Carbon isotope discrimination in leaf juice of Acacia mangium and its relationship to water-use efficiency

Abstract

Using the PMS pressure chamber and isotope mass spectrometer (MAT-252), the leaf juice of Acacia mangium was obtained, and the carbon isotope discrimination (D) representing the most recently fixed carbon in the juice was determined. At the same time, the water-use efficiency of A. mangium was estimated. The results indicated that the carbon isotope ratio in the air of forest canopy (d_a), 10 m high above ground averaged -7.57±1.41‰ in cloudy days, and -8.54±0.67‰ in sunny days, respectively. The diurnal change of the carbon isotope ratio in the photosynthetic products of the leaf juice (d_p) was of saddle type in cloudy days, but dropped down from morning to later afternoon in sunny days. A strong negative correlation between d_p and leaf-to-air vapor pressure deficit (D) was observed in sunny days, but a slight change in d_p was found in cloudy days. The d_p also decreased with decreasing leaf water potential (Ψ), reflecting that water stress could cause the decrease of d_p. The carbon isotope discrimination of the leaf juice was positively correlated with the ratio between intercellular (P_i) and atmospheric (P_a) partial pressure of CO₂. For A. mangium, the isotope effect on diffusion of atmospheric CO₂ via stomata was denoted by a = 4.6‰, and that in net C₃ diffusion with respect to P_i was indicated by b = 28.2‰. The results were in reasonable accord with the theoretically diffusive and biochemical fractionation of carbon isotope. It was defined that carbon isotope discrimination of photosynthetic products in A. mangium leaf juice was in proportion to that from photosynthetic products in dry material. The water-use efficiency estimated by the carbon isotope discrimination in leaf juice, fit well with that measured by gas exchange system (R² = 0.86, p< 0.0001). The application of leaf juice in measuring the stable carbon isotope discrimination would reduce the effects of fluctuating environmental factors during the synthesis of dry matter, and improve the eco-physiological studies on carbon and water balance when scaling from the plant to canopy in the fields.

Keywords

carbon isotope discrimination / water-use efficiency / photosynthetic products in leaf juice

Introduction

The fourth assessment report of the IPCC (Intergovernmental Panel on Climate Change, 2007) pointed out that the global climate warming was an undisputed fact, and the temperature of the earth’s surface would increase by 1.7-1.4°C on average to the end of 21st century (IPCC, 2007). Chinese researchers forecasted that the temperature from 2020 to 2030 in China would increase 1.3°C, and 2.2°C to 2050, in comparison with that in 2000 (Qin, 2003). The climate warming would enhance the evaporation of the earth’s surface and transpiration in plant leaves and then increase water losses from plant and water uptake from soil by roots, making the soil dry. Usually, plants take up water from soil by soil–plant hydraulic system and balance the vapor pressure deficit by transpiration via leaf stomata and cuticle. The stomata are diffusion pathways for CO₂ and H₂O in plant leaves. Atmospheric CO₂ enters mesophyll and transfers to the site where carboxylation takes place. Photosynthesis (A) goes on at the same time the water evaporates through stomata and cuticle (E). The A/E ratio reflects the ratio of CO₂ assimilation rate to transpiration rate, namely water-use efficiency (WUE). The WUE of plant species differs markedly due to their different genetic and physiological characteristics, as well as different habitats. A gas exchange system is often employed to measure leaf WUE, but it fails when it is measured on a larger scale in the fields (Farquhar et al., 1989).

CO₂ presents in the forms of ¹³CO₂ and ¹²CO₂ in the atmosphere, and they are discriminated by plants when photosynthesis fixes CO₂. ¹³C can be discriminated by enzyme Rubisco which assimilates CO₂ in C₃ plants (Park and Epstein, 1960). The mass of C atoms influences CO₂ diffusion via the stomata, making dissimilar allocations of ¹³C and ¹²C in photosynthetic products (O’Leary, 1981). Farquhar et al. (1982) pointed out that there was a linear relationship between carbon isotope discrimination (D) of C₃ plants and the ratio of intercellular CO₂ partial pressure to atmospheric CO₂ partial pressure. Furthermore, Farquhar (1984) proved that the WUE of C₃ plants correlated with the D. Potted plants e.g. groundnut (Hubick et al., 1988), cotton (Martin and Thorstenson, 1988), barley (Hubick and Farquhar, 1989) took on such similar relationships. Wright et al. (1988) proved that there was a relationship between D and WUE of products in aboveground organs from eight genotypes of groundnut, whereas it needed more research to find out whether such a relationship is applicable at larger spatial scale. The D of dry matter in aboveground organs was applied to estimate WUE in early studies in that the stable isotope ratio of dry matters was taken as an aggregate of a series of complicated physiological and biochemical reactions for longer time period, and in which the change of stomatal conductance and water loss by transpiration, caused by the fluctuation of vapor pressure deficit, was difficult to estimate. On the assumption that soluble sugar was the main photosynthetic product in leaf and accumulated in mesophyllic cells or transferred to outside, the carbon in leaf juice collected by pressuring could represent the carbon that was newly assimilated, and the fluctuation of microclimate which caused changes of photosynthesis and stomatal conductance could be supposed to be relatively stable at this period of time.

Since mid 1980s A. mangium was introduced to the Guangdong hilly land region located in the low subtropics. A. mangium plantation played an important role in the utilization of spatial energy resource, regulation of microclimate and improvement of ecological environment of hilly land. The soil of the degraded ecosystem in south China is very infertile. Soil erosion is serious as a result of vegetation loss on the land surface. Its physical and chemical characters were deteriorated. During the rainy season, water supply is discontinuous because of the lower water-holding capability of soil, and it is even worse in dry season (from September to December). These conditions above are disadvantageous for the growth of many tree species, and climatic warming would make it worse. The water deficit in the soil caused by a prolonged dry season is an adverse factor for the introduced tree species. In this study, carbon isotope discrimination (D) is employed to estimate the WUE of A. mangium, inspect the effect of environmental change on photosynthesis and water-use, and is suitable and effective experimental method for the study on forest ecosystem carbon and water balance.

Study area and method

Study site

We conducted this study in Heshan Hilly Land Interdisciplinary Experimental Station, Chinese Academy of Sciences, which is located at 22°41′N and 112°54′E, in Heshan city, Guangdong Province. The station is characterized with southern subtropical monsoon climate and crimson soil. The mean annual temperature is 21.7°C, precipitation 1700 mm and evaporation amounts to 1600 mm. Dry season is from September to December. The A. mangium plantation in which the field measurement was conducted is 23 years old and 10 m to 17 m high. The fieldwork was carried out on sunny days and cloudy days in December, 2006. The weather during the consecutive 3 days before experimental measurement was stable.

Carbon isotope ratio measurement

We collected air samples from canopy of the A. mangium plantation (about 10 to 15 m above ground) with an electromagnetic pump and a plastic tube. The samples were then injected right away into sampling bags which were vacuumed beforehand. Air samples were collected once every 1 h and repeated twice. At the same time, an infrared CO₂ analytical instrument (Li-Cor 6400, Inc., Lincoln, NE, USA) was employed to record the atmospheric CO₂ concentration. CO₂ isotope ratio of air samples which were pretreated was determined on the MAT-252 stable isotope mass spectrograph (d_a):

(1){\delta }_{\mathrm{a}}=\left({\displaystyle \frac{R_{\mathrm{a}}}{R_{\mathrm{s}\mathrm{t}\mathrm{d}}}}-\mathrm{1}\right)\times \mathrm{1000}

where R_a is the atmospheric ¹³C/¹²C ratio, and R_std is the ¹³C/¹²C ratio of Pee Dee Belennite (PDB) standard sample.

The collection of plant leaf photosynthetic products samples and the measurement of carbon isotope ratio

We collected mature leaves from the canopy of A. mangium every 2 h at the same time with the air sample collection which were then pressured with PMS pressure chamber (PMS Instruments, Cregan, USA), and then sucked the leaf juice with the mini-sucker. These were then injected into a 10-mL vial at once, frozen in the refrigerator and afterwards, we measured the carbon isotope ratio (d_p) of leaf juice with MAT-252 stable isotope mass spectrograph:

(2){\delta }_{\mathrm{p}}=\left({\displaystyle \frac{R_{\mathrm{p}}}{R_{\mathrm{s}\mathrm{t}\mathrm{d}}}}-\mathrm{1}\right)\times \mathrm{1000}

where R_p is the ¹³C/¹²C ratio of plant samples. According to the method presented by Farquhar and Richards (1984) carbon isotope discrimination (D) of plant photosynthetic products was calculated:

(3)\mathit{\Delta }={\displaystyle \frac{{\delta }_{\mathrm{a}}-{\delta }_{\mathrm{p}}}{\mathrm{1}+{\delta }_{\mathrm{p}}}}

Determination of leaf photosynthetic and transpiration rate

With respect to the sample site slope, we set up a bracket on higher land and employed other Li-Cor 6400 photosynthesis measurement system to measure photosynthetic and transpiration rate of canopy leaf on the lower land, collecting 20 pieces of leaves randomly every 2 h, detecting continuously and recording the temperature, relative humidity and photosynthetic active radiation.

Data processing

WUE calculation

According to Farquhar et al. (1985), the ratio of leaf CO₂ assimilation rate (A) to transpiration rate (E) (i.e., WUE):

(4){\displaystyle \frac{A}{E}}=WUE={\displaystyle \frac{P_{\mathrm{a}}-P_{\mathrm{i}}}{\mathrm{1.6}D}}

(5)\mathit{\Delta }=a+(b-a)\times {\displaystyle \frac{P_{\mathrm{i}}}{P_{\mathrm{a}}}}

(6)WUE={\displaystyle \frac{P_{\mathrm{a}}\times {\displaystyle \frac{b-d-\mathit{\Delta }}{b-a}}\times (\mathrm{1}-{\varphi }_{\mathrm{c}})}{\mathrm{1.6}\times D\times (\mathrm{1}+{\varphi }_{\mathrm{w}})}}

where P_a and P_i are atmospheric and intercellular CO₂ partial pressure, respectively, and D is the leaf-to-air vapor pressure deficit (kPa). When C₃ plants photosynthesize, the carbon isotope discrimination of atmospheric ¹³CO₂ and ¹²CO₂ that diffuse via stomata was denoted by a = 4.4‰ (Craig, 1954), and the carbon isotope discrimination related to intercellular CO₂ partial pressure P_i and carbon fixation is indicated by b = 27‰ (Farquhar et al., 1989), d is the emendation value related to CO₂ assimilation rate and negligible under daytime light radiation conditions (Evans et al., 1986), whereas the effect of respiration in the dark or without illumination on the discrimination is supposed to be 7‰ (Werner et al., 2006). f_c is a parameter related to soil humidity, (1-f_c) = 0.7 in dry season, and (1-f_c) = 1 under irrigative conditions (Hubick and Farquhar, 1989), f_w = 0 among the same plant species.

Leaf-to-air vapor pressure deficit

According to Cambell and Norman (1998), it is obtained based on the calculation of measured leaf temperature and air relative humidity.

Statistical analysis

The data were mean values from three or four measurements. They were analyzed using variable analysis (ANOVA) and least significance detection (LSD) with SPSS (SPSS, INC, Chicago IL), and the least significance level is 0.05.

Results and analysis

The carbon isotope ratio of canopy atmospheric CO₂ in A. mangium plantation

The fluctuation of atmospheric d_a in A. mangium plantation canopy in the experimental site was significant (p < 0.05). Figure 1 shows that atmospheric CO₂ concentration of canopy was higher in the morning and d_a was lower. Keeling et al. (1979) pointed out that atmospheric ¹³CO₂ consumption was slow as ¹³C was discriminated when photosynthesis assimilated CO₂ during daytime. The CO₂ assimilation decreases the concentration of ambient CO₂, then remaining CO₂ containing ¹³C in atmosphere was denser than before, and d_a was higher. The atmospheric CO₂ concentration of A. mangium plantation canopy was less fluctuant, so was d_p which changed along with CO₂ concentration (Fig. 1).

On December 7, 2006, which was a cloudy day, the atmospheric CO₂ concentration in the A. mangium plantation canopy (10 to 15 m above ground) was 445 μmol/mol in the morning, declined with the time and dropped down to 401±18.3 μmol/mol at noon. The minimum was 390 μmol/mol in the afternoon (Fig. 2(a)). During daytime the mean value of atmospheric carbon isotope ratio d_a was -7.57±1.41‰ (n = 14) and lower at noon, the diurnal change was of saddle type (Fig. 2(b)). On December 17, 2006, which was a sunny day, the atmospheric CO₂ concentration of A. mangium plantation canopy was 400.2±4.3 μmol/mol on the average and the atmospheric CO₂ carbon isotope ratio d_a was -8.5±0.67‰, which was significantly lower than in the cloudy day (p < 0.05). During sunny days, the diurnal change of d_a was relatively more stable but lower in the morning (Fig. 2(d)). Farquhar et al. (1989) suggested that the atmospheric d_a was supposed to 8.0‰ when estimating carbon isotope discrimination of plants in the field.

The carbon isotope ratio of photosynthetic products from A. mangium leaves

Figure 2 shows that solar radiation was lower in the morning on December 7 (cloudy day). Mean photosynthetically active radiation was 324 μmol/(m²·s) from 9:00 to 14:00 and then declined gradually. The mean diurnal temperature was 23.5±3.6°C, and leaf-to-air vapor pressure deficit (D) was 0.94 kPa before 8:00, which then increased from 1.89 kPa at noon to 2.17 kPa in the afternoon and declined to 1.22 kPa after 15:00 (Fig. 2(a)). During cloudy days, the carbon isotope ratio of plant photosynthetic products was lower in the morning and afternoon, but took on two peak values at 11:00 and 14:00, and lower at 12:00, the diurnal change was of saddle type. The diurnal mean d_a was -31.75±0.59‰ (Fig. 2(b)). On December 17 (sunny day), the mean photosynthetically active radiation was 650 μmol/(m²·s) from 9:00 to 15:00, and the diurnal mean temperature was 16.12±4.0°C; leaf-to-air vapor pressure deficit (D) was 2.18 kPa at noon, which declined in the morning and afternoon, and diurnal mean D was 1.43±0.47 kPa (Fig. 2(d)). The results show that the carbon isotope ratio d_a of photosynthetic products in A. mangium leaf was -28.89±1.42‰ in sunny days, which was higher than that in cloudy days (p < 0.05).

In sunny days, A. mangium leaf took on higher photosynthetic rate and lower intercellular CO₂ partial pressure (P_i), e.g. P_i was 25.78 Pa in sunny days and 31.06 Pa in cloudy days; the ratio of intercellular CO₂ partial pressure to atmospheric CO₂ partial pressure (P_i/P_a) was 0.648 at noon in sunny days but 0.772 in cloudy days. Therefore d_a of leaves was higher in sunny days than that in cloudy days.

From Fig. 3 it can be seen that the carbon isotope ratio (d_a) of A. mangium leaf presented small change along with the increase of leaf-to-air vapor pressure deficit in cloudy days, whereas in sunny days the d_p of A. mangium leaf was higher, but dropped down markedly with the increase of D. This study indicated that d_p of A. mangium leaf fluctuated more obviously in sunny days under similar D conditions.

When D was 1.4 kPa, the carbon isotope ratio (d_p) of photosynthetic products in A. mangium leaf declined with the decrease of leaf water potential (Ψ) (Fig. 4). The decrease of leaf water potential reflected an increase of water stress, and caused the leaf stomata to close and intercellular CO₂ partial pressure to increase, which then resulted in a decline of d_p (Ehleringer and Cooper, 1986).

The carbon isotope discrimination (D) of photosynthetic products in leaf in relation to water-use efficiency (WUE)

With the increase of intercellular and atmospheric CO₂ partial pressure, D of A. mangium leaf juice increased, and there was significant correlation between D and the P_i/P_a ratio which was measured by gas change system (Fig. 5). It was identical to the result proved by Farquhar et al. (1982) that dry matters of different plant species were significantly correlative with the P_i/P_a ratio.

When the atmospheric CO₂ partial pressure was 38.6 Pa and D was 1.4 kPa, there was negative relationship between D obtained by measurement and calculation with Eq. (3) and leaf water-use efficiency (WUE, P_a/kPa). If the WUE was higher, the D was lower (Fig. 6). Scatter dots figure (Fig. 7) based on two types of WUE obtained from D and gas exchange system show that two results were similar. Using the relationship between D of leaf juice and the P_i/P_a ratio, we calculated that a = 4.6‰ if P_i/P_a = 0, b = 28.2‰ if P_i/P_a = 1. Two types of WUE were close to each other (R² = 0.86, p < 0.0001) (Fig. 7(b)).

Discussion

¹²C and ¹³C are two types of stable carbon isotope existing in nature, and their allocations are uneven in different compounds, which occur in the physic-chemical and metabolism processes when plants take up and assimilate CO₂. The abundance of ¹³C relative to ¹²C in plants tissues is smaller than that in atmospheric CO₂, which reflects that the carbon isotope is discriminated during CO₂ assimilation. Starting with O’Leary’s study (1981), this research field made great progress and researchers uncovered the effect of the photosynthetic metabolism to the environment making use of the carbon isotope discrimination characteristics in plant photosynthesis. In recent years, stable carbon isotope detecting techniques have been applied to studies on the forest ecosystem carbon cycling (Gessler et al., 2001; Ponton et al., 2006; Werner et al., 2006), which also received attention in China (Yi and Zhuang, 2005; Zheng et al., 2005).

Researchers explained the allocations of stable carbon isotope in C₃ plants photosynthesis with multi-modes which were in common with the assumption that the different allocations of carbon isotope were attributed to the difference of ¹²CO₂ and ¹³CO₂ diffusion via the stomatal pathway and the difference of enzyme Rubisco carboxylation capability (Keeling et al., 1979; Vogel, 1980; Farquhar et al., 1989). The C₃ plants leaf carbon isotope discrimination proposed by Farquhar et al. (1982) was simple, which used in the relationship between D of dry matter from different herbaceous species and the P_i/P_a ratio to determine a and b directly. When stomatal conductance relative to the capability of CO₂ fixation was lower, intercellular CO₂ partial pressure (P_i) was lower and carbon isotope discrimination (D) increased to a = 4.4‰. However, when the stomatal conductance was higher and P_i was close to atmospheric CO₂ partial pressure (P_a), D inclined to b = 27‰, i.e. net allocation or Rubisco discrimination resulted from carboxylation. Farquhar et al. (1982) proved that D of dry matters in plant photosynthetic products significantly correlated with the P_i/P_a ratio, and our study also proved that D of A. mangium leaf photosynthetic products positively correlated with the P_i/P_a ratio measured with gas exchange system (Fig. 5). Thus a and b of woody plants were 4.6‰ and 28.2, respectively, which fitted with the WUE calculated from Eq. (6), and accorded with the WUE measured with the gas exchange system. Although the sugar content in the leaf juice of A. mangium was not measured in this study, most fixed carbon came into being soluble sugar in the first instance. This study indicated that the increase of atmospheric CO₂ concentration for a short period of time caused the stomata to close, and then the concentration of cellular CO₂ increased and made the C_i/C_a ratio or P_i/P_a ratio change, where the D of the leaf juice increased and the WUE declined, i.e., the increase of atmospheric CO₂ concentration would change the WUE of plants. Comstock and Ehleringer (1993) also proved that the increase of atmospheric CO₂ concentration for longer periods of time and the fluctuation of leaf temperature would influence d_p and WUE. Brugnoli et al. (1988) proved that the D of soluble sugar in the cotton leaf correlated intimately with the P_i/P_a ratio and calculated D of sugar reflected the allocation of carbon isotope in middle temporal scale. In this study we extracted leaf juice within 1 hour after photosynthesis, which could reflect photosynthetic products in the short term. From Fig. 5, the P_i/P_a ratio was 0.7-0.9, which was in accordance with the results of Eqs. (4) and (5) related to stomatal diffusion and bio-chemical discrimination.

Francey et al. (1985) found that atmospheric carbon isotope ratio (d_a) presented an obvious gradient change in the Lagastrobos franklinii plantation, and in the A. mangium plantation we found d_a fluctuated with time in the diurnal course. Due to the ¹³C discrimination in leaf photosynthesis, the difference of forest canopy photosynthetic activity, soil respiration and litter decomposition caused a slow change of the atmospheric CO₂ isotope ratio. On account of the fluctuation of d_a in A. mangium plantation, and especially the significant difference of d_a during cloudy days and sunny days, relevant d_a needed to be measured when estimating carbon isotope discrimination (D) of plant grown on farm. The modified atmospheric CO₂ carbon isotope in field could reflect atmospheric CO₂ carbon isotope ratio of A. mangium plantation stand in a larger scale. The carbon isotope discrimination measurement system has become an effective method in C₃ plants eco-physiological photosynthesis and relevant water-use research. However, there is much work to be done on how to scale up from individual plant to canopy, e.g. the regulation of water dissipation is not an independent variable and soil evaporation negatively correlates with the relevant leaf area. Vapor pressure deficit fluctuated frequently and is not an independent variable. Vapor pressure deficit changes with the fluctuation of stomatal conductance and canopy aerodynamic data are also needed to ascertain the difference of D at the canopy level. The experimental site of this study is located in the Zhujiang Delta Region where the cloudy days are quite frequent, and we measured the mean carbon isotope ratio of leaf juice in sunny days and cloudy days, respectively, which are in accordance with the local climate. The short-term change of the C_i/C_a ratio can be estimated with the use of leaf juice carbon isotope discrimination measurement system, whereby the water-use efficiency of C₃ plants in a short time (e.g. 1 h) can be calculated. This method can reduce the effect of fluctuating environmental factors on water-use and offers a a new approach to measure the C_i/C_a ratio during the growth season, and it is good for the measurement of water-use efficiency in different season. It is also helpful for further research on the carbon and water balance when scaling from an individual plant to canopy.

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