Effects of elevated ozone, carbon dioxide, and the ...

Environmental Pollution 189 (2014) 9e17

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Effects of elevated ozone, carbon dioxide, and the combination of both on the grain quality of Chinese hybrid rice Yunxia Wang a, Qiling Song a, Michael Frei b, Zaisheng Shao a, Lianxin Yang a, * a b

Key Lab of Crop Genetics & Physiology of Jiangsu Province, Yangzhou University, Yangzhou 225009, China Institute of Crop Science and Resource Conservation (INRES), University of Bonn, Bonn, Germany

a r t i c l e i n f o

a b s t r a c t

Article history: Received 19 November 2013 Received in revised form 13 February 2014 Accepted 15 February 2014

The effects of CO2 and/or O3 elevation on rice grain quality were investigated in chamber experiments with gas fumigation performed from transplanting until maturity in 2011 and 2012. Compared with the control (current CO2 and O3 concentration), elevated CO2 caused a tendency of an increase in grain chalkiness and a decrease in mineral nutrient concentrations. In contrast, elevated O3 significantly increased grain chalkiness and the concentrations of essential nutrients, while changes in starch pasting properties indicated a trend of deterioration in the cooking and eating quality. In the combination of elevated CO2 and O3 treatment, only chalkiness degree was significantly affected. It is concluded that the O3 concentration projected for the coming few decades will have more substantial effects on grain quality of Chinese hybrid rice than the projected high CO2 concentration alone, and the combination of two gases caused fewer significant changes in grain quality than individual gas treatments. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Global change Ozone Carbon dioxide Quality Oryza sativa

1. Introduction Human activities are causing rapid changes in the concentration of key trace gases in the earth’s atmosphere. Foremost among these changes are the increase of atmospheric carbon dioxide concentration (CO2) and tropospheric ozone concentration (O3). The global CO2 concentration has risen from approximately 280 mL L1 in preindustrial times to present 394 mL L1(http://www.esrl.noaa.gov/ gmd/ccgg/trends/, accessed 5 November 2013), and is projected to rise further for the next 50 years despite the various efforts to reduce carbon emissions (Fisher et al., 2007). Concomitant with the increase of the CO2 concentration, the ground-level ozone concentration (O3) has also risen rapidly, especially in regions with high population density and vibrant economic growth (Chan and Yao, 2008; Engardt, 2008; Wang et al., 2009, 2011b). Recent model studies concluded that surface O3 concentration will increase in South China by 2050 due to climate change, even if very stringent pollution control keeps the emission level at the 2006 level (Liu et al., 2013). Both CO2 and O3 are important anthropogenic greenhouse gas responsible for global temperature change since preindustrial times (Forster et al., 2007), but they have different impacts on crop

* Corresponding author. E-mail address: [email protected] (L. Yang). http://dx.doi.org/10.1016/j.envpol.2014.02.016 0269-7491/Ó 2014 Elsevier Ltd. All rights reserved.

productivity. Many empirical data have shown that an increase in CO2 concentration has positive influences on crop growth and yield by promoting photosynthetic rates and possibly reducing crop water use (Ainsworth and Long, 2005; Long et al., 2006). In contrast, as a strong oxidant, tropospheric O3 is highly phytotoxic. It enters leaves through the stomata, induces oxidative stress in plant cells, and inhibits photosynthesis and other physiological processes, thus resulting in reduced plant growth and yield loss (Fiscus et al., 2005; Ainsworth et al., 2012). While individual effects of atmospheric CO2 or tropospheric O3 on crop performance have been studied and analyzed intensively, there is a critical gap in knowledge about CO2 and O3 interactions (Long et al., 2006; Ainsworth et al., 2012). Of further concern for future food security is the growing evidence that CO2 or O3 impacts on crop quality (DaMatta et al., 2010; Moretti et al., 2010), which is considered to be as important as effects on crop quantity. Elevated CO2 or/and O3 could affect a series of physiochemical processes. These altered processes may eventually affect physical and chemical composition of crops and finally the quality of the harvested products (Wang and Frei, 2011). Rice (Oryza sativa L.) is the main staple food which presently provides 20% or more of the daily food energy to half the world’s population (Timmer et al., 2010), and the demand for high quality rice will continue to rise in the near future because most of the riceconsuming countries are still experiencing a population growth and a shrink of cropland (Bruinsma, 2009). In view of the socio-

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Y. Wang et al. / Environmental Pollution 189 (2014) 9e17

economic importance of rice under the scenario of global environmental change, much research has been conducted to assess the impacts of rising CO2 or O3, separately, on rice production, but almost none have evaluated the two factors in combination (Ainsworth, 2008). To our knowledge, there is no publication on rice quality response to the combined elevation of CO2 and O3 up to date. The effects of single gas enrichment of CO2 (Lieffering et al., 2004; Lee et al., 2013; Taub et al., 2008; Yang et al., 2007) or O3 (Frei et al., 2012; Wang et al., 2012) on rice quality have been inconsistent except changes in protein and nitrogen concentrations. A full-size FACE (Free Air gas Concentration Enrichment) systems was developed for paddy rice for CO2 fumigation in 1997 (Okada et al., 2001) and later for O3 fumigation in 2007 (Tang et al., 2011). However, this technology has not been used to investigate the interactive effects of simultaneous change in CO2 and O3, due to technological limitations. Therefore, we recently set up a naturally sunlit fumigation system, which mimicked paddy field conditions (Zhao et al., 2012). By using this facility, we aimed at elucidating how grain quality of rice would change in a future high-CO2 and O3 world, and to analyze if the effects of CO2 and O3 observed are additive or compensative. The hypotheses were: the effects of elevated O3 on rice grain quality are different from that of elevated CO2; the effects of elevated O3 on rice quality can be offset by elevated CO2. The CO2 (200 mL L1 above ambient, anticipated 2050 levels) and O3 treatments (60% above ambient, projected level in the near future, also within a range currently experienced in polluted areas, Fisher et al., 2007), as well as the soil and cultivation technique, were identical to those used by our in situ CO2-FACE (Yang et al., 2006) and O3-FACE experiment (Shi et al., 2009; Tang et al., 2011). A hybrid rice cultivar Sanyou63 was selected for this study based on the high sensitive of this cultivar both to CO2 (Liu et al., 2008) and O3 (Shi et al., 2009) in our previous FACE experiments. 2. Materials and methods 2.1. Plant culture Soil from paddy field was filled into cement ponds inside the fumigation chambers in which the gas fumigation was performed. The soil is Shajiang Aquic Cambosols with a sandyeloamy texture. The details of soil properties can be found in Shi et al. (2009). Standard cultivation practices as commonly performed in rice field in the region were followed in all experimental chambers. Seeds of an O3/CO2sensitive hybrid rice cultivar were germinated and hand-sown on 21 May 2011 and 22 May 2012, in a nursery field under ambient air, and manually transplanted at a density of 1 plants hill1into all growth chambers on 20 June 2011 and 18 June 2012. Spacing of the hills was 16.7 by 25 cm (equivalent to 24 hills m2). In both years, 9 g N m2, 7 g P2O5 m2 and 7 g K2O m2 was applied as basal dressing just prior to transplanting. Additional 6 g N m2 was applied at panicle initiation on 25 July 2011 and 30 July 2012. The cement ponds were flooded with water with about 4 cm depth from transplanting to mid-tillering, drained dry for several times until panicle initiation, and then flooded intermittently after that. 2.2. Gas fumigation The glasshouse fumigation system used in this study was previously described (Zhao et al., 2012). Four 3 3 1.7 m chambers were built with colorless toughened glass (light transmittance 95%). A temperature and humidity control unit enabled the real time simulation of ambient meteorological conditions inside the chambers while a gas distribution system simulated the targeted trace gas concentration. The main control system (S7-200, Siemens, Nuernberg, Germany) analyzed the data from a temperature and humidity sensor (EE21, E þ E elektronik GmbH, Engerwitzdorf, Austria), a CO2 monitor (LI-820, LI-COR, Lincoln Nebraska, USA) and an ozone monitor (model 49i, Thermo Scientific Co., Franklin, MA, USA), and use computer feedback through a proportional integral derivative algorithm to control each environmental factor or gas concentration to reach the target level in each chamber. Light was not controlled in the chambers, but was monitored continuously at 1-min interval using light sensors (HD2021T, Delta,W & B Instruments, Bayswater, Australia) placed at the plant canopy. The gas fumigation treatments and CO2/O3 concentrations in the gas fumigation chambers are summarized in Table 1. CO2 fumigation started from sunrise and ended at sunset, while O3 fumigation started at 9:00 in the morning and ended at sunset.

Table 1 Gas fumigation treatments and actual seasonal mean gas concentrations in 2011 and 2012 rice seasons. Treatments O3

CO2 a

Ambient Current CO2 Elevated CO2

C-O3 E-O3 C-O3 E-O3

Target gas concentrations

CO2 (mL L1)

O3 (nL L1)

2011

2012

2011

2012

Current Current Current Current Current Current

402 408 411 601 604

389 402 421 602 596

49.2 48.2 78.6 48.8 78.6

46.5 43.4 73.3 42.5 74.4

CO2 and O3 CO2 and O3 O3 160% CO2 þ200 mL L1 CO2 þ200 mL L1 O3 160%

a Ambient refers to the values measured outside the chambers, the gas concentrations inside chambers was based on the values measured simultaneously in the ambient. C-O3 ¼ current O3 concentration; E-O3 ¼ elevated O3 concentration. The seasonal means are daylight averages. For CO2 concentration, daylight refers to the period from sunrise to sunset; for O3 concentration, daylight refers to 9:00 AM to sunset.

CO2 or O3 concentrations were recorded at 1-min interval throughout the experiment. The average values from 1-week after plant transplanting to the complete of grain filling are shown in each year. Ozone was produced by an electric discharge ozone generator (QD0013A, Jiahuan, Guangzhou, China) using pure O2as the source gas, in order to avoid the formation of other pollutants. Ozone was delivered to the chamber after quick mixture with air in a gas mixture unit through a high-speed fan, and the flow rate of the O3-enriched gas was regulated by controlling the output of the ozone generator. A data logger-controller calculated the O3 flow demand based on the O3 concentration measured by an ozone analyzer and the target O3 level, and sent the signal to the ozone generator to regulate the O3 flow rate. All ozone analyzers were calibrated against a transfer standard (Thermo Electron 49i-PS, Thermo Scientific Co., Franklin, MA, USA) on a monthly basis. Pure CO2 was mixed with air in a mixture box before being introduced into each chamber, and the flow rate of CO2 was controlled by a CO2 electromagnetic valve. The CO2 monitor took air samples from each chamber at 1-s interval and analyzed the CO2 concentration, then sent a signal to the data logger-controller to calculate the CO2 flow demand. The signal of CO2 demand was sent to CO2 electromagnetic valve to regulate the CO2 flow rate. Microclimatic conditions were monitored at 1-min interval in each chamber as well as outside the chambers (Ambient), and values did not differ significantly between the chambers and the ambient air in each year. Average temperature was 26 C (24 h mean) in 2011 and 27 C in 2012; Average relative air humidity was 79% (24 h mean) in 2011 and 80% in 2012; average light intensity was 7.1 Klux (24 h mean) in 2011 and 9.9 Klux in 2012. 2.3. Determination of grain quality traits At maturity, 6 subsamples (hills) were obtained from each chamber in each year. After harvest, the grains were threshed carefully and fertile grains were selected using an airflow separator. The evaluation of rice processing quality (brown rice percentage and milled rice percentage) and appearance characteristics (chalky grain percentage, chalkiness area and chalkiness degree) followed the standard methods from China National Standard GB/T 17891-1999 for rice quality evaluation issued by the ministry of Agriculture, PR China (Supervising Department of Quality and Technology of China, 1999). Brown rice percentage is defined as the weight percentage of brown rice obtained from a sample of rough rice, while milled rice percentage is expressed as weight percentage of milled rice obtained from a sample of rough rice. The head rice refers to the milled rice with a length greater or equal to three quarters of the whole kernel, and head rice percentage is expressed as weight percentage of head rice obtained from a sample of rough rice. The chalky grain percentage and chalkiness area of head rice was measured using a chalkiness visualize (China National Rice Research Institute, Hangzhou, China). Chalky grain percentage is expressed as the percentage of number of chalky head rice to number of total head rice, while chalkiness area is expressed as the ratio of the area of chalkiness to the area of the whole head rice. Chalkiness degree was calculated by multiplying the chalky grain percentage by chalkiness area, this parameter assess the chalkiness of whole sample, not individual grains. After evaluation of the appearance, the milled rice samples were oven-dried at 60 C to constant weight. The samples were then ground with a stainless steel grinder with a 100-mesh sieve for subsequent analyses of starch properties and mineral element concentrations. The methods for amylose content and gel consistency of milled rice samples were also based on China National Standard GB/T 17891-1999. Amylose content was determined by using a colorimetric method which is based on the ability of amylose to bind to iodine. Gel consistency was measured by the length of cold milled rice paste in a test tube in horizontal position. Rapid Viscosity Analyzer (RVA) profiles of starch in grains were analyzed with Super 3 Rapid Viscosity Analyzer (Newport Scientific Co., Sydney, Australia) using the Thermal Cycle for Windows software. The RVA paste viscosity was determined

Y. Wang et al. / Environmental Pollution 189 (2014) 9e17 according to the protocol from the American Association of Cereal Chemists (AACC 1995-61-02). The measurements of total starch, resistant starch and digestible starch followed the methods described by Frei et al. (2003). Total nitrogen concentration was measured by using the Kjeldahl method. One gram of milled rice powder was digested in concentrated H2SO4 at 420 C for 1 h with K2SO4 and CuSO4 as catalyst, the N concentration in the resultant solution was determined automatically by the Kejeltec 2300 autoanalyzer (Foss Tecator AB, Hoganas, Sweden). The analyses of non-protein N (NPN) followed the protocol of (Makkar and Becker, 1997). In brief, 2 g of finely ground sample material was homogenized in 10 ml of phosphate buffer (pH 7.0, 0.05 M) for 6 min. After centrifugation (15 min, 3500 g), 5 ml of the supernatant were mixed with 5 ml of 20% trichloroacteic acid and kept overnight in a refrigerator. After further centrifugation (15 min, 3500 g) the protein free supernatant was concentrated by freeze drying and the N in each sample was determined using a Vario max CN-Analyzer (ElementarAnalysenysteme GmbH, Hanau, Germany). This N fraction was referred to as NPN. Protein N (PN) was calculated by subtracting the NPN from Total N. For the measurement of nutrient element concentrations, 0.5 g ground milled rice samples were wet digested with HNO3 in a microwave oven (MARS 5, CEM Corporation, Matthews, USA). After dilution with ultrapure water, the solution was filtered and the concentrations of potassium (K), magnesium (Mg), calcium (Ca), phosphorous (P), iron (Fe), zinc (Zn), manganese (Mn) and copper (Cu) in the filtrate were quantified by ICP-AES (IRIS Intrepid II XSP, Thermol Elemental, Franklin, USA). The rice standard reference material (GBW080684) was used to ensure precision of the analytical procedures.

2.4. Statistical analyses The experiments were carried out in four chambers, i.e. one chamber for each of the four treatments. Therefore, the whole experiment was replicated over time in two seasons (2011 and 2012). Subsamples taken from within one chamber in one year were not treated as replicates. Data were analyzed using a mixed model in PROC MIXED of SAS 9.3 (SAS Institute Inc., Cary, NC). The model included O3 concentration, CO2 concentration and their interaction as fixed factor and year and CO2 by O3 by year interaction as random factors. Treatments were compared by Tukey test and differences were declared as statistically significant if p < 0.05.

3. Results The processing quality closely relates to final milled rice yield and impacts the income of rice growers. The processing suitability of rice grains is often evaluated by brown rice percentage, milled rice percentage and head rice percentage. The effect of elevated CO2 and/or O3 concentrations on the three parameters is presented in Tables 2 and 3. The differences between any combination of CO2 and O3 treatments on processing quality were non-significant. Among three parameters assessing grain chalkiness, the O3 treatment effects were most significant for chalkiness degree, followed by chalky grain percentage, and then chalkiness area (Table 2). There was a trend towards increasing chalky grain percentage, chalkiness area, as well as chalkiness degree of rice grains from the CO2 and/or O3 elevation treatments, with the highest values obtained from the rice grown under elevated O3 alone, followed by the combination of elevated CO2 and O3, elevated CO2 alone, and the control conditions (current CO2 and O3 concentrations) (Fig. 1). The results indicate that both CO2 and O3 had negative effects on appearance quality, with O3 showing more profound impacts. However, compared with the current CO2 conditions, the impact of elevated O3 was mitigated by elevated CO2. The concentrations of potassium, phosphorus, magnesium, manganese, copper and zinc in milled rice from the various CO2/O3 treatments were analyzed (Fig. 2 and Table 2). Compared with the control treatment, elevated CO2 tended to decrease the concentrations of nutrient elements, but differences were only statistically significant for zinc and copper (p < 0.05). In contrast elevated O3 tended to increase the elemental concentrations, which was particularly significant for zinc and copper. Zinc and copper concentrations almost doubled under elevated O3 compared with other treatments. Interestingly, the combination of elevated CO2 and elevated O3 treatments exhibited almost the same value as in current CO2 and O3 treatments for each element, which suggests

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Table 2 Analysis of variance for quality properties of rice grains in response to CO2 and/or O3 enrichment. Parameters

F-value

Brown rice percentage Milled rice percentage Head rice percentage Chalky grain percentage Chalkiness area Chalkiness degree K concentration Mg concentration P concentration Zn concentration Cu concentration Mn concentration N concentration NPN concentration PN concentration Total starch concentration Amylose concentration Amylopectin concentration Ratio of amylose to amylopectin Resistance starch Digestible starch Gel consistency Gelatinization temperature Peak viscosity Hot viscosity Breakdown Final viscosity Setback Consistency Peak time

P-value

CO2

O3

CO2 O3 CO2

O3

CO2 O3

0.21 0.05 3.12 0.21 0.02 0.36 4.60 2.87 3.39 10.95 36.06 9.34 10.81 0.00 10.79 0.48

8.38 4.95 0.45 22.78 11.34 33.41 1.67 1.96 0.98 21.71 31.15 1.23 33.03 0.02 32.99 3.62

4.49 0.46 7.15 9.55 0.93 5.90 0.07 0.00 0.02 7.29 9.43 0.10 2.49 3.44 2.35 0.04

0.0627 0.1125 0.5500 0.0175 0.0435 0.0103 0.2864 0.2564 0.3950 0.0186 0.0114 0.3489 0.0105 0.8930 0.0105 0.1531

0.1244 0.5472 0.0754 0.0537 0.4065 0.0935 0.8146 0.9794 0.8871 0.0738 0.0545 0.7678 0.2128 0.1607 0.2228 0.8505

0.6807 0.8389 0.1755 0.6775 0.9070 0.5899 0.1212 0.1888 0.1628 0.0454 0.0093 0.0552 0.0462 0.9613 0.0463 0.5401

20.57 19.27 2.04 2.33 0.11 1.21

0.0201 0.0219 0.2487 0.2245 0.7585 0.3518

28.87 14.24 4.45

0.0126 0.0326 0.1255

0.23 0.44 4.05 0.03

2.43 3.29 0.03 2.40

0.01 0.04 0.28 0.01

0.6652 0.5558 0.1376 0.8728

0.2166 0.1673 0.8718 0.2192

0.9255 0.8551 0.6348 0.9225

10.04 16.71 6.25 12.12 5.91 2.99 1.27

7.19 8.01 6.41 7.70 4.85 1.61 6.02

1.84 4.44 0.69 2.52 1.07 0.26 0.08

0.0505 0.0264 0.0877 0.0400 0.0933 0.1822 0.3416

0.0749 0.0661 0.0853 0.0692 0.1150 0.2936 0.0914

0.2679 0.1256 0.4659 0.2109 0.3763 0.6443 0.7926

The p values in bold show significant treatment effects at p < 0.05; Data were analyzed by mixed model ANOVA.

that elevated CO2 counteracted the effect of elevated O3 in this aspect. Nitrogen concentration in milled rice is closely related with protein content. The nitrogen in rice grains can be classified as protein N (PN) and non-protein N (NPN). More than 95% of total N in milled rice was protein N (Fig. 3). Averaged over two years, elevated ozone increased total N concentration in milled rice by 49.1% under current CO2 conditions (p ¼ 0.01, Table 2), which was mainly due to the increase in protein N. On the contrary, elevated CO2 reduced the total N as well as the PN concentrations of milled rice (p ¼ 0.05, Table 2). The impact of elevated O3 was moderated by elevated CO2 as shown by less increase in total N or PN by elevated O3 under elevated CO2 conditions, which indicated elevated CO2 counteracted effect of elevated O3 on N concentration in milled rice.

Table 3 Effects of CO2 and/or O3 enrichment on processing quality of rice grains. Elevated CO2

Parameters

Current CO2 C-O3

E-O3

C-O3

E-O3

Brown rice percentage (%) Milled rice percentage (%) Head rice percentage (%)

82.0 0.1

81.2 0.2

81.6 0.0

81.4 0.1

71.9 2.9

70.3 3.5

71.6 2.6

70.8 2.7

57.0 13.0

52.3 13.8

50.8 10.7

53.6 12.5

C-O3 ¼ current O3 concentration; E-O3 ¼ elevated O3 concentration. Values are means SE.

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stickiness of cooked rice. Among all four treatments, the elevated O3 showed the lowest value in the peak viscosity, hot viscosity, final viscosity, breakdown, but the highest value in setback and peak time, while elevated CO2 did not alter RVA profile of rice starch. However, elevated CO2 modified the influence of elevated O3 on starch pasting properties as shown by very similar RVA profiles for the grains from the current CO2 and O3 treatments and the combination of elevated CO2 and elevated O3 treatments. 4. Discussion

Fig. 1. Chalky grain percentage (A), chalkiness area (B) and chalkiness degree (C) in grain samples of Shanyou 63 exposed to different levels of CO2 and/or O3. Bars represent average values with standard errors; Bars not sharing the same letter differ significantly at p < 0.05. C-O3 ¼ current O3 concentration; E-O3 ¼ elevated O3 concentration.

No clear differences were found between treatments on NPN concentration in rice, indicating that the additional N in elevated O3 treated grains was in the form of protein. The total starch of rice can be classified into amylose and amylosepectin based on the structure of starch, or grouped into resistant starch and digestible starch according to the digestibility of starch. When averaged over two years, significant CO2 and O3 treatment effects were only detected for amylose concentration and the ratio of amylose to amylopectin, while no significant effects were found for concentrations of total starch, amylopectin, resistant starch as well as digestible starch (Table 2). However, although statistically not significant, total starch, resistant starch and digestible starch showed a consistent trend towards a decrease under elevated O3 treatments (Table 4). Elevated O3 decreased amylose concentration of milled rice by 15.1% when plants were grown in current CO2, but no significant O3 effects were observed for plants grown in elevated CO2. The elevated CO2 and/or O3 treatments exhibited no significant effects on gel consistency or gelatinization temperature of milled rice (Tables 2 and 4). Starch pasting properties of ground milled rice were characterized by the RVA profile (Fig. 4), which assess the texture and

The chalkiness of rice grains is a key factor determining the appearance quality of rice, since the majority of consumers prefer rice with a transparent appearance. In agreement with previous reports on grain appearance quality (Yang et al., 2007; Wang et al., 2012), the grain chalkiness was increased by elevated CO2 or O3 (Fig. 1). An increase of 13.1% in chalky grain percentage and 25.4% in chalkiness degree by elevated CO2 in the present study was comparable with the FACE results for a japonica cultivar by Yang et al. (2007), where the respective increase of the two parameters was 16.9% and 28.3%. A fluctuating grain-filling rate (excessive grainfilling rate during early grain filling stage and incomplete filling during late grain-filling stage) was suggested to be a possible reason for increased grain chalkiness under elevated CO2 conditions (Yang et al., 2007). In the case of O3, the premature senescence shortened the grain filling duration and might have resulted in incomplete grain filling (Wang et al., 2012). Our FACE study showed that Shanyou 63 grown under O3 stress matured about one week earlier than the control plants (Shi et al., 2009). In the present study on the same hybrid, we also observed an 11.5 days earlier maturation under elevated vs. current O3 concentration (see Table 5). Although both elevated CO2 and O3 increased grain chalkiness, the combination of two gases did not further increase the chalky grain percentage, chalkiness area and chalkiness degree, which ranked between the two single gas treatments. This results indicates the interactive effects of CO2 and O3 on rice appearance quality are not simply additive. In general, the observed changes in mineral element concentrations induced by elevated CO2 or O3 alone (Fig. 2) are in line with previous results: under elevated CO2, the concentrations of nutrient elements showed a tendency to decrease compared with the control treatment (Loladze, 2002; DaMatta et al., 2010; Wang et al., 2011a), while an opposite trend occurred under elevated O3 (Zheng et al., 2013; Frei et al., 2012; Wang et al., 2012). However, the O3 effects were more pronounced than CO2 in terms of micronutrients Zn and Cu. A strong and significant O3 effect on Zn concentration in this study was in line with the earlier observation on wheat (Pleijel, 2012). These results indicated that although the general picture of O3 effect on rice is negative, a positive effect of O3 exist through enriching micronutrient density in grains, which might be beneficial for battling Zn deficiency in rice consumers. Changes in element concentrations under elevated CO2 or O3 conditions have been attributed to changes in dry matter or carbon assimilation (dilution effect) (Lieffering et al., 2004; Wang and Frei, 2011; Loladze, 2002). CO2 elevation tends to increase and O3 pollution tends to decrease rice dry matter or carbon assimilation (Ainsworth, 2008), which may result in dilution or enrichment of mineral elements. The observed negative correlation between total starch and all measured mineral elements (N, P, K, Mn, Mg and Cu: r ¼ 0.31* to 0.49**; Zn: r ¼ 0.28ns) also support the “dilution effect”. Despite “dilution effect”, other mechanisms specifically relevant to particular element may also exist, since the magnitude of each element in response to CO2 or O3 is different (Fig. 2). In a recent report on wheat, Pleijel (2012) found the changes on grain Zn concentration by O3 was almost twice larger than that on grain


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Fig. 2. K concentration (A), Mg concentration (B), P concentration (C), Zn concentration (D), Cu concentration (E) and Mn concentration (F) in milled rice of Shanyou 63 exposed to different levels of CO2 and/or O3. Bars represent average values with standard errors; Bars not sharing the same letter differ significantly at p < 0.05. C-O3 ¼ current O3 concentration; E-O3 ¼ elevated O3 concentration.

protein concentration, which may be attributed to the different availability of N and Zn to plants. While all elements showed the same trends toward elevated CO2 or O3, only a few showed statistically significant effects. Nitrogen or protein is the only element showing consistent trend in the scientific literature so far:elevated CO2 decreased N/protein concentration (Taub and Wang, 2008; Wang et al., 2011a) while elevated O3 increased N/protein concentration (Zheng et al., 2013; Frei et al., 2012; Wang et al., 2012). Our results are in agreement with the previous reports, but the new finding in the present study is that the changes in total N concentration under elevated CO2 or/ and O3 is mainly due to changes in protein N, while no changes in NPN were found (Fig. 3). The NPN could be free amino acids glutamate (Gln) and asparagine (Asn), which are the dominant amino acids found in the endosperm sap of developing rice grains (Hayashi and Chino, 1990), or small peptides, amines, amides, nucleic acids, or inorganic N-compounds such as urea or ammonium salts, which have no nutritional value in human diets (PérezConesa et al., 2005). The results indicate the protein synthesis in rice grains may not have been affected by elevated CO2 or O3. The protein N changes by elevated CO2 or O3 in this study could be attributed to the concentration effect proposed by previous researchers, i.e., the stimulated or inhibited production of carbohydrates by elevated CO2 or O3 dilutes or enriches plant protein

(Gifford et al., 2000; Taub and Wang, 2008; Frei et al., 2012). Similar conclusions were also reached in soybean by using meta-analysis: Rotundo and Westgate (2009) found the increase in protein concentration did not necessarily result from a stimulation of protein synthesis, but rather from a concentration effect due to reduced biomass production under stress. In rice, starch content and grain mass reduction under ozone stress have been associated with the concomitant increase in protein concentration (PN) of grains (Huang et al., 2012; Frei et al., 2012). A non-significant decrease in starch content (Table 4) and a significant decrease in grain mass (Table 5) were also observed in this study, which is partly due to the shortened grain filling period. Across the two years, the maturity with elevated O3 occurred about 10 days earlier compared to under ambient O3 (Table 5) which could have affected leaf lifespan and thus synthesis of carbohydrates for the seeds which depends primarily on concurrent carbon fixation during seed filling (Yamagata et al., 1987; Gebbing and Schnyder, 1999). In addition, ozone stress could also inhibit carbohydrates from entering the grain by relocating more carbon assimilates to the leaves to cope with the oxidative stress (Betzelberger et al., 2010; Wilkinson et al., 2012). In contrast, protein accumulation in the grain was less affected by shortened grain filling period, because accelerated leaf senescence leads to nitrogen remobilization from vegetative tissue, and amino acids derived

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Y. Wang et al. / Environmental Pollution 189 (2014) 9e17 Table 4 Effects of CO2 and/or O3 enrichment on starch property of milled rice. Parameters

Total starch concentration (%) Amylose concentration (%) Amylopectin concentration (%) Ratio of amylose to amylopectin (%) Resistance starch (RS, %) Digestible starch (DS, %) Gel consistency (mm) Gelatinization temperature ( C)

Elevated CO2

Current CO2 C-O3

E-O3

C-O3

E-O3

72.2 1.7

69.7 1.3

73.5 1.5

70.4 1.1

21.2 0.9

18.0 0.1

22.9 0.4

21.2 0.5

51.0 0.8

51.8 1.2

50.6 1.2

49.2 0.9

0.41 0.01

0.35 0.01

0.45 0.00

0.43 0.00

1.37 70.8 69.1 80.1

0.03 1.27 1.7 68.5 1.2 70.2 0.9 82.0

0.00 1.41 1.3 72.1 5.5 76.9 3.6 79.7

0.01 1.30 1.6 69.1 0.9 74.8 0.6 81.9

0.08 1.2 4.3 1.3

C-O3 ¼ current O3 concentration; E-O3 ¼ elevated O3 concentration. Values are means SE.

Fig. 3. Nitrogen concentration (A), non-protein nitrogen (NPN) concentration (B) and protein nitrogen (PN) concentration (C) in milled rice of Shanyou 63 exposed to different levels of CO2 and/or O3. Bars represent average values with standard errors; Bars not sharing the same letter differ significantly at p < 0.05. C-O3 ¼ current O3 concentration; E-O3 ¼ elevated O3 concentration.

from the protein degradation in leaf compensate for the decrease in grain filling time and the nitrogen shortage under stress conditions (Triboi and Triboi-Blondel, 2002). As a consequence of this enhanced amino acid remobilization, protein synthesis may less affected by stress than other major components such as starch in cereals and lipids in legumes (Rotundo and Westgate, 2009; Wang and Frei, 2011). In addition, no limiting in the availability of micronutrients such as Zn and Cu under ozone stress may also beneficial for protein synthesis in developing seeds. However, the overall nitrogen acquisition of plants are usually reduced due to the adverse effects of O3 on plant vitality, which results in lower protein yield at the harvest (Pleijel and Uddling, 2012; Frei et al., 2012). The concentration effect also applied to combined CO2 and O3 experiments. In an earlier OTC experiment conducted across four countries, a negative linear correlation between grain yield and grain PN was found when spring wheat were exposed to different concentrations of CO2 and O3 (Pleijel et al., 1999). A strong negative correlation between total starch and PN in this study (r ¼ 0.49**) verifies the close association between PN and starch accumulation in cereal grains.

The cooking and eating quality of rice is closely related with the physicochemical properties of starch in the endosperm. Although we did not find significant treatment effects in gel consistency and gelatinization temperature of rice starch, the lower amylose concentration or the amylose to amylopectin ratio of the O3-treated grains (Tables 2 and 4) suggested softer texture of rice after cooking (Juliano, 1979). However, the lower peak viscosity, hot viscosity, final viscosity, breakdown, but higher setback in starch RVA profile of grains from elevated O3 treatments indicated increased firmness and deterioration of sensory acceptability of cooked rice (Chrastil, 1994; Okadome et al., 1999; Allahgholipour et al., 2006). Similar apparently contradicting effects on starch pasting properties by amylose and RVA profile were also found in the previous FACE study (Wang et al., 2012), which was explained with the influence of protein on starch viscosity, implying that protein could mask the effects of amylose. It is well documented that high N/protein concentration is associated with the deterioration of taste properties (Terao et al., 2005; Juliano et al., 1965; Ishima et al., 1974; Matsue et al., 1997; Yamashita and Fujimoto, 1974). In the present study, correlation analysis also show a strong positive correlation between N/protein concentration and setback (r ¼ 0.88**) but negative correlations between N/protein concentration and peak viscosity (r ¼ 0.88**), hot viscosity (r ¼ 0.78**), breakdown (r ¼ 0.88**) and final viscosity (r ¼ 0.85**). In contrast to ozone, the starch-pasting properties of rice exposed to elevated CO2 showed an opposite trend (i.e., breakdown þ14%, setback 25%), though effects were not statistically significant. Similar results were also observed for a japonica cultivar in our previous FACE study (Yang et al., 2007). The combination of two gas treatments exhibited no differences on the traits relating to cooking and eating quality from that of control plants, indicating that elevated CO2 might nullify the negative effects of elevated O3 on eating and cooking quality. There is growing evidence that rising atmospheric CO2 concentration will reduce or prevent reductions in the growth and productivity of C3 crops attributable to O3 pollution (Polle and Pell, 1999; Morgan et al., 2003; Biswas et al., 2013). Our results contribute to this concept from the crop quality aspect. Although no significant CO2/O3 interaction occurred at the p < 0.05 level of confidence, some parameters exhibited significant CO2/O3 interaction at p < 0.1 (Table 2). Moreover, from multiple comparisons between the four treatments as shown in the figures, it is clear that O3 affect many quality traits (chalkiness, PN, Zn, Cu, hot viscosity, setback) significantly at current CO2, but becomes non-significant at elevated CO2. These results indicate that elevated CO2 can modify the ozone impacts on rice quality. The mechanisms


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Fig. 4. The values of peak viscosity (A), hot viscosity (B), breakdown (C), final viscosity (D), setback (E) and peak time (F) in the RVA profile of milled rice of Shanyou 63 exposed to different levels of CO2 and/or O3. Bars represent average values with standard errors; Bars not sharing the same letter differ significantly at p < 0.05. C-O3 ¼ current O3 concentration; E-O3 ¼ elevated O3 concentration.

underlying the amelioration of O3-induced damage by elevated CO2 are not well understood, but ozone exclusion or reduced ozone uptake through stomatal closure by elevated CO2 have been considered as the major factor responsible for the protection against O3 (Cardoso-Vilhena et al., 2004; McKee et al., 1997; Broadmeadow and Jackson, 2000; Olszyk and Wise, 1997). Decreased stomatal conductance due to elevated CO2 decreases ozone uptake (McKee et al., 1995; Medlyn et al., 2001); Less ozone uptake will lead to less ozone damage on leaves, and thus more healthy green leaves for carbon assimilation and for biomass growth and/or grain filling (Wilkinson et al., 2012). The cumulative

Table 5 Effects of CO2 and/or O3 enrichment on phenological development and individual grain mass. Treatments CO2

O3

Current CO2

C-O3 E-O3 C-O3 E-O3 CO2 O3 CO2 O3

Elevated CO2 ANOVA results

Whole growth duration (days) 111.5 100.0 108.5 102.0 0.836 0.016 0.331

1.5 3.0 0.5 3.0

Individual grain mass (mg) 24.2 22.0 25.5 23.4 0.103 0.029 0.971

0.3 0.9 0.7 0.5

C-O3 ¼ current O3 concentration; E-O3 ¼ elevated O3 concentration. Whole growth duration means growth period from transplanting to maturity. Values are means SE. Data were analyzed by mixed model ANOVA.

protection by high CO2 against O3 damage over the whole growth season may account for the observed few changes in rice quality when comparing effects of elevated CO2 and O3 with current CO2 and O3 treatments, since grain quality changes due to environmental stress is a cumulative process involving numerous physiochemical changes at different levels (Wang and Frei, 2011). Therefore, we need to keep in mind that the mechanisms responsible for rice grain quality changes under elevated CO2 are not simply the opposite of that under elevated O3. A recent strong evidence provided by Pleijel and Uddling (2012) through a large range of experiments for wheat revealed that in addition to “dilution effect”, elevated CO2 (not O3) has a direct negative effect on grain protein accumulation independent of yield effect. In conclusion, the projected levels of atmospheric CO2 and tropospheric O3 had different effects on several quality characteristics including mineral element concentrations, RVA profiles, and the responses being more pronounced in the case of O3. Grain chalkiness was an exception as it was increased by both factors. The combination of elevated CO2 and O3 caused fewer significant changes in grain quality than individual gas treatments, leading us to conclude that predictions of rice quality in a future atmosphere need to consider both factors. Acknowledgments This work was funded jointly by the National Natural Science Foundation of China (Grants Nos. 31171460, 31371563, 31071359),

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the Science Bridge Asia Program of the Robert Bosch Foundation (2011), and the PPP program jointly supported by CSC and DAAD (2012), the Major Fundamental Research Program of Natural Science Foundation of Jiangsu Higher Education Institutions of China (11KJA210003), and the Priority Academic Program Development of Jiangsu Higher Education Institutions.

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