The effects of elevated CO 2 (0.5%) on chloroplasts in the tetraploid black locust (Robinia pseudoacacia L.)

Abstract Some ploidy plants demonstrate environmental stress tolerance. Tetraploid (4×) black locust (Robinia pseudoacacia L.) exhibits less chlorosis in response to high CO 2 than do the corresponding diploid (2×) plants of this species. We investigated the plant growth, anatomy, photosynthetic ability, chlorophyll (chl) fluorescence, and antioxidase activities in 2× and 4× black locusts cultivated under high CO 2 (0.5%). Elevated CO 2 (0.5%) induced a global decrease in the contents of total chl, chl a, and chl b in 2× leaves, while few changes were found in the chl content of 4× leaves. Analyses of the chl fluorescence intensity, maximum quantum yield of photosystem II (PSII) photochemistry (Fv/Fm), K‐step (V k), and J‐step (VJ) revealed that 0.5% CO 2 had a negative effect on the photosynthetic capacity and growth of the 2× plants, especially the performance of PSII. In contrast, there was no significant effect of high CO 2 on the growth of the 4× plants. These analyses indicate that the decreased inhibition of the growth of 4× plants by high CO 2 (0.5%) may be attributed to an improved photosynthetic capacity, pigment content, and ultrastructure of the chloroplast compared to 2× plants.

because of its rapid growth and good wood texture but also can be used as a superior feed for domestic fowl and livestock because its fleshy leaves are rich in vitamins and minerals (Meng, Pang, Huang, Liu, & Wang, 2012;Wang, Wang, Liu, & Meng, 2013). Moreover, TBL has a wide range of adaptability to adverse abiotic and biotic environmental conditions (Li et al., 2009;Podda et al., 2013;Zhang & Forde, 2000). Therefore, TBL has higher economic and ecological value due to these properties.
At present, polyploidy induction has become an important method to gain insight into physiological mechanisms of plants in the response to environmental stress (Comai, 2005;Woode et al., 2009). In fact, many polyploidy plants have a superior tolerance to environmental stresses compared to their corresponding diploid (2×) plants (Huang et al. 2002;Zhang, Hu, & Yao, 2010). Chloroplasts, one of the primary organelles, are more sensitive to various environmental stresses than other organelles because photosynthesis and other biochemical and biophysical processes occur within these structures (Barry & John, 1981;Wang et al., 2013). However, the mechanisms by which EC affects chloroplasts are not completely understood. In particular, few studies have explored the special response mechanisms of chloroplasts from polyploidy woody plants to specific EC conditions. Indeed, changes in the response to EC of the chloroplasts from polyploidy plants could have a significant ecological impact.
The aim of this study was to determine the effects of a particular EC concentration (0.5%) on two black locust species (2× and 4×).
Based on the response of TBL to other abiotic stresses (Meng et al., 2012;Wang et al., 2013), we proposed the following hypotheses: (1) TBL (fast-growing trees) may adapt better to EC conditions compared to its 2× counterpart, and (2) TBL may adapt to an EC environment via adjustments at the chloroplast level. Accordingly, to determine the physiological and biochemical responses of the 2× and 4× plants to a particular EC condition (0.5%), we measured and observed the morphology, anatomy, photosynthetic ability, antioxidase activities, and ultrastructure and other parameters of the chloroplasts.

| Plant growth
All of the materials were introduced into China directly from Korea by Beijing Forestry University. The 2× and 4× black locust (Robinia pseudoacacia L.) seedlings were from same germplasm and had the same genetic base . In June 2014, 30 plants (1 year old) from among the 2× and 4× species were selected for size uniformity and were grown in plastic pots (10 cm in diameter and 10 cm in depth) filled with a 2:1 (v/v) mixture of soil and sand. This experiment was carried out in a high-performance CO 2 -controlled growth chamber (Huanghua Faithful Instrument Co., Ltd., Hebei, China) under 0.5% CO 2 concentrations as treated with a light/dark cycle of 16 hr/8 hr, at temperatures of 30/25°C, 50/60% humidity, and 250 μmol photons m −2 s −1 light. The seedlings were harvested and measured followed by treatment as control (day 0) and then harvested and measured again at 6 days after the treatment (day 6).

| Determination of leaf morphology, leaf nitrogen (N), and carbon isotope composition (δ 13 C)
On day harvested, the leaves morphology was photographed. For the determination of nitrate concentrations and carbon isotope composition (δ 13 C), the leaves were immediately weighed and dried at 80°C for 48 hr to a constant weight. Nitrate concentrations were determined according to the method of Yin et al. (2012). δ 13 C was analyzed using a mass spectrometer (Finnegan MAT Delta-E, Germany) (Li et al., 2009).

| Determination of chlorophyll pigment
Total chlorophyll, chlorophyll a, and chlorophyll b contents were estimated according to the methods of Meng et al. (2012). 300 mg of fresh leaves was ground in 80% cold acetone and centrifuged at 12,000 g for 20 min. Then, the supernatant was collected and fixed in 10 ml of 80% acetone and detected at 470, 645, and 663 nm.

| Isolation of chloroplasts
To isolate and purify chloroplasts from the leaves, we followed protocols described by Yin et al. (2011) with minor modifications. All of the steps were performed at 4°C. In total, 30 g of leaves was harvested and ground in 200 ml of isolation buffer I containing 50 mmol/L HEPES (hydroxyethyl piperazine ethanesulfonic acid)/KOH (pH 7.5), 5 mmol/L hexanoic acid, 0.3% bovine serum albumin (BSA) (w/v), 0.3 mol/L sucrose, 10 mmol/L β-mercaptoethanol, 20 mmol/L ethylenediaminetetraacetic acid (EDTA), 30 mmol/L Na-ascorbate, and 1% (w/v) polyvinylpyrrolidone (PVP). Then, the homogenate was filtered through six layers of mesh nylon cloth (40 × 40 μm) and centrifuged at 4,000 g for 10 min. The supernatant was centrifuged at 20,000 g for 10 min. The precipitate was carefully suspended in buffer II containing 20 mmol/L HEPES/KOH (pH 7.5), 330 mmol/L sorbitol, 10 mmol/L NaCl, 2 mmol/L EDTA, and 5 mmol/L Na-ascorbate and washed twice. Subsequently, the resuspended chloroplasts were loaded onto a Percoll gradient consisting of a 6:6:6:3 ratio, top to bottom, of 10%, 40%, 70% and 90% Percoll. The mixture was centrifuged for 0.5 hR at 40,000 g, and the chloroplasts were present between the 40% and 70% interface. Then, the intact chloroplasts were collected, washed, and centrifuged at 10,000 g for 15 min in buffer II medium.

| Measurement of the lipid peroxides and hydrogen peroxide of chloroplasts
Lipid peroxides were estimated using a modified method of Janik-Papis et al. (2009). In total, 0.5 ml of chloroplast supernatant was added to a test tube containing 2 ml of a mixture of 20% TCA (Trichloroacetic acid), 0.01% butylated hydroxytoluene, and 0.6% thiobarbituric acid. The mixture was heated in boiling water for 30 min and then quickly cooled on ice. After centrifugation at 12,000 g for 10 min, the absorbance of the supernatant was determined at 450, 532 and 600 nm. Hydrogen peroxide (H 2 O 2 ) was detected spectrophotometrically according to the method of Sergiev et al. (1997). A volume of 0.5 ml of chloroplast supernatant was homogenized in 0.1% TCA in an ice bath. After centrifugation at 12,000 g for 10 min, 0.5 ml of the extracted solution was mixed with 0.5 ml of potassium phosphate buffer (pH 7.5) and 1 ml of potassium iodide (1 mol/L). The absorbance of the supernatant was measured at 390 nm, and the concentration of H 2 O 2 was obtained using a standard curve.
Enzymatic activity was detected spectrophotometrically at 420 nm.

| Changes in morphology and MDA and H 2 O 2 levels
High EC led to chlorosis in the leaves of both 2× and 4× seedlings compared with their corresponding controls ( Figure 1a). Furthermore, the 2× leaves exhibited more obvious and severe chlorosis than the leaves from the 4× seedlings ( Figure 1b

| Chlorophyll content, N content, and carbon isotope composition (δ 13 C)
The contents of total chlorophyll (chl), chl a and chl b of 2× and 4× plants are presented in Table 1. In the 2× species, statistically significantly lower contents of total chl (52.86%), chl a (51.11%), and chl b (57.14%) were detected after 0.5% CO 2 treatment, whereas no significant changes were found in total chl, chl a, and chl b of the 4× plants under 0.5% CO 2 conditions. In contrast, 0.5% CO 2 did not significantly affect the carbon isotope composition (δ 13 C) or N content of the 2× and 4× plants.

| Chloroplast ultrastructure
The ultrastructures of the 2× and 4× species are depicted in Figure 2.
The ultrastructure and arrangement of the chloroplasts of both the 2× and 4× seedlings were normal under control conditions, but injuries were apparent in the 2× plant under 0.5% CO 2 conditions ( Figure 2A). Under control conditions, grana (Gr) of both the 2× and 4× seedlings were well developed and highly stacked with normal thylakoids (Figure 2). In the 2× plants, 0.5% CO 2 induced a disturbance of the chloroplast structures.
Swollen, instead of smooth and spindly, chloroplasts and marked swelling of the Gr and an incompact structure of thylakoids in the chloroplasts were found. The effects of starch and osmiophilic globule accumulation were also observed relative to controls (Figure 2A). In contrast, the 4× plants maintained structural integrity of the chloroplasts compared with those of the 2× plants after 0.5% treatment ( Figure 2B). An important difference between diploid and tetraploid chloroplasts is the amount of osmiophilic bodies-in the diploid. About 0.5% CO 2 induced increased numbers of osmiophilic globules in 2× plants (Figure 2A, e and f).

| Measurement of gas exchanges
The photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), and intercellular CO 2 (Ci) in the 2× and 4× leaves decreased after exposure to 0.5% CO 2 (Table 2). However, the decreases in the 2× leaves were much greater than those in the 4× leaves. Notably, the Pn and Ci in the 2× leaves decreased dramatically after high CO 2 treatment.

| Measurement of chl fluorescence
The 2×  both the 2× and 4× species increased after 0.5% CO 2 treatment, while that of the 4× was higher compared with that of the 2× species (Figure 3f).

| SOD, GR, POD, and APX activities
The application of 5% CO 2 decreased the activities of SOD, GR, and POD in the 2× plants by 27.49%, 52.21%, and 6.05%, respectively, while the APX activity exhibited a significant increase (38.38%) ( Figure 4). However, in the 4× plants, the SOD and GR activities were marginally decreased, while the APX activity was greatly stimulated by 0.5% CO 2 (Figure 4). In addition, the SOD, GR, POD, and APX activities in the 4× plants were much higher than those in the 2× plants both in control and treatment.

| DISCUSSION
Chloroplasts are one of the vital organelles in plant cells because photosynthesis occurs in these structures. Many plant species display ultrastructural damage from environmental stress (Liu, Zhang, Qi, & Li, 2014).
Accordingly, this damage to the chloroplast may lead to a significant decrease in photosynthesis (Zhang et al., 2010). This decrease in photosynthesis may result from rearrangements of the light-harvesting complex I and light-harvesting complex II (Garstka et al., 2005). In the current study, 0.5% CO 2 induced a disturbance in the chloroplast structure by T A B L E 1 Chlorophyll a, chlorophyll b, total chlorophyll contents, N content, and carbon isotope composition (δ 13 C) in leaves of 2× and 4× affected by high CO 2 condition  Both mineral nitrogen (N) and CO 2 are involved in plant growth (Peterson et al., 1998). Our present study suggested that 0.5% CO 2 increased the leaf N content of the 2× and 4× seedlings by 0.06% and 0.01%, respectively; however, this effect was not statistically significant (Table 1). This finding led us to conclude that elevated CO 2 does not have a significant effect on N content. In fact, N is also a part of chl (Yin et al., 2012). However, elevated CO 2 induced a decrease in chl a, chl b, and total chl of the 2× plants by approximately 50%, which could partly explain the decrease in the Pn (  (1982) reported that the Tr can be estimated by measuring the δ 13 C in leaves during photosynthesis. Similar results have also been obtained in some legume crops, such as bean, cowpea, groundnut, and soybean (Kashiwagi et al. 2006). Therefore, there is a close relationship between the δ 13 C and Tr. However, no significant correlation was observed between the δ 13 C and Tr when the 2× and 4× plants were grown under 0.5% CO 2 conditions (Tables 1 and 2). A similar result has been reported in sunflower (Virgona, Hubick, Rawson, Farquhar, & Downes, 1990). This indicated that the δ 13 C discrimination was limited in describing the Tr under elevated CO 2 conditions. Alternatively, our results on black locust may indicate that the differences in the Tr are attributed to changes in Gs rather than by changes in mesophyll  efficiency. These findings were extended, indicating that elevated CO 2 negatively affects the Pn or Tr at N and δ 13 C levels.
Generally, an OJIP curve reflects the photochemical state in PSII (Tóth, Schansker, Garab, & Strasser, 2007). It is accepted that the shape of the initial fluorescence rise is reflected by the energetic connectivity or probability of exciton exchange between the PSII units (Yordanov et al., 2008). In higher plants, the OJIP curve possesses a sigmoidal shape under optimal conditions and is sensitive to various abiotic stresses (Lu, Qiu, Wang, & Zhang, 2003;Pierangelini, Stojkovic, Orr, & Beardall, 2014;Zhang et al., 2011). In our experiment, a decreased rate of the J-step and I-step was found in the 2× and 4× species after 0.5% CO 2 treatment (Figure 3a,b). One may speculate that this effect could be related to the low absorption cross section of PSII and higher segregation of the photosystems, thereby reducing energetic connectivity between individual PSII units (Govindjee, 1995;Schansker, Tóth, & Strasser, 2005). The OJIP curves of the CO 2 -treated 2× and 4× plants declined significantly (especially for the 2× sample), which indicated that an impairment of electron flow from PSII to the PQ pool occurred (Antal et al., 2006). In addition, a prominent decline in the P point was found in the 4× plant after 0.5% CO 2 treatment, which may be due to the influence of nonphotochemical quenching (Antal et al., 2009).
To further characterize the function of the photosynthetic apparatus in response to the strain under the special EC condition (0.5% CO 2 ), we also analyzed selected parameters derived from the OJIP curves.
Generally, an increased rate of Vk in the O-J phase is a convenient indicator of the degree of damage of the oxygen-evolving complex (OEC) activity (Pospíšil & Dau, 2000). The OEC in the membrane-bound protein complex PSII catalyzes the water oxidation reaction that takes place in oxygenic photosynthetic organisms (Carina et al., 2013). In this study, the Vk value in the O-J phase in both the 2× and 4× plants tended to decrease under elevated CO 2 (0.5%) compared to the 2× plants ( Figure 3c).
The magnitude of this drop was higher in the 4× than in the 2× plants, indicating that the special CO 2 may induce OEC activity to protect PSII.
However, these changes were not confirmed by the structural changes of thylakoids in the 2× chloroplasts (Figure 2) because the inhibition of the OEC activity can lead to lipid peroxidation damage, an increase in membrane permeability, and the release of more liposomes (Takahiro, Mitsue, & Yasusi, 2004). Generally, the degradation of thylakoids can result in the formation of liposomes (Ma, Zhang, Wang, Song, & Zhang, 2013). Therefore, in the 2× plants, an incompact structure of thylakoids was found in the chloroplasts with more liposomes compared with the 4× chloroplasts, which suggests that the damage to the OEC in the 2× plants by 0.5% CO 2 treatment was not more significant than that in the 4× plants ( Figure 2). We also noted that the L value (V L , 0.15 ms) in the O-K phase of the 4× sample was decreased after 0.5% CO 2 treatment; however, this value only changed a little (Figure 3d). In general, the absorption, transmission, and transformation of light energy are carried out in the thylakoid membranes (Chow, Haehnel, & Anderson, 2006).
The increase of the V L value is an indicator of thylakoid dissociation. The structure of thylakoids is related to PSII function (Kirchhoff et al., 2007).
Accordingly, the 4× samples had improved function of the thylakoids after 0.5% CO 2 treatment. In addition, The equation ϕ po = Fv/Fm is widely used to reflect the maximum potential efficiency of PSII or the intrinsic quantum efficiency of the PSII units (Kitajima & Butler, 1975). As a multiparametric expression of the three main functional steps of photosynthetic activity by a PSII reaction center complex, the overall photosynthetic performance index (PI ABS ) is also suitable to distinguish the photosynthetic performance of plants under various environmental conditions (Lepeduš et al., 2012). As shown in Figure 3, when exposed to 0.5% CO 2 , the 2× and 4× seedlings all exhibited decreases in Fv/Fm, indicating that 0.5% CO 2 affected the behavior of PSII. At the same time, the different Fv/ Fm ratio with 0.5% CO 2 suggested that the photochemical reaction in 4× seedlings was different from that of 2× seedlings. Furthermore, the results of this study clearly reveal that the 4× species maintained a higher level of PI ABS than did the 2× species under 0.5% CO 2 conditions. Therefore, the photosynthetic system was damaged more severely in the 2× than in the 4× plants by 0.5% CO 2 treatment.
As a consequence of the inhibition of PSII, excess light energy will accordingly lead to photoinhibition and oxidative stress, which can ) or lipid peroxidation (Asada, 1999;Yin, Pang, & Chen, 2009).
Furthermore, an increase in ROS will result in chl loss and a decrease in photosynthetic CO 2 assimilation (Ahmed et al., 2013). As evidence of ROS and lipid peroxidation generation, significantly increased H 2 O 2 and MDA were observed in the 2× seedlings after 0.5% CO 2 treatment ( Figure 1). Additionally, Pn and chl contents were decreased in the 2× plants by 0.5% CO 2 treatment (Figure 1, Tables 1 and 2). These observations confirmed that more serious oxidative stress occurred in the 2× than in the 4× plants after 0.5% CO 2 treatment; that is, the 4× species had less oxidative stress.
To alleviate the damage initiated by oxidative stress, plants can modulate antioxidative enzymes such as SOD, POD, GR, etc. (Smirnoff and Wheeler 2000). The SOD, APX, and GR activities in leaves increase when exposed to elevated CO 2 (Guo, Zhou, & Zhang, 2015). In our experiments, SOD, GR, and POD activities were decreased in the chloroplasts from the 2× leaves under the 0.5% CO 2 condition ( Figure 4). In contrast, the activities of POD and APX of the 4× leaves were remarkably stimulated by 0.5% CO 2 (Figure 4).
This result suggested that high CO 2 stimulated an increase in the antioxidant enzyme activities of the 4× leaves to alleviate oxidative damage and indicted that the 4× species has a highly efficient defense system under elevated CO 2 conditions to some extent.
Although SOD, GR, and POD activities showed different changes between diploid and tetraploid black locust under specially elevated CO 2 (0.5%), some sensitive enzymes have not been identified in the photosynthetic carbon metabolism. For instance, Umeton et al. (2012) found that 11 sensitive enzymes are confirmed to play a key role in terms of maximal CO 2 uptake and minimal nitrogen consumption. Thus, in the future, these parameters or molecular methods should be used to gain a systematic and comprehensive understanding of polyploidy plants under elevated CO 2.
In some studies, increased numbers of osmiophilic globules in the cytoplasm and nucleolus-associated bodies as well as electron dense material in vacuoles were observed in cadmium tolerant cells (Gzyl, Przymusiński, & Gwóźdź, 2009). And Bréhélin also found that the tocopherols stored in osmiophilic globules would be delivered to thylakoid membranes to scavenge ROS under oxidative stress (Bréhélin, Kessler, & van Wijk, 2007). In Tillandsia albida (Bromeliaceae), Papini found an increase in plastoglobules in plastids involved in autophagy (Papini, Mosti, & van Doorn, 2014). And Sallas et al. also reported that a non significant decrease in the number of plastoglobules in response to elevated CO 2 in Norway spruce while an increase in number of chloroplast plastoglobules in Scots pine (Sallas, Luomala, Utriainen, Kainulainen, & Holopainen, 2003). It indicated that the response of plastoglobules depends on the plant species.

ACKNOWLEDGMENTS
This study was supported by the Fundamental Research Funds for the Central Universities (No. 2572016EAJ4 and No. 2572015DA03) and University student innovation project of northeast forestry university (201710225303).

CONFLICT OF INTERESTS
None declared.