Physiological and Biochemical Responses of Hydrilla verticillata under

Cu and Zn Stress

Guiqing Gao

, Weihan Yang

, Bo Fang

and Qingwu Chen

School of Civil and Architecture Engineering, Nanchang Institute of Technology, Nanchang, China

Keywords: The Heavy Metal Stress, Hydrilla verticillata, Superoxide Dismutase, Peroxidase, Malondialdehyde.

Abstract: In order to understand the physiological and biochemical responses of Hydrilla verticillata under heavy

metal stress, single and combined Cu or Zn were used for stress cultivation. The results showed that the

activities of SOD, POD and MDA in the first treatment group (Cu<0.2 mg/L, Zn<1.6 mg/L, Cu+Zn<0.1

mg/L + 0.8mg/L) were slightly lower than those in a blank control group. SOD, POD and MDA increased

significantly with the increase of heavy metal concentration within 7 days. From the 7th day to the 21st day

of culture, they increased first, then decreased. The change range of enzyme activity was the largest under

combined stress. The inhibitory effects of heavy metals on H. Verticillata were: combined stress > Cu stress

> Zn stress.

1 INTRODUCTION

Cu, Zn, Pb and Cd are common heavy metal

pollutants in water (Xing, 2013). Heavy metals play

a key role in the balance of active oxygen

metabolism system in plants (Ji, 2017; Gao, 2019).

The types and sources of heavy metals are different,

which lead to the complexity of water pollution

(Shahid,2017). Therefore, heavy metal polluted

water also generally appears in the form of

combined stress. Cu and Zn are trace elements for

plant growth, which can promote its development

and growth at low concentration. However, high

concentrations of Cu and Zn will affect the

absorption of other nutrients by plants, break the

balance of cell metabolism, and seriously curb their

physiological growth (Jian, 2016).

Hydrilla verticillata is the dominant species in

most lakes in China. Because of its rapid growth,

wide distribution and good effect on the enrichment

of heavy metals, it has gradually become a pioneer

species to solve the problem of heavy metal

pollution (Wang, 2020). In this study, the

physiological and biochemical characteristics of

H.verticillata under Cu and Zn stress were discussed

https://orcid.org/0000-0002-8468-3405

https://orcid.org/0000-0001-5871-2543

https://orcid.org/0000-0002-7352-8266

https://orcid.org/0000-0002-3754-6993

in order to provide some basis for the ecological

restoration of heavy metal polluted water.

2 EXPERIMENT MATERIAL AND

METHOD

2.1 Material

H.verticillata and sediment were collected in Poyang

Lake. After removing sundries, the sediment with a

thickness of 8 cm was evenly laid in plastic square

boxes (34.0 cm long, 22.5 cm wide and 10 cm high).

The apical tips of H.verticillata with a height of 20

cm were planted in the plastic boxes after washing

off the surface attachments, with 6 tips in each box.

Then, the plastic boxes were placed into the glass jar

(40 cm long, 40 cm wide and 50 cm high). Air-dried

tap water was slowly added into the jars to the

height of 48 cm for preculture. After the plant

growth was stable, it was used in the later

experiment.

2.2 Experimental Design

The toxicity of Cu and Zn to H.verticillata was

about 8:1 from pre-experiment. Naturally, the

concentrations of Cu and Zn should be configured

according to 1: 8 in water body.

Gao, G., Yang, W., Fang, B. and Chen, Q.

Physiological and Biochemical Responses of Hydrilla verticillata under Cu and Zn Stress.

DOI: 10.5220/0011189000003443

In Proceedings of the 4th International Conference on Biomedical Engineering and Bioinformatics (ICBEB 2022), pages 111-115

ISBN: 978-989-758-595-1

111

Different concentrations of CuSO

and ZnSO

combined stress treatment, and 5 treatment groups

(T1, T2, T3, T4 and T5) were set respectively (Table

1). At the same time, a blank control group (CK)

was set, and 3 replicates were set in each treatment

group. The indexes of stress culture were measured

every 7 days, and the experimental period was 21

days.

Table 1: Concentration setting (mg/L).

Cu Zn Cu+Zn

0 0 0+0

T1 0.2 1.6 0.1+0.8

T2 0.4 3.2 0.2+1.6

T3 0.6 4.8 0.3+2.4

T4 0.8 6.4 0.4+3.2

T5 1.0 8.0 0.5+4.0

2.3 Method

Malondialdehyde (MDA) was determined by

thiobarbituric acid colorimetry. Superoxide

dismutase (SOD) was determined by nitrogen blue

tetrazole colorimetry. Peroxidase (POD) was

determined by guaiacol.

2.4 Data Analysis

The experimental results were expressed in the terms

of the mean and standard deviation more than three

parallel data. Excel 2017 was used for the processing

and mapping of the original test data. SPSS 19.0

software was used for one-way analysis of variance,

and SNK method was used for multiple comparative

analysis. P < 0.05 indicated significant difference.

3 RESULTS AND ANALYSIS

3.1 Effects of Cu and Zn Stress on SOD

Single and combined stress of Cu and Zn had

significant effects on SOD of H.verticillata (P <

0.05). From T1 to T5, the SOD activity showed an

upward trend and reached the maximum at T5

(Figure 1).

There was no significant change in T1 and T2

compared with CK, and the activity of SOD at T1

was lower than that at CK. When the stress duration

was 14 days, the maximum of SOD under Cu single

stress and combined stress were 45.91 U/mg and

45.80 U/mg respectively, and appeared at T4. The

maximum of SOD under Zn single stress was 45.55

U/mg and appeared at T5.

Figure 1: Effects of different heavy metals stress on

SOD.

Under combined stress, the activity of SOD at T5

decreased below than that at CK. After 21 days’

stress culture, the maximum of SOD all appeared at

T3 under three stress modes, which increased

50.47%, 40.38% and 54.35% respectively compared

with CK, and then decreased gradually. On the

whole, the inhibition of Cu and Zn Stress on SOD

was as follows: combined stress > Cu Stress > Zn

stress.

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

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3.2 Effects of Cu and Zn Stress on

POD

After 7 days’ stress, the activity of POD gradually

increased with the increase of heavy metal

concentration (Figure 2).

The change of POD at T1 was not significant

compared with CK, which indicated that this

concentration range had a weaker effect on

H.verticillata. The values of POD at T5 reached the

maximum, which were 30.11 U/mg, 30.82 U/mg and

35.05 U/mg respectively. After 14 days’ stress

culture, the values of POD increased from T1 to T4,

then decreased at T5. The maximum values of POD

increased 91.12%, 84.05% and 117.28%

respectively compared with CK. When the culture

time reached 21 days, they reached the maximum at

T3 under three treatments, which were 31.87 U/mg,

29.66 U/mg and 33.64 U/mg respectively. The

values at T1 were smaller than those at CK under

three stress modes. On the whole, there were

significant differences in POD activity under heavy

metal stress.

Figure 2: Effects of different heavy metals stress on POD.

3.3 Effects of Cu and Zn stress on

MDA

Different concentrations of heavy metals had

significant effects on MDA (P < 0.05). After 7 days’

stress, the activity of MDA increased significantly

with the increase of heavy metal concentration

(Figure 3).

Physiological and Biochemical Responses of Hydrilla verticillata under Cu and Zn Stress

113

Figure 3: Effects of different heavy metals stress on MDA.

The maximum values of MDA were 17.41

μmol/g, 16.71μmol/g, 20.56 μmol/g respectively at

T5 under three stress modes. There was no

significant change at T1 compared with CK. After

14 days’ stress, the values of MDA still maintained

an upward trend under single stress of Cu and Zn,

and reached the maximum values at T5. However, it

reached the maximum of 24.51 μmol/g at T4

combined stress and began to decrease at T5. After

21 days’ stress, the maximum values of Cu and Zn

single stress were 20.58 μmol/g and 20.25 μmol/g

respectively at T4. The maximum value of combined

stress was 20.35 μmol/g at T3. In the three modes,

the change range of combined stress was greater

than that of single stress.

4 DISCUSSION

SOD plays an important role in improving plant

resistance to stress. In general, the toxicity of heavy

metals leads to excessive O

in plants. In order to

eliminate this effect, plants catalyze the conversion

of O

to H

by increasing SOD activity to protect

cells from damage (Parveen, 2017). Therefore, the

activity of SOD increased gradually with the

increase of heavy metals concentration. In this study,

the activity of SOD after 7 days’ stress culture

accorded with this characteristic. But the activities at

T5 on the 14th day, T4 and T5 on the 21st day

showed downward trends, which might be because

the stress of heavy metals has exceeded the tolerance

of H.verticillata and inhibited the enzyme activity of

the plant itself. Cu and Zn are elements required for

plant growth and can participate in their normal

physiological activities, and are conducive to the

exchange of cellular materials inside and outside the

cells. This is also the reason why the values of SOD

at T1 in the three treatments are slightly lower than

those of CK. In addition, the change range of SOD

under combined stress is greater than that under

single stress, which indicates that the existence of Zn

promotes the absorption of Cu by plant cells and

aggravates the toxicity of heavy metals.

POD catalyzes the decomposition of H

into

O and O

(Cao, 2004). The change law of POD is

very similar to that of SOD in this study. In the early

stage of the experiment, POD increased significantly

with the increase of heavy metals, but began to

decrease under the influence of high concentration

for a long time. On the 21st day, a significant

decrease of POD was observed in T4 and T5, which

indicated that high concentration of heavy metal

stress destroyed the normal function of enzyme,

weakened the ability of enzyme system to scavenge

reactive oxygen. It is similar to the reason for the

decrease of SOD.

The content of MDA represents the degree of

cell membrane oxidation (Tang, 2010), which is

used to reflect the damage degree of membrane lipid

in the process of plant stress or aging. The higher the

content of MDA, the more serious the inhibition of

plants (Wang, 2004). The content of MDA in

H.verticillata increased significantly with the

concentration of heavy metals and the duration of

poisoning, which indicated that the life activities of

plants were significantly inhibited by heavy metals.

On the 21st day of treatment, the value of MDA

reached the maximum under the single stress of Cu

or Zn at T4, but the combined stress reduced its

activity, which may be due to the increased toxicity

caused by the interaction between Cu and Zn, and

the toxic degree increased significantly, resulting in

the destruction of the enzyme protection system of

the plant itself. Therefore, the content of MDA

decreased significantly in T5 treatment group. The

change range of SOD, POD and MDA under

combined stress is the greatest, followed by Cu

single stress which may be that Cu is a very active

oxidative and reduced transition metal element, and

Cu can absorb or release an electron (Cu

/Cu

) to

produce free radicals causing damage (Li, 1993),

while the chemical properties of Zn are much

weaker than that of Cu.

5 CONCLUSIONS

The activities of SOD, POD and MDA increased

significantly with the increase of heavy metal stress

in the early stage (within 7 days). From the 7th day

to the 21st day, the indexes in high concentration

treatment group showed a significant downward

ICBEB 2022 - The International Conference on Biomedical Engineering and Bioinformatics

114

trend. The inhibition on H.verticillata was the

largest under Cu and Zn combined stress, followed

by Cu stress. It is the smallest under Zn stress.

ACKNOWLEDGEMENTS

This study was financially supported by the

General Project of Jiangxi Science and Technology

Department (20212BAB204402), and the 18

“Challenge Cup” Science and Technology

Competition of of Nanchang Institute of

Technology.

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