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# Effects of cadmium stress on growth and physiological characteristics of sassafras seedlings

## Introduction

In recent years, environmental pollution and ecological damage have become increasingly serious due to the rapid development of industrialization. Among these issues, soil heavy metal pollution has become one of the most prominent environmental problems in the world. Cadmium (Cd) is a toxic, silver-white metal element. Because of its water solubility, fluidity, and toxicity, Cd can be easily absorbed by plant roots, and it can alter plant structural and functional properties, inhibit seed germination and root elongation1. Cd directly or indirectly inhibits physiological processes such as respiration, photosynthesis, water movement and gas exchange, leading to impairment in plant metabolism2. Additionally, Cd can affect the metabolism, chlorophyll synthesis3 of pants, and disturbs antioxidant defense system by increasing the production of reactive oxygen species (ROS)4.

The absorption and accumulation of Cd can affect the normal growth of plants. Cd stress leads to a decline in biomass and photosynthetic rate of plants, also causes oxidative damage and imbalance of nutrient uptake5,6. Apart from inhibiting the formation of photosynthetic pigments, reducing the efficiency of photosynthesis, Cd stress can also increase the accumulation of active oxygen and enhance peroxidation7,8. Plants can reduce the stress caused by heavy metals through the generation and synthesis of various enzymatic antioxidants, non-enzymatic antioxidants, osmolytes and chelating agents9. Because that Cd can accumulate in plants and enter human body through the food chain, causing chronic poisoning and endangering human health10, it is of great importance to find an effective method to remediate Cd-contaminated soils.

Woody plants have proved an effective means for removing or stabilizing toxic metals from contaminated soils, with their high accumulation of heavy metals, perennial trait, high biomass production, and fast growth6,11,12,13. Sassafras tzumu Hemsl. belongs to the genus Sassafras and the family Lauraceae. It is often used for shipbuilding and making high-quality furniture. Sassafras trees are beautiful and have red leaves in autumn, which makes them excellent ornamental trees for gardens14,15. Sassafras has good prospects in terms of timber production and garden use. Therefore, most studies on sassafras in China have focused on genetic breeding and reproduction technology, cultivation and afforestation technology, disease and pest control, etc.16,17,18. However, few studies have been performed on the physiological conditions of sassafras under heavy metal stress. In view of this, this study aimed to investigate the effects of Cd stress on the growth, photosynthetic and physiological characteristics of sassafras and to analyze the physiological response of sassafras to Cd stress in order to provide a theoretical basis for further research on the resistance of sassafras to Cd stress.

## Materials and methods

### Experimental design

The plants used in this experiment were 1-year sassafras seedlings, which are common native tree species in Sichuan. The seedlings were obtained from the same source and were pests and diseases free, which were collected in accordance with relevant national and international guidelines and legislations. The tested soil was a yellow soil taken from a forest farm in Ya’an city, Sichuan Province. The pH of the test soil was 5.75, and the total nitrogen, phosphorus and potassium contents were 9.10 g/kg, 0.64 g/kg, and 12.59 g/kg, respectively.

The experiment was conducted at the teaching and research station of Sichuan Agricultural University. Its geographical coordinates are east longitude 103°51′29″ and north latitude 30°42′18″. Approximately 12 kg of mixed soil was weighed and placed into each pot. The content of Cd2+ (mg/kg) in the soil of each pot was calculated according to the dry weight of the soil, and Cd2+ was added to the pots in the form of a CdCl2 aqueous solution. A single-factor test design was used to establish 5 cadmium treatment levels: CT (0 mg/kg), Cd5 (5 mg/kg), Cd20 (20 mg/kg), Cd50 (50 mg/kg), and Cd100 (100 mg/kg). Five replicates were established for each treatment group, and all treated plants were placed in a greenhouse.

The experiment began in early May 2019. CdCl2 was applied 5 times, with an interval of 15 d; the same amount of pure water was added to CT. At the time of application, the prepared CdCl2 solution was poured evenly onto the soil surface in the basin. Any CdCl2 solution that exuded from the pot was collected at the bottom of the basin with a tray pad and poured back into the soil. The CdCl2 applications ended in mid-July 2019. After 30 d of plant growth, the plant photosynthetic and physiological characteristics were measured. The plants were harvested at the end of December 2019, and the plant roots, branches and leaves were harvested separately.

### Determination of the plant growth index

The height and ground diameter of each plant were measured before and after the experiment, and the subtraction method was used to calculate the results. The height of the seedlings was measured with a ruler (precision: 0.1 cm). An electronic Vernier caliper (precision: 0.1 mm) was used to measure the ground diameter from two perpendicular directions at the root neck, and the average value was calculated. The plant samples were washed with deionized water, and then the plant organs, i.e., roots, branches and leaves, were harvested separately. In the lab, the plant organs were put in an oven at 105 °C for 30 min, after which they were dried at 70 °C to constant weight and weighed. The biomass of each dried organ was then calculated.

### Determination of the physiological characteristics

In the 5 replicates of each treatment, five mature fresh functional leaves were randomly selected from the middle to upper part of the tree canopy and placed separately into an ice box for the measurement of each index.

The content of free proline (Pro) was extracted with sulfonyl salicylic acid and determined with acidic ninhydrin colorimetry19. The content of soluble protein (SP) was determined by the Coomassie brilliant blue method20. The malondialdehyde (MDA) and soluble sugar contents (SS) were determined by the thiobarbituric acid heating colorimetric method20,21.

The activity of superoxide dismutase (SOD), Peroxidase (POD) and catalase (CAT) were determined by the spectrophotometer. 0.2 g of the fresh leaves were weighed and placed in a grinding bowl, 2 ml phosphate buffer solution was added and placed in an ice bath for rapid grinding, then centrifuge at 10,000 r/min at 4 °C for 15 min, the supernatant was collected as the test sample. According to the method of Donahue et al.22, the OD value at the wavelength of 560 nm causes 50% inhibition of the reduction rate of nitro blue tetrazolium (NBT) to measure the SOD activity. POD activity was measured by the Guaiacol oxidation method23, and the increase in absorbance at a wavelength of 470 nm was determined. CAT activity was quantified as the decrease in the absorbance at a wavelength of 240 nm caused by the consumption of the substrate H2O224. The H2O2 content was determined with a method based on H2O2 and titanium ions forming a colored [TiO(H2O2)]2+ coordination compound (the specific absorption peak is 410 nm). All enzyme activities were determined with kits produced by Nanjing Jiancheng Biological Research Company.

### Determination of the chlorophyll content

Two to three mature functional leaves of the plants in each treatment were randomly selected for the determination of chlorophyll content. During the determination, after removing the veins of the leaf, cut the remaining parts into pieces, and store the samples in refrigerator for preservation. An amount of 0.1 g leaves was accurately weighed and placed into a 10 mL centrifuge tube. An amount of 9 ml chlorophyll extraction solution (80% acetone and anhydrous ethanol 1:1 mix) was added, and the mixture was placed in the dark and left for more than 24 h until the leaves were completely white26. Spectral measurements were performed at wavelengths of 663 nm and 646 nm.

### Determination of the photosynthetic parameters

The net photosynthetic rate (Pn), stomatal conductance (Gs), transpiration rate (Tr), and intracellular CO2 concentration (Ci) were determined with an LI-6800 portable photosynthesis system (Li-Cor Inc, USA). Three plants were selected from each treatment, three leaves were selected from each plant, and ten data points were recorded for each leaf.

### Determination of the photosynthetic light-response curve

The LI-6800 portable photosynthesis system (Li-Cor Inc, USA) was used to measure the photosynthetic light-response curves of the leaves selected for the previous determination. The photosynthetically active radiation (PAR) gradient values were 1800, 1600, 1000, 800, 600, 400, 200, 100, 75, 50, 25, and 0 μmol/m2/s, the CO2 concentration was set to 400 ppm, and the room temperature was set to 30 °C. Each leaf was photoinduced for 20 min before the determination, and in this test, the photoinduction strength was 800 μmol/m2/s.

The photosynthetic light-response curves of sassafras leaves treated with different concentrations of cadmium were fitted by using Ye’s27 modified linear hyperbolic model. The fitting equation of the modified linear hyperbolic model is as follows:

$$Pn = \alpha \frac{1 - \beta I}{{1 - \gamma I}}I - Rd$$

When Pn = 0, the light compensation point (LCP) can be obtained:

$$LCP = \frac{{\alpha - Rd\gamma - \sqrt {\left( {\alpha - Rd} \right)^{2} - 4\alpha \beta Rd} }}{2\alpha \beta }$$

With dPn/dI = 0, the light saturation point (LSP) can be obtained:

$$LSP = \frac{{\sqrt {\left( {\beta + \gamma } \right)/\beta } - 1}}{\gamma }$$

When I = Is, Pnmax can be obtained:

$$Pn\max = \alpha \frac{1 - \beta Is}{{1 + \gamma Is}}Is - Rd$$

where Pn is the net photosynthetic rate; Pnmax is the maximum net photosynthetic rate at light saturation; α is the initial quantum efficiency; I is the photosynthetically active radiation; Rd is the dark respiration rate; β is the light suppression coefficient, which is the correction coefficient; and γ is the light saturation coefficient, which is the ratio of the initial slope of the light-response curve of the plant leaves to the maximum net photosynthetic rate.

### Statistical analysis

All data were collected in Excel 2016 and statistically analyzed with SPSS 24.0. The significance of the differences among means was assessed by one-way analysis of variance (ANOVA). Multiple comparisons were performed mostly with Duncan’s test to compare the mean values between treatments (p < 0.05). The figures were drawn in Origin 8.5.

## Results

### The effects of Cd stress on plant growth

The net growth in plant height decreased with increasing Cd concentration, but there were no significant differences among treatments (P < 0.05). The net growth in ground diameter showed an increasing trend, but there were no significant differences among treatments (P < 0.05). Significant reductions were recorded in root biomass by 18.18%, 27.35%, 27.57% and 28.96%, respectively among treatments compared to CT. The biomass of leaves and branches decreased with the aggravation of cadmium stress, and there were significant differences between treatments and CT for branches (except in Cd5); however, no significant differences were found for leaf biomass (P < 0.05). Declines were found in total biomass by 11.01%, 18.92%, 21.14% and 27.72%, respectively in sassafras under different Cd treatments. (Table 1).

### The effect of Cd stress on physiological characteristics

#### The contents of H2O2 and MDA in sassafras leaves under Cd stress

The H2O2 content decreased first by 10.96%, 11.82% and 7.02% respectively in Cd 5, Cd 20 and Cd50, and then increased by 2.36% in Cd100, but there were no significant differences among treatments. The MDA content showed the opposite trend to the H2O2 content as the cadmium concentration increased; increases were found in Cd 5, Cd 20 and Cd50 by 15.47%, 16.07% and 7.85%, respectively, and declined by 15.24% in Cd100; however, there were still no significant differences among treatments. (Fig. 1).

#### Response of antioxidant enzyme activity to Cd stress

SOD activity fluctuated with the increasing cadmium concentration, while the CT treatment showed the highest SOD activity, decreases were found by 17.05%, 10.68%, 20.85% and 8.91%, respectively among treatments. CAT activity showed a fluctuation trend, there were increases found in Cd5, Cd20 and Cd 100 by 47.19%, 32.45% and 9.06%, and decreased by 58.77% in Cd50. POD activity showed a slowly increasing trend, and the highest activity was observed in the Cd100 treatment. The increases of POD activity were recorded in sassafras by 5.41%, 3.66%, 2.81% and 8.83%, respectively in Cd5, Cd20, Cd50 and Cd100. (Fig. 2).

#### Changes in the contents of osmotic regulation substances in response to Cd stress

Upward trends were observed in Pro content by 90.76%, 74.36%, 99.73% and 126.01%, respectively among treatments; the Cd100 treatment had the maximum Pro content, but there were no significant differences among treatments. The content of SS was the highest in the CT treatment, while the SS content in the other treatments decreased compared to CT. Decreases were found in SS content by 32.84%, 5.85%, 25.55% and 38.69%, respectively. The SP content first increased and then decreased with increasing cadmium concentration and reached a maximum in the Cd50 treatment. Compared to CT, there were increases recorded in SP content by 2.34%, 21.36%, 53.13% and 24.22%, respectively; however, the differences between treatments were not significant. (Fig. 3).

### The effects of Cd stress on photosynthetic characteristics

#### Chlorophyll content in sassafras leaves under cadmium stress

With the increase in the cadmium concentration, the chlorophyll a and b contents and the total chlorophyll content in sassafras leaves first increased and then decreased, reaching a maximum in Cd20. There were no significant differences in the chlorophyll a content or the total chlorophyll content under the Cd5, Cd20 and Cd50 treatments, while the Cd100 treatment was significantly different from CT (P < 0.05). The chlorophyll b content showed a similar change trend as the chlorophyll a content, but a significant difference between the chlorophyll a and b contents appeared in Cd50 (P < 0.05). (Fig. 4).

#### Changes in photosynthetic gas exchange parameters in sassafras under cadmium stress

With the increase in Cd concentration, the Pn in the sassafras’ leaves showed a downward trend. Significant reductions were found in the Pn of sassafras’ leaves by 1.60%, 14.35%, 44.74% and 55.57%, respectively among treatments. The different concentrations of Cd did not significantly affect Gs, Ci or Tr. There were decreases found in Gs by 15.08%, 17.08%, 21.08% and 30.31%, respectively. Declines were also recorded in Tr by 14.29%, 30.95%, 42.86% and 38.10%, respectively. (Fig. 5).

#### The effect of Cd stress on the photosynthetic light-response curve

When the PAR was weak (PAR ≤ 200 μmol/m2/s), the Pn of leaves showed a nearly linear increasing trend, the increase in the Pn in CT was significantly higher than that in the cadmium treatments, and the increase in the Pn of Cd5, Cd20, Cd50 and Cd100 decreased with the increase in cadmium concentration. The increasing trend of the Pn of leaves slowed under different Cd concentrations when the PAR exceeded 200 μmol/m2/s. When the PAR was between 800 ~ 1800 μmol/m2/s, the Pn of leaves tended to be stable and eventually reached the light saturation point when leaf photosynthesis exhibited photoinhibition (Fig. 6).

The modified orthogonal hyperbolic model from Ye27 was used to fit the photosynthetic response curves of the sassafras leaves at the different cadmium concentrations, and the characteristic parameters of the light response were calculated (Table 2).

The maximum net photosynthetic rate (Pnmax) of the leaves decreased significantly with increasing Cd concentration. The highest Pnmax appeared in CT, at 9.19 μmol/m2/s; then, Pnmax decreased in the different treatments, by 37.11% (Cd5), 50.38% (Cd20), 80.20% (Cd50) and 84.22% (Cd100). The dark respiration rates (Rd) of the sassafras leaves under the different Cd concentrations were not significantly different from that of CT (Table 2).

The light compensation point (LCP) of the leaves was the highest in the Cd50 treatment and the lowest in the Cd100 treatment; however, there were no significant differences among the treatments. The light saturation point (LSP) of the leaves was the highest at Cd5 and the lowest at Cd100, and there were also no significant differences among the treatments (Table 2).

## Discussion

Cadmium (Cd) is highly mobile in plant tissues and affects plant physiological growth28. This study demonstrated that Cd stress in soil had a negative influence on sassafras growth and reduced the net growth in plant height and leaf, branch and root biomass. The decrease of the net growth in plant height and biomass may have been due to the toxicity of Cd to photosynthetic organs29 and plant structure30. Under Cd stress, the biomass allocation pattern of sassafras also changed; the degree of the decline in aboveground biomass was higher than that of the decline in underground biomass, probably because cadmium stress limited the acquisition of mineral nutrients required for plants31. These effects intensified the competition for nutrients among different plant organs; therefore, the plants retained their underground biomass at the expense of aboveground biomass in order to obtain more soil nutrients and maintain normal growth. In addition, cadmium stress can limit and inhibit plant photosynthesis, which weakens their photosynthetic production capacity and ultimately reduces their biomass; these impacts were more evident on aboveground biomass than on underground biomass32.

Photosynthesis is an important physiological process in plants. Many studies have shown that cadmium stress can inhibit plant photosynthesis and is significantly related to the degree of heavy metal stress55,56. Its effects on plant photosynthesis are mainly reflected in the destruction of chlorophyll structure and the reduction in photosynthetic pigment content, which can affect the ability of plants to photosynthesize57. The chlorophyll content is directly related to the intensity of photosynthesis and can reflect the ability of leaves to absorb and transform light energy. As the main pigment involved in photosynthesis, chlorophyll a is responsible for converting light energy into chemical energy in the light reaction center, while chlorophyll b is responsible for capturing and transmitting light energy58. In this research, the chlorophyll content first increased and then declined at increasing cadmium concentrations, which indicated that a low concentration of cadmium promotes an increase in chlorophyll content, while a high concentration of cadmium inhibits the formation of chlorophyll. This was probably due to the low concentration of Cd complexes accelerating the absorption of Mg, Fe, K and P nutrients from the soil, promoting the formation of leaf porphyrin rings, and thus increasing the chlorophyll content59.

Studies have shown that the Pn of plants generally decreases with increasing heavy metal concentration60, which is in line with our experimental results. Factors that affect the photosynthetic rate of plant leaves can be divided into stomatal factors and nonstomatal factors. Farquhar et al. proposed that to judge the factors that affect the photosynthetic rate of plants, changes in stomatal conductance and intercellular carbon dioxide concentration should be observed at the same time61. In this experiment, the Pn and Gs of sassafras leaves decreased, while the Ci increased, indicating that the photosynthetic rate of sassafras leaves was limited by nonstomatal factors.

Photosynthetic light-response parameters can reflect the photosynthetic potential, light energy utilization and light inhibition of plants under adverse conditions27,62. Pnmax can reflect the potential photosynthetic capacity of plants. The greater the value of Pnmax, the greater the photosynthetic potential of plant leaves, indicating that leaves can synthesize more photosynthetic products under the appropriate light conditions63. In this study, the Pnmax of sassafras leaves decreased significantly with increasing Cd stress, indicating that heavy metal stress significantly affected the photosynthetic capacity of the sassafras leaves. The LCP and LSP reflect the ability of plants to utilize weak light and strong light, respectively64. In this study, with the increase in Cd concentration, the LCP and LSP of sassafras leaves generally showed a decreasing trend, indicating that Cd stress weakened the ability of sassafras leaves to use both weak light and strong light. Qi’s research showed that reducing the LCP was an adaptive response of plants to a low light environment and was conducive to maintaining the carbon balance of plants under low light intensity65. Lower Rd and LCP in leaves are more conducive to reducing the consumption of photosynthetic products in leaves, thus increasing the net photosynthetic accumulation to obtain the maximum carbon accumulation63. Under cadmium stress, sassafras leaves maintained normal photosynthesis by reducing the Rd and LCP, which reflected the mechanism of the response of sassafras to cadmium stress.

## Conclusions

Cd stress had negative influences on sassafras growth, as it reduced the net growth of plant height and the biomass of leaf, branch, and root. The contents of H2O2 decreased first then increased while MDA showed the opposite trend with increasing cadmium concentration, which indicated that cadmium stress had a certain effect on the peroxidation of the inner membrane of sassafras seedlings, resulting in damage to the cell membrane structure. SOD activity declined among treatment compare to CT, while POD activity increased steadily with increasing cadmium concentration, indicating that POD was likely the main protective enzyme involved in the reactive oxygen removal system in sassafras. The increase in Pro content indicated that cadmium stress induced sassafras to synthesize more Pro to resist osmotic stress. Compared to that in CT, the SS content declined under the different Cd treatments. The SP content increased with the increasing levels of cadmium stress. At increasing levels of cadmium stress, the chlorophyll content of the seedlings first increased and then decreased, and it was higher in the Cd5 and Cd20 treatments than in the CT treatment. These results reflected that Cd had photosynthesis-promoting effects at low Cd concentrations and photosynthesis-suppressing effects at high Cd concentrations. The photosynthetic gas exchange parameters and photosynthetic light-response parameters showed downward trends in the Cd treatments compared with those in CT, which reflected the inhibition of photosynthesis in sassafras due to Cd stress.

## Abbreviations

Cd:

Chl a:

Chlorophyll a

Chl b:

Chlorophyll b

Car:

Carotenoids

Chl:

Chlorophyll

PAR:

P n :

Net photosynthetic rate

Gs :

Stomatal conductance

T r :

Transpiration rate

C i :

Intercellular CO2 concentration

LSP :

Light saturation point

Rd :

Dark respiration rate

LCP :

Light compensation point

SOD:

Superoxide dismutase

POD:

Peroxidase

Pro:

Proline

SS:

Soluble sugar

SP:

Soluble protein

CAT:

Catalase

MDA:

Malondialdehyde

ROS:

Reactive oxygen species

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## Acknowledgments

The authors would like to thank Prof. Hu and Prof. Zhang for critical reading of the manuscript and fruitful corrections. We would also thank all the participants in this project.

## Funding

The project has been funded by the Breeding Research Project of Sichuan Province during the 13th Five-year Plan period (2016 NZ0098-10), Key Project of Sichuan Education Department (13ZA0246).

## Author information

Authors

### Contributions

H.Z. developed the project and write the manuscript, which was read and revised by all co-authors. Fieldwork was performed by H.Z., J.G., Q.L. and X.Z. Laboratory analyses were performed by H.Z. and X.Z. H.Z., J.G.and Q.L. performed the statistics and data evaluation. H.H. and J.Z. were both corresponding authors who gave advices and provided funding for this project. All authors listed have made substantial, direct and intellectual contributions to the work, and have approved it for publication.

### Corresponding authors

Correspondence to Hongling Hu or Jian Zhang.

## Ethics declarations

### Competing interests

The authors declare no competing interests.

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Reprints and Permissions

Zhao, H., Guan, J., Liang, Q. et al. Effects of cadmium stress on growth and physiological characteristics of sassafras seedlings. Sci Rep 11, 9913 (2021). https://doi.org/10.1038/s41598-021-89322-0

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