Effect of H2O2 on Growth of Collard (Brassis oleracea L.) Seedlings Under Salt Stress

Wei LI Junjie GUO Hongyan LI

AbstractCollard variety (Brassis oleracea L. var. acephala f. tricolor Hort.) as a research material was treated with exogenous H2O2 and H2O2 scavenger dimethyl thiourea under 100 mmol/L NaCl stress. Two days later, growth rate, dry weight, fresh weight and relative water content of the plants were determined. After 6 h of treatment, the activity and gene expression of three antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX) in plants, were measured. The results showed that the growth rate, dry weight, fresh weight, relative water content, and the activity and gene expression of the three antioxidant enzymes in collard seedlings were higher in the treatment of salt stress with the addition of 0.05 mmol/L exogenous H2O2 than in the simple salt stress treatment; and when endogenous H2O2 was removed, the growth rate, dry weight, fresh weight, relative water content, and the activity and gene expression of the three antioxidant enzymes in plant seedlings were lower than those under simple salt stress. It is speculated that under salt stress, H2O2 is involved in the regulation of antioxidant defense gene expression, and it might be an important regulator of saltinduced antioxidant system in collard leaves.

Key wordsBrassica oleracea L. var. acephala f. tricolor Hort.; H2O2; Salt stress; Antioxidase; Gene expression

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Received: August 21, 2018Accepted: November 12, 2018

Supported by Science and Technology Development Planning Project of Henan Province (182102110305).

Wei LI (1988-), male, P. R. China, lecturer, master, devoted to research about landscape plants and landscape design.

*Corresponding author. Email: 357719846@qq.com.

Salt stress is the most common stress condition causing severe crop yield reduction. Improving plant resistance to environmental stress, especially resistance to salt stress, is the first target for stable plant growth. When plants are exposed to environmental stress, a large amount of active oxygen is produced, including hydrogen peroxide (H2O2), singlet oxygen (1O2), hydroxyl radical (?OH), superoxide anion (O-2•) and lipid peroxy radical (ROO•)［1-4］. Salt stress can rapidly induce the accumulation of reactive oxygen species in plants, causing oxidative damage to proteins and lipids［5］. There are two kinds of scavenging protection mechanisms in plants, namely enzymatic system and nonenzymatic system, of which the enzymatic system includes superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase (APX). Studies have shown that salt stress can increase the activity of active oxygen in plants［6-8］. In recent years, many experiments have shown that H2O2 is a kind of important signaling molecule that can be produced by plant cells when responding to various stresses and further regulates a series of signal transduction of stress response［9-14］. There have been evidences showing that the accumulation of endogenous H2O2 can increase the stress resistance of plants under adverse conditions, and excessive ROS such as H2O2 can cause oxidative damage to macromolecules in cells. Wahid et al.［13］ showed that hydrogen peroxide pretreatment can keep cell membrane system intact, and in turn, improve the antioxidant capacity of wheat seeds and enhance the salt tolerance in wheat during germination. SOS1 (salt overly sensitive), which plays a key role in the salttolerant response of plants, is a Na+/H+ antiporter of Arabidopsis thaliana, and H2O2 can regulate the stability of its mRNA. However, under salt stress induction, the relation between H2O2 and the activity and expression mechanism of oxidases still remains to be fully understood.

In this study, the effects of applying exogenous H2O2 or removing invivo H2O2 on growth characteristics, antioxidant enzyme activity and related gene expression of collard (Brassica oleracea L. var. acephala f. tricolor Hort.) seedlings under salt stress were investigated, so as to reveal the action mechanism of H2O2 in the salttolerant response of plants.

Materials and Methods

Experimental materials

The experiment was carried out in the laboratory of Huanghuai University from August to October, 2015. Collard was selected as the research material and was provided by Nanhai Park in Zhumadian City. The H2O2 donor was 30% H2O2, the H2O2 scavenger was dimethyl thiourea, and both of them were domestic, analytically pure.

Experimental treatments

Collard seeds were sterilized and rinsed for several times with distilled water, and water on the seeds was absorbed with filter paper. The treated seeds were placed in a petri dish with filter paper, and cultured in a greenhouse［temperature: (24±2) ℃, light/darkness: 16 h/8 h, illuminance: 2 000 lx］. When the radicles of collard were 3 to 5 mm long, seedlings with uniform radicles were selected and potted. When seedlings grew to a length of about 10 cm, they were placed in a Hoagland solution and precultured in a greenhouse for 3 d. Three days later, the seedlings were divided into four treatments: control (CK): Hoagland solution, salt treatment (N): 100 mmol /L NaCl+Hoagland solution, NaCl+H2O2 treatment (N+H): 100 mmol/L NaCl+0.05 mmol/L H2O2+Hoagland solution, and NaCl+DMTU treatment (N+DMTU): 100 mmol/L NaCl+5 mmol/L DMTU+Hoagland solution. After 6 h of treatment, analysis and determination of SOD, CAT and APX activity and gene expression (SOD, GenBank: JQ321587.1; CAT, GenBank: JF720325.1; APX, GenBank: XM_013733887.1) were performed. Total RNA in leaves was extracted by guanidine thiocyanate method, and ABI7700 was used for quantitative PCR with actin (GenBank accession number: AF111812.1) as the internal reference (Table 1) according to reference［15］. Each treatment had three replicates. In order to maintain the consistency of treatment, the treatment liquid was changed every day during the treatment. After 48 h of treatment, the growth rate of collard seedlings was determined, and the dry weight, fresh weight and relative water content were determined, according to reference［16］.

Results and Analysis

Effects of applying exogenous or scavenging endogenous H2O2 on collar seedling growth under salt stress

Studies on growth of collard seedlings showed that there was no significant difference between the collard seedlings treated with 50 mmol/L NaCl and seedlings in the CK, and only some old leaves withered. Under the stress of 100 mmol/L NaCl, the damage of collard seedlings increased as the upper leaves turned yellow and lower leaves wilted. When the NaCl concentration reached 150 and 200 mmol/L, leaves were seriously dehydrated, resulting in death of whole collard seedling. Therefore, in this study, 100 mmol/L NaCl was selected for the salt treatment. At this concentration, collard seedlings exhibit certain salt damage symptoms, but death of plants would not be caused. The trial test showed that the H2O2 concentration of 0.05 mmol/L had a remarkable effect on growth of collard seedlings under the stress of 100 mmol/L NaCl. Therefore, the H2O2 concentration of 0.05 mmol/L was selected in this experiment.

The growth of collard seedlings treated with 100 mmol/L NaCl was significantly inhibited, and the plant growth rate and the fresh weight, dry weight and relative water content of the aboveground part decreased significantly compared with the CK. Exogenous H2O2 significantly increased the growth rate, fresh weight, dry weight and relative water content of saltstressed collar seedlings compared with the simple salt stress treatment (Fig. 1). Under salt stress, the treatment with H2O2 scavenger significantly reduced leaf growth rate, plant fresh weight, dry weight and relative water content compared with the simple salt stress treatment, indicating that the growth of collard under salt stress was inhibited after H2O2 removal.



CK: Control, 1/2 Hoagland solution; N: 100 mmol/L NaCl; N+H: 100 mmol/L NaCl +0.05 mmol/L H2O2; N+DMTU: 100 mmol/L NaCl+5 mmol/L DMTU. Similarly hereinafter.

Fig. 1Effect of H2O2 on growth of collard seedlings under salt stress

Table 1Primer sequences

Gene name Primer sequence (3′→5′)

SOD F: CGCCATCAAGTTCAACGG

R: CAGCACCTTCAGCACTCATC

CATF: CCCACAGGACTACAGGCACA

R: AATAGCAGGGCAGAAAGCAA

APXF: TCCCACAGGACTACAGGCACA

R: ACAATAGCAGGGCAGAAAGCA

αctinF: CCCTCAGCACTTTCCAACAGATGT

R: CACACTCACCACCACGAACCAG

Effects of applying exogenous or scavenging endogenous H2O2 on activity of antioxidant enzymes in collar seedlings under salt stress

When plants are in a stressful environment, the activity of antioxidant enzymes usually increases to enhance the adaptation to adversity. Under 100 mmol/L NaCl stress, the activity of SOD in collard seedlings increased significantly, with a significant difference from the CK. The treatment with exogenous 0.05 mmol/L H2O2 could significantly increase SOD activity compared with the simple salt stress (Fig. 2). It indicates that a low concentration of exogenous H2O2 can enhance the activity of antioxidant enzymes. CAT and APX were similar to SOD.



Fig. 2Effects of H2O2 on SOD, CAT and APX activity in collard seedlings under salt stress

Effects of applying exogenous or scavenging endogenous H2O2 on gene expression of antioxidant enzymes in collar seedlings under salt stress

Compared with the CK, under the salt stress condition, the expression of SOD, CAT and APX genes increased, and showed the same trend. When exogenous H2O2 was added, the expression levels of the three genes increased, exhibiting significantly differences from the CK. When using the H2O2 scavenger, the expression of the three antioxidant protective genes in collard under salt stress were determined, and the results showed that the expression levels of the three genes were significantly lower than those under the simple salt stress (Fig. 3).



Fig. 3Quantitative analysis of SOD, CAT and AXP genes

Wei LI et al. Effect of H2O2 on Growth of Collard (Brassis oleracea) Seedlings Under Salt Stress

Discussion

When plants are subjected to salt stress, the active oxygen metabolism in plants is imbalanced, and accumulated reactive oxygen species causes salt damage［17-18］. Therefore, in this study, high concentration H2O2 treatment caused increased active oxygen in collard seedlings and decreased adaptability to salt stress, thereby resulting in increased plant damage and severely inhibited growth. Recent studies have shown that active oxygen such as H2O2 can also play a signal transduction role in plants. Trace oxygen peroxide and other reactive oxygen species play an important role in regulating certain physiological phenomena, especially in cell signal transduction. The 0.05 mmol/L H2O2 used in this study may play a certain signal role in the response to salt stress in collard, and participate in or affect the salt stress signal transduction process, thus alleviating the inhibition of salt stress on growth of collard seedlings to some extent. Salt stress can inhibit the respiratory metabolism of plants, during which intracellular ROS (such as H2O2) increases greatly, causing secondary damage to plants, such as lipid peroxidation of cell membranes. In order to eliminate ROS in plants, the activity of antioxidant enzymes in plants would increase accordingly, so as to increase salt tolerance in plants［19-20］. Under salt stress, antioxidant enzymes such as plant SOD, CAT and APX act synergistically to remove excess reactive oxygen species, which makes the active oxygen metabolism in equilibrium and protect the integrity of biofilm which thus can endure salt stress within a certain range. However, when the production of reactive oxygen species exceeds the scavenging capacity of the antioxidant system, plants are harmed［21］. SOD catalyzes the disproportionation of O-2? to avoid toxic damage of O-2• to cells, while H2O2 produced during disproportionation is scavenged by CAT, APX, etc.［22-23］. It has also been suggested that H2O2 is an important endogenous signal molecule in the active oxygen signal transduction pathway and plays an important role in plant resistance to biotic and abiotic stresses［24］. The results of this study showed that under salt stress, the activity of the three antioxidant enzymes was enhanced compared with the CK. However, under high salt stress, the appropriate concentration of H2O2 could increase the activity of SOD, CAT and APX in collard leaves, while under the action of the H2O2 scavenger, the three antioxidant enzymes decreased significantly compared with the simple salt stress treatment. It indicates that a low concentration of H2O2 plays a role of signal transduction under salt stress.

H2O2 as a signaling molecule also participates in the regulation of stressrelated gene expression. Under certain conditions, H2O2 as a signaling molecule can regulate the expression of a series of related genes［25-26］. For example, when plants are infected by pathogens, H2O2 in plants induces the expression of PR1 and PAL genes［27］. Some people treated the legumes with 10 mmol/L H2O2, and found that the NOS activity in the immune response increased by nearly 8.3 times. Soybean (Glycine max L.) cells were treated with exogenous H2O2, and the cytoplasmic APX transcription level was significantly improved［28］. If cultured rice (Oryza sativa L.) cells are treated with the APX inhibitor hydroxyurea or the CAT inhibitor aminotriazole, the production of H2O2 will significantly increase, which significantly improves the transcription level of APX［29］. Other studies have shown that H2O2 activates mitogenactivated protein kinase (MAPK) to further enhance antioxidant defense enzyme activity［30］. The results of this study showed that under salt stress, the expression of the three antioxidant enzymes was positively regulated by hydrogen peroxide (Fig. 3), indicating that the improvement of plant tolerance to salt stress by the antioxidant defense system is related to the accumulation of endogenous hydrogen peroxide.

In summary, under salt stress, H2O2 is involved in the regulation of antioxidant enzyme activity in collard, and also participates in the regulation of antioxidant enzyme gene expression. It might be an important regulator of saltinduced antioxidant system in collard leaves.

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