Short‐term effect of elevated CO 2 concentration (0.5%) on mitochondria in diploid and tetraploid black locust (Robinia pseudoacacia L.)

Abstract Recent increases in atmospheric CO 2 concentration have affected the growth and physiology of plants. In this study, plants were grown with 0.5% CO 2 for 0, 3, and 6 days. The anatomy, fluorescence intensity of H2O2, respiration rate, and antioxidant activities of the mitochondria were analyzed in diploid (2×) and tetraploid (4×) black locust (Robinia pseudoacacia L.). Exposure to 0.5% CO 2 resulted in clear structural alterations and stomatal closure in the mitochondria. Reduced membrane integrity and increased structural damage were observed in 2× plants at 6 days. However, after 0.5% CO 2 treatment, little structural damage was observed in 4× plants. Under severe stress, H2O2 and malondialdehyde were dramatically induced in both 2× and 4× plants. Proline remains unchanged at an elevated CO 2 concentration in 4× plants. Moreover, the total respiration and alternative respiration rates decreased in both 2× and 4× plants. In contrast, the cytochrome pathway showed no decrease in 2× plants and even increased slightly in 4× plants. The antioxidant enzymes and nonenzymatic antioxidants, which are related to the ascorbate–glutathione pathway, were inhibited following CO 2 exposure. These analyses indicated that 4× and 2× plants were damaged by 0.5% CO 2 but the former were more resistant than the latter, and this may be due to increases in antioxidant enzymes and nonenzymatic antioxidants and stabilized membrane structure.

CO 2 in the atmosphere is inadequate to saturate the ribulose-1,5-bisphosphate carboxylase (RuBisCO) enzyme that drives photosynthesis in C 3 plants (Taiz & Zeiger, 1991). In comparison, C 4 -type plants are likely to respond less to elevated CO 2 levels as they possess an innate concentrating mechanism that increases CO 2 level at the site of RuBisCO to 2,000 ppm. Moreover, studies have indicated that EC affects rise of tiller number, net photosynthetic rate, and morphology, as well as yield enhancement (Hasegawa et al., 2013;Liu et al., 2008;Shimono et al., 2009). To date, variability in plant respiration has been observed and the underlying mechanism has been elucidated (Hu et al., 2006). While plant growth, development, and function in increased CO 2 concentrations have been extensively researched (Zinta et al., 2014), the effects of elevated CO 2 on the ultrastructure and function of mitochondria in polyploid plants are relatively unknown.
Tetraploid black locust (Robinia pseudoacacia L.) (TBL) is native to Korea. This species is cultivated for its wood throughout the world (Isely & Peabody, 1985). Its leaves are rich in many types of vitamins and minerals, which can be used for the food industry. Moreover, TBL can tolerate abiotic stresses, such as drought, salt, and low temperature (Joshi, Bourges-Sévenier, Russell, & Mo, 2012). Its bark and roots can be used for drug development and disease treatment due to its abundance of flavonoids (Garlock, Yi, Balan, & Dale, 2012). Because of its rapid growth and pleasant fragrance, TBL has been cultivated as a useful component of secondary forests in gardens and on roads.
However, the response of TBL to high concentrations of CO 2 has not been elucidated.
Many polyploid plants have a higher tolerance to environmental stresses than their diploid counterparts (Meng, Pang, Huang, Liu, & Wang, 2012). Conversely, research has shown that the volume of organelles in polyploid plants is greater than that in their corresponding diploid relatives. Nevertheless, mitochondria are ultimately responsible for oxidative phosphorylation in metabolic processes (Plaxton & Podestá, 2006) and are sensitive to various abiotic stresses (Halliwell & Gutteridge, 1981). Mitochondria are the primary cellular organelles that respond to elevated CO 2 levels, which has been the topic of extensive research. While the acute response of plants to elevated CO 2 has been studied, the precise mechanism by which EC affects mitochondria in polyploid woody plants is presently unknown. In particular, understanding the basis for this variation in polyploid woody plants exposed to rising CO 2 concentrations is important for further selection and development of elevated [CO 2 ]-responsive crop lines. CO 2 as a abiotic factor can affect plant growth and development.
Previous studies have suggested that changes in morphology and photosynthesis may be related to high CO 2 . However, little or no work has been performed to investigate black locust responses to high CO 2 . In this study, the effect of elevated concentrations of CO 2 on the anatomy, respiration, and antioxidant activity of diploid (2×) and tetraploid (4×) plants was studied after the plants were exposed to 0.5% CO 2 for 0 (up) and 6 (down) days. Based on this analysis, we increased our understanding of the mechanism underlying the CO 2 response and tolerance of plants to environmental stress.

| Stomatalapertureandchloroplast ultrastructure
Fresh leaf sections (1 cm 2 ) were immediately fixed in 3% glutaraldehyde in 0.1 M phosphate buffer at 4°C for 2 hr and thoroughly washed in cacodylate buffer (0.1 M, pH 6.8) twice, with 10-min intervals between each washing. They were then dehydrated in a graded ethanol series (30%, 50%, 70%, 80%, 90%, 90.5%, and 100%) with 10 min each time, and the 100% ethanol wash was repeated twice. After dehydration, the samples were further dried in acetone and embedded in an Epon-Araldite mixture. For scanning electron microscopy (SEM), the samples were pasted to copper stubs with colloidal silver and were sprayed with 50 nm gold. Then, the samples were observed and photographed using a scanning electron microscope (JSM-5310LV, Japan).

| DeterminationofH 2 O 2 in guard cells
Detection of H 2 O 2 in guard cells was performed as previously described by Comai (2005). H 2 O 2 generation in the stomata was assessed with H 2 DCF (2, 7-dichlorodihydrofluorescein diacetate), a specific fluorescence probe for H 2 O 2 . The young leaves were harvested after 0, 3, and 6 days of 0.5% CO 2 treatments from both 2× and 4× plants. The young epidermal strips were incubated in 10 mM MES-KCL buffer (pH 7.2).
Then, 50 μM H 2 DCF was added at room temperature and incubated for 20 min in the dark. The leaves were rinsed with MES buffer twice to remove the additional fluorescence detector. The fluorescence of the H 2 O 2 probe was measured with an Axioskop 2 plus microscope (Zeiss) (excitation wavelengths of 488 nm).

| Respirationmeasurements
Leaf respiration was measured at room temperature using a Clark oxygen electrode (Hansatech, England) that was inserted into a 2-ml cuvette on a magnetic stirrer. The data were collected by a computer.
The slices were continuously stirred to dissolve the oxygen. Alternative respiration (alt) was sensitive to SHAM, and cytochrome respiration (cyt) was sensitive to NaN 3 . To distinguish between alt respiration and the cyt respiration, we added 5 mM SHAM and 0.1 mM NaN 3 to the suspension, respectively. The rate of respiration was surveyed at 5 min in the presence of inhibitors. We then calculated the percentage inhibition of the total respiration rates.
Then, the homogenate was centrifuged at 4,000 r/min for 10 min. The supernatant was centrifuged at 10,000 r/min for 10 min. The pellet was resuspended in washing buffer 20 mM HEPES/KOH (pH 7.8), 330 mM sorbitol, 10 mM NaCl, 2 mM EDTA, and 5 mM Na ascorbate] and centrifuged for 20 min at 4000 r/min. Then, the supernatant was centrifuged at 10,000 r/min for 10 min. The intact mitochondria were collected, washed, and centrifuged at 12,000 r/min for 20 min in PBS.

| AnalysisofH 2 O 2 ,MDA,andproline
H 2 O 2 was analyzed according to Sergiev et al. (1997). The absorbance of the supernatant was measured at 390 nm, and the H 2 O 2 concentration was obtained using a standard curve. For measurement of malondialdehyde (MDA) content, 1.5 g fresh leaf was homogenized with 0.1% trichloroacetic acid (TCA). After centrifugation at 12,000 r/min for 10 min, 1 ml of the supernatant was collected and mixed with 2 ml 0.5% thiobarbituric acid (TBA). Then, the mixtures were heated in boiling water (100°C) for 30 min. The homogenate was centrifuged at 12,000 r/min for 5 min. The absorbance changes at 450, 532, and 600 nm were monitored at 25°C.
Proline content was measured following the method of Kong et al. (2014), with minor modifications. The leaves (1.5 g) were extracted in 3% sulfosalicylic acid in a mortar and pestle at 4°C. After incubation at 100°C for 10 min, 1.5 ml of ninhydrin reagent (2.5% ninhydrin, 60% glacial acetic acid, and 40% 6 M phosphoric acid) and 1 ml of glacial acetic acid were added to 1 ml of the leaf extract at room temperature. The mixture was heated again in boiling water (100°C) for 30 min. Then, 3 ml of toluene was added, and the sample was incubated on an ice bath for 1 hr. The absorbance change was monitored at 520 nm.

| Measurementsofnonenzymaticantioxidants
Total AsA content (AsA+DHA) was determined using a modified protocol from Law, Charles, and Halliwell (1983). Briefly, 1.5 g of leaf sample was homogenized with 0.5% sulfosalicylic acid, and the supernatants were obtained by centrifugation for 20 min (12,000 r/min; 4°C). Then, 10 mM DTT and 0.5% N-ethylmaleimide were added to 0.1 ml extract. After the sample was mixed for one minute, 0.7 ml double-distilled water, 4% α′-dipyridyl in 70% ethanol, 10% trichloroacetic acid and 44% phosphoric acid were successively added to the mixture. Then, the mixture was heated to 40°C for 40 min. Then, 3% FeCl 3 was added. The absorbance was measured at 525 nm. AsA content was determined as total AsA, with the exception of 10 mM DTT and 10 mM N-ethylmaleimide.
The total content of reduced glutathione (GSH+GSSG) was measured as described previously Ellman (1959). Briefly, 100 μl of the extract was incubated with 100 mM PBS, 0.6 mM DTNB, and 2 mM NADPH for 10 min at 25°C. The reduced glutathione content (GSH) was measured at 412 nm as total reduced glutathione, with the exception of 2 mM NADPH.

| Stomatamorphology
Figure 2a,d shows a typical SEM image of the 2× and 4× plant stomata structure. Stomatal openings in the leaves of 2× plants that were exposed to CO 2 for 3 days were fewer than those of the control (Figures 2b, 3). At the same time, exposure to the gaseous CO 2 for 6 days resulted in total closure of the 2× plant leaves ( Figure 2c). Interestingly, in the 4× plants, 0.5% CO 2 induced the complete closure of guard cells after 3 days (Figure 2e,f). Figure 3 shows significant changes in the degree of stomatal opening in the 4× plants. In contrast, there was no significant change in the 2× plants.

| FluorescenceintensityofH 2 O 2
As expected, after 0.5% CO 2 exposure, accumulation of H 2 O 2 in the 4× plants was greater than that in the controls (Figure 4). During the treatment, H 2 O 2 levels increased gradually at 3 and 6 days in the 2× plants ( Figure 4). The relative fluorescence intensities of the 2× plants after 6 days of CO 2 stress was approximately 3.6-fold that of the control ( Figure 4). Moreover, accumulation of H 2 O 2 was significantly increased after 3 days of exposure to 0.5% CO 2 in 4× plants compared to that of 2× plants (Figure 4).

| ChangesinH 2 O 2 ,MDA,andproline
MDA was induced by 48.24% and 31.4% after 3 and 6 days of stress, respectively (Figure 5b). Proline content in the 2× plants was significantly enhanced by 70.2% after CO 2 stress for 6 days ( Figure 5c); however, these values were similar in the 4× plants between the control and treatment (Figure 5b,c). Moreover, the accumulation of H 2 O 2 in 2× plants after 6 days increased to 31.1% that of the CK (Figure 5a).

| Leafrespirationrate
To investigate the effects of high concentrations of CO 2 on plant respiration, we measured leaf respiration at 0, 3, and 6 days. As shown in Figure 6a, total respiration of the plants was temporarily increased at 3 days under elevated CO 2 compared to that at 6 days in 4× plants. In contrast, this ratio was decreased in 2× black locust plants under stress conditions (Figure 6a). The total respiration decreased to 51.9% and 41.6% in 2× and 4× plants, respectively ( Figure 6a).
Conversely, the alt respiration of 4× plants was slightly induced initially and clearly decreased at 6 days; however, 2× plants that were grown under CO 2 stress conditions showed a significant decrease in alt respiration rate from 0 to 6 days ( Figure 6b). The responses of plant cyt respiration to CO 2 were also explored ( Figure 6c). There were no significant changes in 2× plants compared with those in 4× plants, which showed a substantial alteration. The change in trend of residual respiration was similar to that of alt respiration in black locust leaves (Figure 6d).

| DISCUSSION
Due to anthropogenic carbon emissions and ecosystem processes, excessive CO 2 has been released into the atmosphere. This is predominantly due to cement production, fossil fuel burning, and landuse change (Leakey et al., 2009) resulting in climate change and dramatic increases in the CO 2 concentration. The changing CO 2 concentration has a vital role in plant growth and development.
Many reports have shown that TBL is highly tolerant to environmental stresses (Li et al., 2009;Podda et al., 2013;Yuan, Liu, Fang, Yang, & Mu, 2009 Generally, plants exposed to high concentrations of CO 2 generate excess excitation pressure, which can supply electrons in excess of that required for CO 2 fixation (Asada, 1999;Murchie & Niyogi, 2011). can control the activity of outward/inward-rectifying K + channels, enhance guard cell Ca 2+ concentrations, and induce cytosolic alkalinization, which results in stomatal closure through trigger water efflux (Brearley, Venis, & Blatt, 1997;Felle & Hanstein, 2002;Raschke, Shabahang, & Wolf, 2003). Stomata on the leaf surface continuously regulate gas exchange by a sophisticated mechanism in response to environmental changes. In this study, 2× plant stomatal closure was observed at 6 days and 4× plant closure was observed after 3 days ( Figure 2c,e). Stomatal closure may represent a protective response to abiotic stress. We found that 4× plants responded more rapidly to the stress than did 2× plants, which can decrease the CO 2 flowing into the leaves (Figure 3). The degree of stomatal opening significantly decreased in 4× plants at 3 days compared to that of 2× plants (Figure 3).

Mitochondria act as sensors, initiate stress responses in plant,
and are major producers of ROS (Nomura et al., 2012;Suzuki, Koussevitzky, Mittler, & Miller, 2011;Vanlerberghe, 2013). Under abiotic stress conditions, ROS were generated and accumulated in the guard cells, inducing both cellular damage and protective responses (Wu et al., 2013). In this report, the 0.5% CO 2 stress treatments induced notable disturbances in mitochondrial structure  (Hu et al., 2006). In the present study, 0.5% CO 2 treatment partly induced stomatal closure in both 2× and 4× plants. Therefore, the rate of total respiration decreased to 51.90% and 41.59% in both   (Moller, 2001;Siedow & Umbach, 1995).
These results indicate that the alt pathway can suppress the production of ROS due to short-term CO 2 stress (3 days; Figure 6b).
Temporary increases in the alt pathway have been suggested as an adaptive feature of plants exposed to stress (Mcnulty & Cummins, 1987). However, the mitochondrial membrane was damaged by exposure to 0.5% CO 2 for a longer period of time (6 days) in both 2× and 4× plants (Figure 1c,f). Furthermore, excessive ROS in the mitochondria damage one of the most vital cellular components: proteins/enzymes, which have an important role in plant growth and the antioxidant system. After the treatment, cyt respiration was not affected and was thus less sensitive to CO 2 than was alt respiration in black locust leaves (Figure 6c).
Under stress conditions, the AsA/GSH pathway is one of the major ROS detoxifying systems in the cytosol. This cycle consists of APX-, AsA-, GSH-, and the AsA/GSH-regenerating enzymes, including MDHAR, DHAR, and GR (Noctor, 2009) (Figure 7a,f).
Inhibition of APX activity may promote accumulation of H 2 O 2 , which can be determined by MDA content. AsA content was notably reduced after 3 days of stress ( Figure 7c). Moreover, the inhibition of GR activity also resulted in a decreased pool of GSH in both 2× and 4× plants (Figure 7f,g). DHAR activity was not changed in 4× plants and decreased in 2× plants following CO 2 stress (Figure 7e). The GSH/ GSSG ratio was increased in 4× plants but did not change in 2× plants ( Figure 7h). We also observed that this is a consistent protective response to stress. AsA is a vital nonenzymatic antioxidant in plant cells that eliminates excessive ROS and thus maintains the activity of antioxidant enzymes and stabilizes membrane structure. All results suggested that the 4× plants were more tolerant to CO 2 than were the 2× plants.
Another study showed that AsA and GSH levels substantially deceased under heat and drought stresses and that the AsA redox status was also higher in elevated CO 2 -grown plants. Conversely, the antioxidant system was not significantly changed following CO 2 treatments (730 ppm) (Zinta et al., 2014). In our experiments, the AsA/GSH cycle was inhibited under CO 2 stress, which was less pronounced under stress conditions. Moreover, in our previous study, 1% and 0.5% CO 2 were used to treat 2× and 4× plants, respectively, which resulted in the reduction of SOD and GR activity; however, not all antioxidant defense systems responded similarly to the elevated CO 2 conditions (Zinta et al., 2014). Recent reports have suggested that the inhibition of these antioxidant enzymes may be related to the enhanced oxidative damage in proteins (Aravind & Vara, 2005;Singh, Singh, Kumar, & Prasad, 2015). In conclusion, 4× and 2× plants were damaged by 0.5% CO 2 but the former was more resistant than the latter, which may be attributable to duplicate gene expression. Ultimately, the mechanisms underlying this difference require further investigation.

ACKNOWLEDGMENTS
This study was supported by the Fundamental Research Funds for the Central Universities (2572016EAJ4; 2572015DA03) and the National Natural Science Foundation of China (31360073).

CONFLICTOFINTEREST
The authors declare that they have no competing interests.