized (WF).

Temperature and soil moisture measurement

Air (20 cm above soil surface) and soil temperatures (10 cm depth) were measured in four blocks at 30 min intervals from April to October, 2007, using DS1921G Thermochron iButton data loggers (DS1921G-F5#, Maxim/Dallas Semiconductor Inc., USA). Soil moisture was measured in soil core samples (0–10 cm) collected twice in 1 month at all plots. The soil dried at 105°C for 12 h to determine soil moisture.

Biomass analysis

In late October 2007, 16 randomly selected seedlings from each treatment (mean value of four seedlings from the same plots considered as a replicate) were harvested. And then these plants were divided into root, leaf and stem components. All plant parts were dried to a constant mass at 70°C before measurement of dry weight. Dry mater partitioning was derived based on the measured data.

Photosynthetic parameters

To characterize warming and nitrogen fertilization induced shifts in carbon acquisition, Pn–PAR response curve was measured on fully expanded, exposed current-year leaves under controlled optimal conditions using an open-mode portable photosynthesis system (Model LI-6400, Li-Cor, Inc., Lincoln, NE, USA). Two individuals per replicate for each treatment were randomly selected for sampling, and the mean value of two individuals was used as the replicate for statistical analysis. Response to PAR were measured at 0, 20, 50, 80, 100, 200, 300, 400, 600, 800, 1,000, 1,200, 1,600, 1,800, and 2,000 μmol m−2 s−1. The light–response curve of photosynthesis was fitted with a non-rectangular hyperbola (Hirose and Werger 1987):
$$ P_{\text{n}} = \frac{{\Upphi I + P_{ \max } - \left[ {\left( {\Upphi I + P_{\max } } \right)^{2} - 4\Upphi I\theta P_{\max } } \right]^{ 0.5} }}{2\theta } - R_{\text{d}} $$
where Pn is the net photosynthetic rate, Φ is the initial slope of the curve, I is the PPFD, Pmax is the light-saturated rate of photosynthesis, θ is the convexity and Rd is the dark respiration rate. First, from linear regression of the photosynthetic rate on PPFD at 0–200 μmol m−2 s−1, Φ, Rd and LCP were obtained as the slope, y-intercept and the x-intercept of these regressions, respectively. Then, a non-rectangular hyperbola was fitted to the whole curve using the Φ and Rd values to obtain Pmax and θ (Hikosaka et al. 2004). After the measurements, the measured leaves were collected and their projected areas were determined with a scanner and a UTHSCSA ImageTool analysis system. All the measurements were completed over 7 days, from 08:00 to 11:30 each day.

The content of proline, ascorbic acid (ASA), and membrane stability index (MSI)

The free proline content was determined according to the method described by Bates et al. (1973). 1 g current-year needles were homogenized using a pestle and mortar with 5 cm3 of sulfosalicylic acid (3% w/v). After centrifugation (5 min at 20 000g), 0.5 cm3 of the supernatant was incubated at 100°C for 60 min with 0.5 cm3 of glacial acetic acid and 0.5 cm3 of ninhydrin reagent. After cooling, 1 cm3 of toluene was added to the mixture and the absorbance of the chromophore containing toluene was recorded at 520 nm.

ASA was determined as described by Hodges et al. (1996). Current-year needles (0.5 g) were homogenized in 5 ml of cold 5% (w/v) m-phosphoric acid and centrifuged at 10,000g for 15 min. About 300 µl of supernatant was incubated for 5 min in a 700 µl total volume of 100 mM KH2PO4 and 3.6 mM EDTA. Color was developed with 400 µl of 44% o-phosphoric acid, 400 µl of 65 mM α,α′–dipyridyl in 70% ethanol, and 200 μl of 110 mM FeCl3. The reaction mixtures were then incubated at 40°C for 1 h and quantified at 525 nm.

Membrane stability was estimated by measuring the leakage of electrolytes (conductivity) according to Shanahan et al. (1990). 1 g current-year needles was taken in 10 ml of double distilled water in glass vials and kept at 10°C for 24 h with shaking. The initial conductivity (C1) was recorded after bringing sample to 25°C by using conductivity meter. The sample were then autoclaved at 0.1 MPa for 10 min, cooled to 25°C, final conductivity (C2) and distilled water conductivity (C0) were recorded. Membrane stability index (MSI) was calculated as: MSI = [1 − (C1/C2)] × 100.

The rate of superoxide anion radical (O2) production, hydrogen peroxide (H2O2) and Malodiadehyde (MDA) content

The rate of superoxide radical production (O2) was measured as described by Ke et al. (2002) by monitoring the nitrite formation from hydroxylamine in the presence of O2. With 1.5 ml of 65 mM potassium phosphate (pH 7.8), 0.5 g samples were grinded and centrifuged at 5,000g for 10 min. Then, 0.5 ml of the supernatant was incubated with 0.45 ml of 65 mM phosphate buffer (pH 7.8) and 0.5 ml of 10 mM hydroxylamine hydrochloride at 25°C for 20 min. After incubation, 8.5 mM sulfanilamide and 3.5 mM α-naphthylamine were added to the incubation mixture. After reaction at 25°C for 20 min, the absorbance in the aqueous solution was read at 530 nm. A standard curve with NO2 was used to calculate the production rate of O2 from the chemical reaction of O2 and hydroxylamine.

Hydrogen peroxide (H2O2) content was determined as described by Prochazkova et al. (2001). 0.5 g needles were homogenized with 5 ml cooled acetone in a cold room (10°C), filtered and then mixed with 2 ml titanium reagent and 5 ml ammonium solution to precipitate the titanium–hydrogen peroxide complex. Reaction mixture was centrifuged at 10,000g for 10 min and precipitate was dissolved with 5 ml 2 M H2SO4. The above mixture was re-centrifuged and supernatant was read at 415 nm.

The thiobarbituric acid (TBA) test was used to determine the lipid peroxidation. 0.5 g needles were ground with 5 ml of 20% (w/v) trichloroacetic acid (TCA) and the homogenate was centrifuged at 3,500g for 20 min. Then 2 ml of the aliquot of the supernatant was mixed with 2 ml of 20% TCA containing 0.5% (w/v) TBA and 100 μl 4% (w/v) butylated hydroxytoluene in ethanol. The mixture was heated at 95°C for 30 min and then quickly cooled on ice. The contents were centrifuged at 10,000g for 15 min and the absorbance was measured at 532 nm. The value for non-specific absorption at 600 nm was subtracted. The concentration of MDA was calculated using an extinction coefficient of 155 mM−1 cm−1. Results were expressed as nmol g−1 FW.

Antioxidant enzymes activities

1.0 g needles were homogenized under ice-cold conditions in 3 ml of extraction buffer 50 mM phosphate buffer (pH 7.4), 1 mM EDTA, 1 g polyvinylpyrrolidone (PVP) and 0.5% (v/v) Triton X-100]. The homogenates were centrifuged at 10,000g for 30 min at 4°C, and the supernatant was used for the following assays.

SOD (EC 1.15.1.1) activity was determined by the inhibition of the photochemical reduction of nitroblue tetrazolium (NBT), as described by Becana et al. (1986). The reaction mixture [50 μl of enzyme extract and 3.0 ml O2 generating mixture solution containing 50 mM potassium phosphate (pH 7.8), 0.1 mM Na2EDTA, 13 mM methionine, 75 μM NBT, and 16.7 μM riboflavin] in test tubes was shaken and placed 30 cm from light bank consisting of six 15-W fluorescent lamps. The reaction lasts for 10 min and stopped by shutting off the light. The absorbance was read at 560 nm. Without illumination and enzyme, respectively, blanks and controls were run in the same way. One unit of SOD was defined as the amount of enzyme which produced a 50% inhibition of NBT reduction under the assay conditions (Costa et al. 2002).

CAT (EC 1.11.1.6) activity was determined by measuring the decrease in absorption at 240 nm in a reaction solution of 50 mM potassium phosphate buffer (pH 7.2), 10 mM H2O2 and 50 μl enzyme extract (Kato and Shimizu 1987). CAT activity was calculated using the extinction coefficient (40 mM−1 cm−1) for H2O2.

POD (EC 1.11.1.7) activity was based on the determination of guaiacol oxidation (extinction coefficient 26.6 mM−1 cm−1) at 470 nm by H2O2 (Ekmekci and Terzioglu 2005). The reaction mixture contained 50 mM potassium phosphate buffer (pH 7.0), 20. 1 mM guaiacol, 12.3 mM H2O2, and enzyme extract in a 3 ml volume.

APX (EC 1.11.1.11) activity was measured using fresh extracts by measuring the reduction of ascorbic acid (ASA) being oxidized by APX in the presence of H2O2 (Nakano and Asada 1981). The reaction medium consisted of 50 mM potassium phosphate buffer (pH 7.0), 0.1 mM H2O2, 0.5 mM ascorbate and 0.1 mM EDTA. The reduction of ASA was obtained by reading the absorbance decrease at 290 nm (extinction coefficient 2.8 mM−1 cm−1).

Soluble protein contents were determined followed by methods of Bradford (1976), using bovine serum albumin as a calibration standard.

Statistical analysis

Analysis of variance (ANOVA) for a blocked split-plot design was used to detect the effects of warming, N-fertilization and their interactions. Multiple comparisons were also performed to permit separation of effect means using the least significant difference test at significant level of P = 0.05. All statistical analyses were done using the software statistical package for the social science (SPSS) version 11.5.

Results

Warming effects of infrared heaters

Both mean air (at 20 cm aboveground) and mean soil (at 10 cm depths) temperature were significantly elevated by artificial warming, by 2.1 and 2.6°C above those of control plots, respectively (Table 1). Monthly soil moisture showed no statistical significant differences between warmed plots and control plots (Table 1).
Table 1

Monthly mean air temperature (°C), soil temperature (°C) and soil moisture (%) in control plots (C) and warmed plots (W) from April to October 2007

Month

C

W

Tair (°C)

Tsoil (°C)

Soil moisture (%)

Tair (°C)

Tsoil (°C)

Soil moisture (%)

Apr.

11.32

10.61

19.01

13.17

14.25

19.56

May

16.76

15.92

21.03

19.22

19.01

20.73

Jun.

17.13

16.79

23.73

19.31

19.63

22.29

Jul.

20.54

18.89

21.08

22.49

21.76

23.34

Aug.

19.79

18.34

20.56

22.56

21.21

21.09

Sep.

15.21

14.69

21.88

17.36

16.33

23.41

Oct.

11.60

10.77

20.16

13.00

12.29

18.75

Average

16.05

15.14

21.06

18.16

17.78

21.31

Tair mean air temperature (20 cm above soil surface), Tsoil mean soil temperature (10 cm depth)

Growth and biomass analysis

Warming significantly caused an advance of biomass accumulation in unfertilized subplots and a retreat in fertilized plots (Fig. 1a). On the other hand, nitrogen fertilization caused a notable increase of total biomass in unwarmed plots and no notable alteration in warmed plots. In addition, nitrogen fertilization also markedly changed in biomass allocation in both unwarmed and warmed plots (Fig. 1b, c, d). But no warming effect or interactive effect of warming and fertilization was detected in biomass partition.
https://static-content.springer.com/image/art%3A10.1007%2Fs11738-008-0217-8/MediaObjects/11738_2008_217_Fig1_HTML.gif
Fig. 1

Total biomass (a), leaf mass ratio (LMR) (b), stem mass ratio (SMR) (c), and root mass ratio (RMR) (d) of Picea asperata seedlings affected by experiment soil warming and nitrogen fertilization. The bars with different letters are significantly different from each other (P < 0.05). Values are means of four replicates ± SE. FW warming effect, FF nitrogen effect, FW*F interaction effect of warming and nitrogen, Block block effect. ns not significant (P > 0.05); *, **, *** indicate a significant difference between treatments at P < 0.05, P < 0.01, P < 0.001, respectively

Photosynthetic parameters

The maximum net photosynthetic rate (Amax), the apparent quantum yield (Φ), the dark respiration rate (Rd), and the photosynthetic light compensation point (LCP) were remarkably elevated by artificial warming in unfertilized subplots (Fig. 2a, b, c, d). On the contrary, warming resulted in obvious depression in Amax and Φ under additional nitrogen condition (Fig. 2a, b). Similarly, Amax and Φ were increased by nitrogen addition in unwarmed plots but decreased in warmed plots (Fig. 2c, d). Interactions between warming and nitrogen in all these photosynthetic parameters except Rd were statistically significant.
https://static-content.springer.com/image/art%3A10.1007%2Fs11738-008-0217-8/MediaObjects/11738_2008_217_Fig2_HTML.gif
Fig. 2

Maximum net photosynthetic rate (Amax) (a), apparent quantum yield (Φ) (b), dark respiration rate (Rd) (c), and photosynthetic light compensation point (LCP) (d) of Picea asperata seedlings affected by experiment soil warming and nitrogen fertilization. The bars with different letters are significantly different from each other (P < 0.05). Values are means of four replicates ± SE. FW warming effect, FF nitrogen effect, FW*F interaction effect of warming and nitrogen, Block block effect. ns not significant (P > 0.05); *, **, *** indicate a significant difference between treatments at P < 0.05, P < 0.01, P < 0.001, respectively

AOS and MDA

Table 2 showed that experimental warming without supplemental nitrogen resulted in a significant debasement in the rate of O2 production, and the content of H2O2 and MDA; however, a marked positive effect of warming on H2O2 accumulation, the rate of O2 production and MDA content was recorded in seedlings under additional nitrogen condition. Nitrogen addition also brought a reduction in AOS and MDA level under ambient temperature and an enhancement under artificial warming (Table 2). The interactive effects of warming and fertilization significantly affected the rate of O2 production, H2O2 and MDA content.
Table 2

The rate of O2 production and content of H2O2 and MDA in needles of Picea asperata seedlings as influenced by warming and nitrogen fertilization

Treatment

H2O2 (μmol g−1 FW)

O2(nmol min−1 g−1 FW)

MDA (nmol g−1 FW)

UU

16.37 ± 0.51a

7.86 ± 0.12a

23.17 ± 0.44a

WU

12.75 ± 0.38b

4.30 ± 0.31d

19.25 ± 0.94b

UF

12.56 ± 0.44b

5.57 ± 0.29c

19.59 ± 1.10b

WF

15.721.03a

6.57 ± 0.28b

22.28 ± 0.21a

W

ns

*

ns

F

ns

ns

ns

W*F

**

***

**

Block

ns

ns

ns

Means ± SE (n = 4) followed by different letters within rows are significantly different at the P < 0.05 level. Significant effects of the two factors as well as of the interaction are indicated in the last three columns

W warming effect, F nitrogen effect, W*F interaction effect of warming and nitrogen, Block block effect, ns not significant (> 0.05)

*, **, *** indicate a significant difference between treatments at P < 0.05, P < 0.01, P < 0.001, respectively

Antioxidant enzymes systems

The interaction between artificial warming and nitrogen fertilization was founded on these antioxidant enzymes except SOD (Table 3). Warming significantly decreased the activities of SOD, POD, CAT and APX in unfertilized subplots, but no prominent influences on POD and CAT were observed under N-additional condition (Table 3). N-fertilization resulted in an improvement in SOD and POD activities, and a decline in CAT in unwarmed plots; however, in warmed plots the activities of SOD, CAT and APX were not changed by fertilization.
Table 3

Activities of SOD, POD, CAT, and APX in needles of Picea asperata seedlings as influenced by warming and nitrogen fertilization

Treatment

SOD (U mg−1protein)

POD (mmolmin−1 mg−1protein)

CAT (mmolmin−1 mg−1protein)

APX (μmolmin−1 mg−1protein)

UU

19.44 ± 0.44ab

36.53 ± 1.79b

8.76 ± 0.15a

26.21 ± 0.84a

WU

17.68 ± 1.11b

31.15 ± 1.61c

7.12 ± 0.73b

21.14 ± 0.97b

UF

22.53 ± 2.17a

41.49 ± 0.52a

5.98 ± 0.13b

28.31 ± 1.39a

WF

17.93 ± 0.68b

42.81 ± 1.23a

6.95 ± 0.19b

18.09 ± 1.09b

W

*

ns

ns

**

F

ns

***

**

ns

W*F

ns

*

*

*

Block

ns

ns

ns

ns

Means ± SE (n = 4) followed by different letters within rows are significantly different at the P < 0.05 level. Significant effects of the two factors as well as of the interaction are indicated in the last three columns

W warming effect, F nitrogen effect, W*F interaction effect of warming and nitrogen, Block block effect, ns not significant (> 0.05)

*, **, *** indicate a significant difference between treatments at P < 0.05, P < 0.01, P < 0.001, respectively

ASA and proline content and MSI

Warming with additional nitrogen led to a prominent decline in content of ASA and proline, as well as MSI (Table 4). In the next place, supplemental nitrogen distinctly increased proline, ASA and MSI level in unwarmed plots. In addition, significant combination effects of warming × fertilizing were observed in proline, ASA and MSI of P. asperata seedlings.
Table 4

Proline and ASA content and MSI of needles of Picea asperata seedlings as influenced by warming and nitrogen fertilization

Treatment

Proline (μg g−1 FW)

ASA (mg g−1 FW)

MSI (%)

UU

5.74 ± 0.17b

1.57 ± 0.02b

72.85 ± 1.02b

WU

5.77 ± 0.15b

1.55 ± 0.03b

75.00 ± 2.16b

UF

7.97 ± 0.19a

1.69 ± 0.04a

84.33 ± 2.81a

WF

4.55 ± 0.30c

1.39 ± 0.05c

62.27 ± 1.56c

W

*

ns

ns

F

ns

*

**

W*F

*

*

**

Block

ns

ns

ns

Means ± SE (n = 4) followed by different letters within rows are significantly different at the P < 0.05 level. Significant effects of the two factors as well as of the interaction are indicated in the last three columns

W warming effect, F nitrogen effect, W*F interaction effect of warming and nitrogen, Block block effect, ns not significant (> 0.05)

*, **, *** indicate a significant difference between treatments at P < 0.05, P < 0.01, P < 0.001, respectively

Discussion

Warming effects of infrared heaters

Various facilities were employed in field experiments, in order to study the possible effect of future climate warming on terrestrial ecosystem, such as open-top chamber, greenhouse, soil-heating pipes and infrared heaters. Among these warming facilities, infrared heaters were reported to most closely simulate processes of climate warming by enhancing downward infrared radiation (Harte and Shaw 1995). Similar with the previous studies, our results showed that infrared heaters significantly increased both soil and air temperature (Luo et al. 2001; Wan et al. 2002); and the warming effects of infrared heaters over the soil surface were spatially equal, because the parabolic reflectors above the heating rod uniformly delivered infrared radiation over the plots (Loik and Harte 1997; Wan et al. 2002). In previous study, soil moisture was not significantly affected by infrared heaters, but other study showed contrary result (Loik and Harte 1997). These differences might contribute to our watering manipulation during experimental period.

The effects of experimental warming on P. asperata seedlings

Agree well with the similar studies (Yin et al. 2008; Danby and Hik 2007), artificial warming clearly increased total biomass accumulation of P. asperata seedlings without supplemental nitrogen (Table 2). However, there were other studies showing different results, such as the report of Mortensen (1994). On the other hand, warming brought on less biomass accumulation of seedlings with additional nitrogen, indicating that the profitable effects of future climate warming in plant growth would be sheltered in the area with heavy nitrogen load. Biomass allocation was an important response of plant to environment changes (Domisch et al. 2002). Several studies found a decrease in R/S ratio caused by artificial warming (Larigauderie et al. 1991; Landhausser and Lieffers 1998). Our results showed warming had no significant influences on LMR, SMR and RMR both in nitrogen fertilized subplots and unfertilized subplots. The discrepancies are probably due to differences in the differential response of leaf, stem and root among different tree species (Peng and Dang 2003).

According to Awada et al. (2003), elevated temperature may stimulate photosynthesis in plants by increasing photosynthetic pigment concentration and apparent quantum yield (Φ), which contributed to increasing the efficiency of harvesting and using light. In our study, warming under unfertilized condition also significantly improved the maximum net photosynthetic rate (Amax) and apparent quantum yield (Φ) (Fig. 2 a, b). Amax reflected the rate of diffusion of CO2 to Rubisco, the activity of Rubisco, and the rate of regeneration of RuBP (Farquhar et al. 1980), and Φ is the efficiency of light utilization in photosynthesis. The positive effects of warming on photosynthesis have also been reported by other studies (Yin et al. 2008; Saxe et al. 2001). However, the positive effects of warming on photosynthesis only found under ambient nitrogen conditions.

Reactive oxygen species can cause lipid peroxidation, in turn damage cell membrane (Smirnoff 1993; Foyer et al. 1994).In our study, experimental warming significantly reduced AOS and MDA level in needles of P. asperata seedlings without additional nitrogen (Table 3). In order to balance and control the oxygen toxicity, plant had developed antioxidative systems. SOD, CAT, POD and APX are important enzymes in plants that scave AOS. Our result suggested that elevated temperature was favorable to alleviate natural oxidative damage for P. asperata seedlings under unfertilized condition, judged from decreasing AOS and MDA cumulate and increasing SOD, CAT, POD and APX activities. On the other hand, warming induced an enhancement in AOS and MDA content, and reduction in activities of these antioxidative enzymes in nitrogen fertilized subplots. Therefore, the effects of warming in plant antioxidative systems had relation to nutrient availability of soil.

Proline could function as a hydroxyl radical scavenger to prevent membrane damage and protein denaturation (Ain-Lhout et al. 2001). ASA was another non-enzymatic antioxidant acting as a substrate for extracellular enzymes for attenuating AOS levels (Burkey et al. 2006). In the present study, no evident influences of artificial warming on proline and ASA content and MSI were detected in unfertilized subplots; whereas, clear reduction in these parameters were resulted from elevated temperature in nitrogen fertilized subplots.

The effects of nitrogen fertilization on P. asperata seedlings

Our results showed that nitrogen addition significantly accelerated the accumulation of plant dry mass in unwarmed plots, which agreed well with the previous study (Fownes and Harrington 2004). However, in warmed plots, these positive effects on growth of P. asperata seedlings were shielded. Previous studies suggested that soil nitrogen deeply affected plant biomass allocation (Nakaji et al. 2001; Grechi et al. 2007). Similar with these results, nitrogen fertilization also prominently increased LMR and SMR, and decreased RMR under both warmed and unwarmed conditions. Therefore, plants could maximize growth potential by concentrating the investment of resource in assimilating parts.

Sugiharto et al. (1990) found a significant and positive correlation between photosynthetic capacity and nitrogen concentration of plant leaves suggesting that most of the nitrogen is used for synthesis of components of the photosynthetic apparatus. In present study, nitrogen addition significantly improved Amax of seedlings in unwarmed plots (Fig. 2a). This result was agreed well with Chen et al. (2005), however, Φ was decreased in their research, which was contrary to ours. Present results showed that light compensation point (LCP) affected by nitrogen only when temperature was elevated. But little effect of nitrogen on LCP was observed under ambient temperature by other study (Chen et al. 2005).

H2O2 content, the rate of O2 production and MDA content were evidently decreased by nitrogen addition in unwarmed plots. This maybe related to the enhancement of antioxidant enzymes activities (SOD, POD and APX) under nitrogen supply (Table 2), since nitrogen nutrition could improve synthesis and physiological activities of antioxidant enzymes. Xiao et al. (1998) reported that nitrogen nutrition can improve light reaction and dark reaction of photosynthetic organization, and reduced deoxidization capacity, which lead to the reduced rate of AOS production and less AOS accumulation. However, obvious increases of H2O2 content, the rate of O2 production and MDA content were detected under warming condition due to supplemental nitrogen, suggesting that the favorable effects of nitrogen on deoxidization capacity were inhibited by experimental warming.

Similar to Sánchez et al. (2002), seedlings with nitrogen supply had higher proline content under ambient temperature. Proline accumulation stimulated by nitrogen addition may be because it is a nitrogen-storage compound (Ahmad and Hellebust 1988) and that synthesis and accumulation of proline are simulated by nitrogen supply (Sánchez et al. 2002). In present study, nitrogen supplement induced an increase in ASA content of these seedlings without warming. MSI was also distinctly enhanced by nitrogen fertilization, suggesting that nitrogen addition play an important role in maintaining membrane stability under ambient temperature. However, in warmed plots, proline and ASA content as well as MSI of plants were significantly reduced by additional nitrogen.

The interactive effects of experimental warming and nitrogen fertilization on P. asperata seedlings

Mäkipää et al. (1999) reported that the combined effect of N-fertilization and artificial warming further increased the growth of Scots pine. However, in present study, the interaction of these two treatments induced less biomass accumulation than N-fertilization alone (Fig. 1a). The possible reason for the different results may be that both artificial warming and nitrogen deposition can influence plant growth indirectly by increased N-mineralization and availability, soil acidification, nutritional imbalances and leaching of nutrients (Mäkipää et al. 1999; Nakaji et al. 2001; Wan et al. 2005). The enhanced N mineralization and availability by warming and N-fertilization would stimulate plant N uptake and accumulation, which may lead to nutrient imbalances of P relative to N in seedlings and consequently reduction in dry mater production (Nakaji et al. 2001). This paper, clearly showed that the effects of warming was on growth and antioxidant defense parameters were dependent up on its nitrogen nutrition. At the same time, growth and antioxidant defense parameters affected by N-fertilization were also dependent on environmental temperature. Nitrogen fertilization significantly improved plant growth in unwarmed plots, by stimulating total biomass, maximum net photosynthetic rate, antioxidant compounds, as well as reducing the content of AOS and MDA. Experimental warming alone significantly increased seedling biomass accumulation and the maximum net photosynthetic rate, and decreased AOS and MDA level under ambient nitrogen conditions, whereas warming under N-fertilizered condition tended to depress the positive effects of nitrogen. Obviously, the favorable effects of warming and N-fertilization on growth and physiology of P. asperata seedlings would not be magnified by the interaction of warming and nitrogen.

Acknowledgments

This study was supported by the Key Program of the National Natural Science Foundation of China (No. 30530630), the Talent Plan of the Chinese Academy of Sciences and “Knowledge Innovation Engineering” of the Chinese Academy of Sciences, and Maoxian Ecological Station, Chengdu Institute of Biology, Chinese Academy of Sciences.

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© Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2008