The role of antioxidant mechanism in photosynthesis under heavy metals Cd or Zn exposure in tobacco leaves

ABSTRACT We examined the effects of Cd and Zn exposure on the photosynthetic function and the tolerant mechanisms of ROS metabolism in tobacco leaves. The results showed that the photosynthesis inhibition caused by Cd exposure was due to the limitation of both stomatal and non-stomatal factors, while Zn exposure only showed a significant effect on the Gs of tobacco leaves. Cd increased the generation rate of O2•– and the content of H2O2, but Zn did not lead to the ROS burst. Cd enhanced the POD activity, but inhibited SOD and CAT activities. The activities of SOD and POD significantly increased under Zn exposure. Cd inhibited the activity and expression of APX. The up regulation of GPX, GR and GST expression and the increase of their activities also play a positive role in the adaptation to Cd. Although Zn inhibited the activities of APX and GPX, it had little effect on other enzyme activities and protein expression in AsA-GSH cycle. TPX and TrxR activities, Trx-Prx pathway-related proteins are very sensitive to Cd. However, only PrxQ and 2-Cys Prx BAS1, which are located in chloroplast, and Trx-HFC164 were significantly down-regulated in Trx-Prx pathway under Zn exposure.


Introduction
Globally, the degree of heavy metals (HMs) pollution in soils varies. Sources of HMs are not only limited to soil parent material, HMs, including Cd, Pb, Zn, Cu and Hg, also derive from air and water sources related to anthropogenic activities, such as industry and farming (Wójcik et al. 2015;Zhang et al. 2020a). Among the different types of HMs, Cd and Zn are most common, being potentially harmful to animals and people (Smolková et al. 2019;He et al. 2020;Jahandari 2020). Excessive concentrations of HMs have also been shown to be toxic to plants by inhibiting root growth (Qin et al. 2018), affecting the absorption of water and nutrients (Sánchez-Pardo et al. 2013;Zhang et al. 2017), destroying the structure of chloroplast and inhibiting chlorophyll synthesis (Kalaji and Loboda 2007;Zhang et al. 2018;Zhang et al. 2020b), and hindering photosynthetic electron transfer and the fixation of CO 2 (Azhar et al. 2019;He et al. 2021). Under stress conditions, electrons can leak from the photosynthesis or respiratory electron transfer chain and attack free O 2 in cells, resulting in the production of superoxide anions (O 2

•-
), H 2 O 2 and hydroxyl radicals (OH) can also be generated through specific chemical reactions (Gupta et al. 2011). Excessive ROS can cause oxidative damage to plants (Møller et al. 2007;Wu et al. 2015). The metabolic balance of ROS therefore represents an important mechanism for plants to adapt to stress.
In order to avoid cells and tissues from ROS oxidative damage, plants will enhance the function of antioxidant system to remove excessive ROS. When HMs exposure such as Cd or Zn disturbs the metabolism balance of ROS, plants can correspondingly improve the function of the antioxidant system to eliminate excessive ROS (Afef 2019;Meng et al. 2019). Khanna et al. (2019) recorded Cd exposure with a concentration of 0.4 mM to result in the accumulation of O 2 •-, H 2 O 2 and malondialdehyde (MDA) in Lycopersicon esculentum leaves, and an increase in catalase (CAT, EC: 1.11.1.6), glutathione peroxidase (GPX, EC: 1.11.1.9), dehydroascorbate reductase (DHAR, EC: 2.5.1.18), glutathione reductase (GR, EC: 1.8.1.7) and glutathione S-transferase (GST, EC: 2.5.1.18) activities with induction. When 9.0 mM Cd stress, an increase in superoxide dismutase (SOD, EC: 1.15.1.1) activity was induced and sodA expression up-regulated in Solarium nigrum L. was recorded (Ullah et al. 2019). However, it has also been shown that high concentrations of Cd can inhibit enzyme activity or protein expression of SOD (Wu et al. 2003), CAT (Wójcik et al. 2006) and L-ascorbate peroxidase (APX, EC: 1.11.1.11) (Li et al. 2014). Although Zn is an essential nutrient for plant growth, playing an important role in growth and development (King 2018), Zn is an indispensable component of plant Cu/Zn-SOD, playing a vital role in maintaining the function of SOD. However, excessive Zn can still lead to the imbalance of ROS in plants. As recorded by Madhava Rao and Sresty (2000), Zn exposure could induce an increase in peroxidase (POD, EC: 1.11.1.7) enzyme activity in pigeon pea (Cajanus cajan L. Millspaugh) leaves but inhibit CAT activity. Although Zn stress was also recorded to inhibit APX activity in Phaseolus vulgaris leaves (Cuypers et al. 2001), Madhava Rao and Sresty (2000) reported Zn exposure to enhance enzyme activity of APX in pigeon pea leaves.
Although numerous investigations have been undertaken in plants under Cd and Zn exposure conditions examining photosynthesis (Tang et al. 2016;Momchil et al. 2018;Szopinski et al. 2019) andROS metabolism (Gomes-Junior et al. 2006;Wójcik et al. 2006;Nazar et al. 2012), presently only a few studies have examined differences in photosynthetic function and antioxidant mechanisms of plants under Cd or Zn exposure using proteomics technology. As in-depth analysis of the antioxidant mechanism of plants under different HMs exposure will provide basic data for improving the tolerance of plants to HMs exposure, we examined the effects of Cd and Zn exposure on photosynthetic gas exchange and chlorophyll fluorescence parameters, enzyme activity and protein expression related to ROS scavenging mechanisms of tobacco leaves. Our goal is to reveal the adaptive mechanisms of photosynthesis and ROS metabolism in tobacco under Cd and Zn stress, and to guide the rational planting of tobacco in heavy metal contaminated areas.

Plant materials and treatment
The tobacco (Nicotiana tabacum L.) cultivar 'Longjiang 911' was used as experimental material. Seeds were provided by the Heilongjiang Tobacco Research Institute. All experiments were performed in Harbin, Heilongjiang Province, China. Plants were seeded into culture medium comprised of a 2:1 (v/v) mixture of peat soil and quartz sand. Tobacco plants were cultivated in a growth chamber set to 25/23°C (light/dark), light intensity of 400 μmol·m −2 ·s −1 , 12-h photoperiod, and relative humidity of approximately 75%. Plants were watered with diluted Hoagland nutrient solution once a week.
After the seedlings grew to the four-leaf stage, individual seedlings were transplanted into individual culture pots (12cm diameter, 15-cm height) filled with sterilized quartz sand. Thirty days after transplantation, a total of 30 seedlings were selected and divided into three groups: the control group (CK), the Cd exposure treatment group and the Zn exposure treatment group. According to the treatment method of heavy metal concentration in our previous experiment (2020c), A ½ Hoagland nutrient solution containing CdCl 2 concentration of 100 μmol·L −1 (Cd exposure) and ZnCl 2 concentration of 200 μmol·L −1 (Zn exposure) were respectively poured into each pot. The exposure concentrations of Cd and Zn in the culture medium were 2.24 and 5.36 mg·kg −1 , and trays were placed under each pot; the same amount of ½ Hoagland nutrient solution was applied to the CK treatment. After 10 days of treatment, the differences of plants phenotypes under different treatments were observed and this data was used to calculate the following indexes.

Determination of parameters and methods
Measurement of gas exchange and chlorophyll fluorescence parameters: The fully expanded leaves of tobacco seedlings in different treatments were measured using a LICOR-6400 photosynthetic measurement system (LI-COR, Lincoln, NE, USA). Using a CO 2 cylinder and built-in light source, the CO 2 concentration and light intensity PFD were set to 400 μL·L −1 and 1,000 μmol·m −2 ·s −1 , respectively. Under these conditions, the net photosynthetic rate (P n ), stomatal conductance (G s ), transpiration rate (T r ), and intercellular CO 2 concentration (C i ) under different treatments were measured. After a 30-min dark adaptation period, the penultimate fully expanded leaves under different treatments were measured with a pulse modulation fluorimeter (FMS-2, Hansatech, Lynn's Gate, UK). The initial fluorescence (F o ) and maximum fluorescence (F m ) were measured in order to calculate the PSII maximum photochemical efficiency (F v Then, the maximum fluorescence (F m ′ ) and steady-state fluorescence (F s ) of tobacco plants were measured after a 3-min application of 1000 μmol·m −2 ·s −1 activating light (PFD) to calculate the electron transfer rate (ETR) with the formula ETR = 0.5 × 0.85 × (F m ′ -F s )/F m ′ ×PFD, where, 0.5 means that the proportion of light energy distribution between PSII and PSI, 0.85 means that 85% of the absorbed light energy is transferred to the reaction center. The parameters of photosynthetic gas exchange and chlorophyll fluorescence were repeated three times.
Measurement of physiological indexes such as reactive oxygen species content, lipid peroxidation and antioxidant enzyme activities: The generation rate of O 2 •and the content of H 2 O 2 were determined using the methods described by Zhang et al. (2007) and Alexieva et al. (2001), and slightly changed. Determination of the content of H 2 O 2 : 0.5 g leaves were weighed and added into 8 mL acetone solution for ice bath grinding, centrifuged at 3000 r/min for 10 min, and the supernatant was used as sample extract. 1 mL of supernatant was absorbed, and 0.1 mL of 5% TiCl 4 solution and 0.2 mL of concentrated ammonia were added. After the mixture was precipitated, the mixture was centrifuged at 3000 r·min −1 for 10 min, and the supernatant was discarded. Washed with acetone 3-5 times until the plant pigment is removed. Added 5 mL of 2 mol·L −1 concentrated sulfuric acid to the precipitate, and added distilled water to the precipitate to 10 mL, and determined the absorbance value at 415 nm wavelength. Determination of the generation rate of O 2 •-: 0.5 g leaves were weighed and added into 8 mL PBS solution for ice bath grinding, and centrifuge at 4000 r·min −1 for 15 min. 1 mL of supernatant was taken, added with 1 mL of PBS and 2 mL of hydroxylamine hydrochloride, and the solution was stationary at 25°C for 20 min. Then 2 mL p-aminobenzene sulfonic acid and 2 ml a-naphthylamine were added, and the absorbance at 530 nm was measured after standing at 25°C for 20 min. MDA content of lipid peroxidation product was determined using the method described by Wang et al. (2003) and slightly changed. The content of MDA was determined by thiobarbituric acid colorimetry. 0.5 g of the leaves were weighed, and 10 mL of 10% trichloroacetic acid (TCA) was added and ground to homogenate, centrifuge for 10 min at 4000 r·min −1 , and the supernatant is the sample extraction solution. Absorb 2 mL of centrifuged supernatant, add 2 mL of 0.6% TBA solution, and the mixture was reacted in a boiling water bath for 15 min. After rapid cooling, the absorbance values at 532, 600 and 450 nm were determined. The activities of SOD, POD and CAT, the relevant enzymes in the AsA-GSH include APX, MDHAR, DHAR, GR, GPX and GS activities, the relevant enzymes in the Trx-Prx pathway include thioredoxin peroxidase (TPX, EC: 1.11.1.24) and TrxR: thioredoxin reductase (TrxR, EC: 1.8.1.9) activities were measured using kits produced by Suzhou Comin Biotechnology Co., Ltd. (Jiangsu, China). The activity (1U) of SOD is defined as the amount of enzymes required to reduce NBT to half of that of the control group; the activity (1U) of CAT is expressed by the amount of H 2 O 2 (μmol) reduced per g fresh sample per min; activity (1U) of POD is expressed by the absorbance at 470 nm (Δ 470 ) increased by 0.5 per g fresh sample per min; The activity of APX is expressed by the amount of AsA (μmol) oxidized per g fresh sample per min; the activity of DHAR is expressed by the amount of AsA (μmol) per fresh g sample per min; GPX, GR and MDHAR activities were expressed by the amount of NADPH (μmol) per g fresh sample per minute (μmol); the activity of GST was expressed by the amount of 1-chloro-2,4-dinitrobenzene (CDNB, μmol·L −1 ) combined with GSH per g fresh sample per minute; the activity of TPX is expressed by the amount of dithiothreitol (μmol) oxidized per g fresh sample per min; the activity of TrxR is expressed by the reduction of 5,5ʹ-dithiobis-(2-nitrobenzoic acid) (μmol) per g fresh sample per min. The above biological indexes were measured for three biological repeats.
Measurement of Proteomic determination and analysis: The tobacco leaves of the same leaf age of different treatment plants were selected, including the control group, were sampled and rapidly frozen in liquid nitrogen. The samples were assayed by Personalgene Corporation (Shanghai, China). Antioxidant machinery-related proteins were identified that differed significantly in expression between the treatment and control groups (P < 0.05), using three biological replicates. For detailed procedures, please refer to Zhang et al. (2020c).
The leaves of the same leaf age of different treatment plants were selected.

Statistical analysis
Excel and SPSS (22.0) were used to analyze the data. All data were as the mean ± standard error (SE) of three biological replicates (n = 3). A one-way analysis of variance (ANOVA) and a least-significant difference (LSD) test with α = 0.05 were used to determine whether there was a significant (P < 0.05) among treatments.

Photosynthetic gas exchange and chlorophyll fluorescence parameters
As shown in Figure 1, under Cd exposure, P n , G s and T r decreased by 58.72% (P < 0.05), 66.56% (P < 0.05) and 62.09% (P < 0.05), respectively (Figure 1(A-C)), C i increased by 32.66% (P<0.05) compared with CK (Figure 1(D)). However, under Zn exposure, G s and T r also decreased compared with CK, but the decreasing range was significantly lower than Cd exposure, P n and C i were slightly decreased, but the difference was not significant compared with CK. Cd exposure reduced F v /F m and ETR by 6.93% (P < 0.05) and 24.76% (P < 0.05) compared with CK, respectively, but Zn exposure had no significant effect on F v /F m and ETR (Figure 1(E,F)).

Figure 2 shows that O 2
•generation rate and H 2 O 2 content increased by 86.47% (P < 0.05), 86.66% (P < 0.05), and MDA content increased by 2.59 times compared with CK plants under Cd exposure, but these three parameters in CK and Zn exposure plants were not significantly different. Cd exposure decreased the activity of SOD and CAT, but the expression of POD activity increased by 31.89% (P < 0.05) compared with CK. Under Zn exposure, the activities of SOD and POD increased by 32.17 (P < 0.05) and 33.05% (P < 0.05), there was no significant change CAT activity under Zn exposure (Figure 2(D-F)).
Under Cd exposure, there was no significant difference between the expression of GPX compared with CK, but the expression of GR (A0A1S3YKW7, P80461) expression increased by 29.58% (P < 0.05) and 16.84% (P < 0.05) respectively. Zn exposure reduced GPX expression, but GR expression did not change significantly. Under Cd exposure, GST (A0A1S4DMX8), GST U17 (A0A1S3XWK1, A0A1S3XW37), GST parA, GST parC, GST L3 X1 and MGST3 expression increased very significantly compared with CK (P < 0.05). However, under Zn exposure, all GST expressions were not significantly different from CK ( Figure 5(B)).

TPX and TrxR activities and the expression of Trx-Prx pathway-related proteins
It can be seen from Figure 6(A,B) that the TPX activity was decreased by 61.93% (P < 0.05) and 58.06% (P < 0.05) under Cd and Zn exposure, respectively, and the TrxR activity was significantly decreased under Cd exposure, but the TrxR activity did not change significantly under Zn exposure compared with CK.

Discussion
HMs exposure, such as Cd and Zn, can inhibit photosynthetic capacity in plants, resulting in the inhibition of plant growth. The main channel for absorbing CO 2 and transpiration of water in plants is via the stomata. During HMs exposure, stomatal conductance of plant leaves is reduced, limiting the supply of photosynthesis raw materials, resulting in a reduction of the photosynthetic capacity (Zhang et al. 2020a;He et al. 2021). In our study, tobacco leaves under Cd exposure recorded significantly reduced G s values compared to the CK, resulting in a significant decrease in P n and T r . Although G s and T r were significantly lower than that of CK under Zn exposure, the decrease was significantly smaller than that under Cd exposure. Under Zn exposure, P n recorded no significant change compared with CK. In addition, C i values increased by 32.66% (P < 0.05) under Cd exposure compared with CK; no significant change was recorded under Zn exposure. These results indicate that the utilization ability of CO 2 decreased under Cd exposure, and Zn exposure had no significant influence. It can be inferred that a decrease in photosynthetic capacity caused by Cd could be attributed to co-limitation of both stomatal and non-stomatal factors, this is consistent with our previous research results ); Zn exposure mainly effects stomatal conductance in tobacco leaves, the results of Andrejic et al. (2018) showed that the decrease of stomatal conductance was the main toxicity of excessive Zn stress to Miscanthus×giganteus plants. Cd exposure can lead to photoinhibition in plants (Xue et al. 2018). Zn plays an important role in photosynthetic carbon assimilation in plants, but excessive concentrations of Zn can inhibit photosynthetic capacity in hyperaccumulator Sedum alfredii (Tang et al. 2016) and block photosynthetic electron transfer Note: The data in the figure are from three biological repeats (n = 3), and represent means ± standard error (SE). Significant differences were expressed by different letters (P<0.05). (Magalhes et al. 2004). Our results indicated that F v /F m and ETR were both significantly decreased under Cd exposure, indicating that Cd exposure blocked PSII photosynthetic electron transfer, resulting in photoinhibition of PSII. However, under Zn exposure, PSII activity did not decrease, possibly being an important reason why P n did not significantly decrease under Zn exposure. This result is similar to findings by Momchil et al. (2018) and Szopinski et al. (2019), in which the effects of Cd on CO 2 assimilation rates and PSII activity were significantly greater than effects caused by Zn. Under the influence of photosynthesis inhibition caused by Cd or Zn, the production rate of O 2 •and the content of H 2 O 2 in our experiment significantly increased; the noticeable accumulation of MDA was associated to Cd exposure. The effect of Zn exposure on ROS production and oxidative damage was minimal. Although excess O 2 •in cells is mainly eliminated by SOD (Noctor et al. 2018), it has been recorded that SOD in plants could be inactive under severe oxidative exposure (Fu et al. 2011;Zhu et al. 2019). SOD (A0A1S4DGN5 and A0A1S4DS24) expression significantly up-regulated under Cd exposure compared with CK, SOD (A0A1S4APX1) and Cu-Zn-SOD (A0A1S4BCV5 and A0A1S4D5J3) expression significantly down-regulated, and SOD activity significantly decreased. Cd exposure may therefore inhibit the function of SOD in removing O 2 •to a certain extent, a finding that is in accordance with previous investigations in wheat seedlings (Lin et al. 2007;Chen et al. 2014). In contrast to Cd, Zn is a necessary metal prosthetic group of Cu/Zn-SOD, playing a key role in maintaining the function of SOD (Wang and Jin 2005). Therefore, although five identified SOD expressions were no significant difference compared with CK, and SOD activity was significantly increased compared with CK under Zn exposure. Therefore, it was proposed that an excess of Zn may enhance the scavenging function of SOD on O 2 •-. POD and CAT can reduce excess H 2 O 2 to H 2 O. The variation trend of POD expression under Cd exposure differed. Compared with CK, two PODs (Q94IQ1 and Q9XFL2) recorded a significant increase in expression, and three PODs (A0A1S3X8M7, A0A1S3Y048 and A0A1S4BIW4) recorded a significant decrease in expression; all CAT expressions were significantly decreased. This result indicates that Cd exposure enhanced POD activity whilst also inhibiting CAT activity. Similar to the results of Gong et al. (2017), adaptation to Cd exposure may be related to an enhanced POD function. However, Cd exposure inhibited CAT activity and the expression of related proteins, consistent with previous findings in coffee (Gomes-Junior et al. 2006). Madhava Rao and Sresty (2000) also recorded that POD activity increased in pigeon pea leaves under Zn exposure and CAT activity was inhibited. Although Zn exposure did not significantly affect the activity of CAT and related proteins expression, POD activity significantly increased. This finding is in accordance with enhanced POD activity recorded in rape leaves by Wang et al. (2009) under Zn exposure. Although Zn had little effect on CAT function in tobacco leaves, CAT scavenging H 2 O 2 function was seriously inhibited under Cd exposure.
AsA-GSH cycle is an important ROS scavenging pathway in plants through APX and GPX, and AsA and GSH, the key metabolites in this cycle, are also important antioxidants Note: The data in the figure are from three biological repeats (n = 3), and represent means ± standard error (SE). Significant differences were expressed by different letters (P<0.05). (Zhang et al. 2020d). In the process of the AsA-GSH cycle, although H 2 O 2 can be reduced during oxidation of APX (Stasolla and Yeung 2010), serious oxidative stress often leads to the deactivation of APX in tobacco leaves under drought stress . High concentrations of Cd have been recorded to result in a decrease of APX gene expression in alfalfa (Gu et al. 2018). Findings by Cuypers et al. (2001) recorded Zn exposure to inhibit APX activity in beans, however, Madhava Rao and Sresty (2000) suggested that Zn exposure increased APX activity in mustard and pigeon pea leaves. APX, APX1, APX3 and APX6 expression significantly decreased under Cd exposure, and APX3 and APX6 expression also significantly decreased under Zn exposure compared with CK. Similar to changes in APX expression, both Cd and Zn exposure resulted in a significant decrease of APX activity, with Cd exposure having a larger Note: The data in the figure are from three biological repeats (n = 3), and represent means ± standard error (SE). Significant differences were expressed by different letters (P<0.05).
decrease of APX activity, this is similar to the results of Lin et al. (2007), soil Cd pollution significantly reduced APX activity in heat seedlings. Apart from oxidization through the APX pathway, AsA can also be oxidized via the AO pathway with O 2 participation, resulting in a loss of AsA without a reduction of H 2 O 2 (Zhang et al. 2020d). AsA regeneration can be achieved through the reduction of MDHAR or DHAR after AsA is oxidized to MDHA or DHA, indicating that elevation of the activity or MDHAR/DHAR expression are beneficial to an increase of AsA content and antioxidant capacity of plants (Yin et al. 2010). The activities and expression of DHAR and MDHAR both up-regulated under Cd exposure, suggesting that the promotion of AsA regeneration may be an important mechanism for tobacco to adapt to Cd exposure; no significant changes were observed under Zn exposure. Similar to APX, GPX can also reduce H 2 O 2 . Regeneration of GSH could be achieved by reducing GR, and GSH is an antioxidant that can improve plant tolerance to environmental stresses, such as those posed by HMs (Jia et al. 2016;Ye et al. 2016). Under Cd  exposure, our results indicate that GPX and GR activities significantly up-regulated in parallel with an increased expression of GR compared with CK, indicating that tobacco leaves may improve the tolerance to Cd by enhancing the GSH-GSSG cycle. This finding is in accordance with the results of Karam et al. (2017) and Gu et al. (2018). In contrast to Cd exposure, the activity and expression of GR did not significantly change under Zn exposure compared with CK. However, GPX activity and expression were significantly lower than that of CK, indicating that an excess of Zn may inhibit the function of GPX. GST could promote the binding of Cd 2+ to GSH to form a GS-Cd complex which can alleviate Cd 2+ toxicity (Adamis et al. 2004). Cd  or Zn (Moons 2003) exposure can result in the up-regulated expression of GST related genes or proteins in wheat roots and rice roots. The expression levels of nine GST proteins were identified to be significantly up-regulated under Cd exposure, and their activities were also significantly increased. However, no significant changes of GST expression and activity were found under Zn exposure compared with CK, suggesting that the induction of GST might be an important mechanism responsible for the adaptation to Cd exposure.
in the TrxR expression. Among these proteins, only Trx-HFC164 was down-regulated under Zn exposure, moreover, TrxR activity did not change significantly compared with CK. These results indicate that Cd exposure could result in the Trx function to be affected by a reduction in the supply of electrons due to down-regulated expression of FTR; Zn exposure had little effect on the expression of FTR and Trx. It has also been previously shown that Trx does not directly reduce ROS, instead it regulates Prx to reduce H 2 O 2 through cysteine residue (-Cys) (Meyer et al. 2005). Prx is a protein that removes ROS in organisms through the oxidation of the -Cys ). Our results also indicated that, although the expression of two Prx (A0A1S3ZZI4, A0A1S4DK72) increased under Cd exposure, the expression of PrxQ and 2-Cys Prx BAS1 significantly down-regulated under Cd and Zn exposure, TPX activity decreased significantly under Cd and Zn exposure. PrxQ and 2-Cys Prx were predominantly located in chloroplasts of plants (Baier and Dietz 1999;Baier et al. 2000), and the Trx-Prx pathway in chloroplast was more sensitive to Cd and Zn exposure. We therefore conclude that chloroplast may be an important organelle that is attacked by Cd and Zn exposure, a finding that requires further investigation. In summary, Cd exposure can significantly inhibit the expression of Trx-Prx pathway-related proteins, TPX and TrxR activities, resulting in an inhibition of ROS scavenging processes and a disturbance of the redox state of cells. Although Zn exposure had little effect on FTR and Trx expressions, it still reduced the expressions of PrxQ and 2-Cys Prx BAS1 in chloroplast, the activity of TPX also decreased significantly under Cd and Zn exposure, possibly interfering with the reduction of H 2 O 2 in chloroplast. The related proteins of antioxidant machinery in tobacco leaves under Cd and Zn exposure are summarized in Figure 8.

Conclusion
Cd exposure not only significantly reduced the stomatal conductance of tobacco leaves, but also led to the decrease of PSII photochemical efficiency and CO 2 utilization capacity. Zn exposure also reduced the stomatal conductance, but it had no significant effect on PSII activity and carbon assimilation capacity. Although Cd exposure could induce the activities and expression of POD, DHAR, MDHAR, GPX, GR and GST, but the functions of SOD and CAT were inhibited, especially the expression of Trx-Prx pathway-related proteins were significantly down regulated, TPX and TrxR activities also decreased significantly, resulting in the significant increase of O 2 •production rate and H 2 O 2 content. Zn exposure inhibited the activities of APX and GPX, but it had little effect on other enzyme activities and protein expression in AsA-GSH cycle. In addition, SOD and POD also played an important role in regulating ROS metabolism under Zn exposure. However, TPX activity and chloroplast located PrxQ and 2-Cys Prx BAS1 were sensitive to Zn exposure. In conclusion, tobacco is more sensitive to Cd exposure, but has strong tolerance to Zn exposure. Therefore, Cd content should be considered when planting tobacco in heavy metal contaminated soil.

Disclosure statement
No potential conflict of interest was reported by the author(s). Guanyu Sun is a Professor in Northeast Forestry University. His research interests lie in the area of Plant physiology and molecular biology.
Huihui Zhang is an Associate Professor in Northeast Forestry University. His research interests lie in the area of Plant physiology and molecular biology.