Effects of melatonin on growth and antioxidant capacity of naked oat (Avena nuda L) seedlings under lead stress

Plant materials and seed germination conditions

The naked oat variety “Jinyan 2” (Avena nuda L.) was provided by the Shaanxi Key Laboratory of Biotechnology and stored in a refrigerator at 4 °C. Healthy and full seeds were selected, disinfected for 30s with 75% alcohol, rinsed with distilled water three times, disinfected with 0.1% mercury solution for 7 min, finally rinsed with distilled water six times, moved into a water culture box containing Hoagland nutrient solution (pH 6.5 ± 0.1). Hoagland nutrient solution was replaced every three days, and the water culture box was placed in the culture room at 25 °C with 75% relative humidity, and a 14L/10D photoperiod.

Experimental group design

When the naked oats seedlings grew to the 7th day, the control group was continued to be irrigated with Hoagland nutrient solution, and the treatment group was irrigated with different concentrations of Pb solution (0, 25, 50, 75, 100 mg L⁻¹). According to the statistical observation, the Pb treatment condition was set as 75 mg L⁻¹ in this work. With reference to the previous experiment, the concentration of MT was selected as 50 and 100 µM (Gao et al., 2018). The experiment was divided into six treatment groups: control, Pb, MT₅₀, MT₁₀₀, MT₅₀+Pb and MT₁₀₀+Pb.Naked oat seedlings were cultured for 7 days and then treated with 0 and 75 mg L⁻¹ Pb(NO₃)₂, respectively, the hydroponic solution was renewed every three days. The seedlings grown in Pb-free nutrient solution was used as the control. MT₅₀ and MT₁₀₀ were treated with MT solution of 50 and 100 µM respectively on the basis of the control group. MT₅₀ +Pb and MT₁₀₀+Pb were root-irrigated with MT solution of 50 and 100 µM on the basis of the Pb treatment group. All six treatment groups were sampled seven days after the start of stress. three groups of parallel repeats were set up for sampling, testing and measurement, respectively.

Calculation of plant height, fresh weight and dry weight

After seven days of treatment in different treatment groups, naked oat seedlings were randomly selected from each hydroponic culture box, and the plant height, plant fresh weight and plant dry weight were measured. The plant height is the linear distance from the base of the radicle to the top of the leaf. After the seedlings of naked oats were taken out, they were simply washed with tap water, then rinsed with distilled water three times, gently dried with paper towels, and the fresh plants were quickly weighed with electronic scales. The seedlings of naked oats were dried at 105 °C for 30 min, then dried at 80 °C for 24 h, and the dry weight was measured by electronic balance.

Determination of chlorophyll content and lead content

The leaf samples frozen in liquid nitrogen from -80 °C (fresh weight 0.2 g) were fully ground in liquid nitrogen with 95% anhydrous ethanol, then the volume was fixed to 20 mL, 4 °C and placed in the dark for 24 h. The values of OD649 and OD665 of supernatants were determined after samples were centrifuged for 10 min. The formula for calculating chlorophyll is as follows: C = C_a+ C_b, in which C_a =13.95A₆₆₅−6.88A₆₄₉−7.32A_665, C_b is 24.96A₆₄₉−7.32A₆₆₅. Chlorophyll content = chlorophyll concentration × extraction liquid volume × dilution multiple / sample fresh weight (Porra, Thompson & Kriedemann, 1989). The content of Pb in leaves of naked oat seedlings was determined by graphite furnace atomic absorption spectrophotometer (model Z2700), following the method of Shi, Li & Pan (2009). The sample was dried in an air-forced oven at 60 °C for 48 h. Dry plant material was ground in a stainless-steel blender to pass through a 0.4-mm sieve. Leaf samples were subjected to mixed acid digestion (HNO₃-HClO₄-HF). A certified reference material of sediment IRMM-804 with (0.42 ± 0.07) mg kg⁻¹ Pb was purchased from the National Centre for Certificate Reference Materials, China, and was used with all patch of digestions. Dried plant samples were digested in a 4:1 (V V⁻¹) mixture of HNO₃-HClO₄. Ten milliliter of mixed acids was added to 0.75 g of plant sample in a high-walled beaker and allowed to stand for 12 h at 25 °C. The samples were then heated in a sand bath at 170 °C until clear. After cooling, the solution was diluted to 25 mL with deionized water. The Pb content of the digested solutions was determined by graphite furnace AAS. A reagent blank was incorporated within each batch of analytical samples.

Determination of antioxidant enzyme activity, soluble protein and proline content

The fresh weight of the leaf sample is 0.2 g. PBS buffer (pH7.4) was added to the sample, which was ground to homogenate in an ice bath, and then centrifuged at 8,000 × g for 10 min to collect the supernatant. The supernatant was used for the determination of superoxide dismutase (SOD; EC1.15.1.1), peroxidase (POD; EC1.11.1.7) and catalase (CAT; EC1.11.1.6). The activities of SOD, POD and CAT were recorded at 550 nm, 420 nm and 405 nm, respectively, according to Elavarthi & Martin’ s method (2010).

The content of soluble proteins was detected by kits (Nanjing Institute of Bioengineering, Nanjing, China). 0.1 g of fresh leaves was homogenized with 900 µL buffer and the homogenate was centrifuged for 10 min (3000 g); 50 µL supernatant was added to three mL Coomassie brilliant blue solution. The mixture was incubated at 25 °C for 30 min and the absorbance of soluble protein content was recorded at 595 nm.

Proline content in seedlings of naked oat was measured according to the ninhydrin method described by Bates, Waldren & Teare (1973). The leaf sample was 0.2 g. Proline was extracted with 3% sulphosalicylic acid and then filtered. A portion of the filtrate was supplemented by the addition of one mL of ninhydrin and glacial acetic acid reagent. The reaction mixture was boiled (95 °C) for 1 h. Then the mixture was placed on ice to stop the reaction, the absorbance of the sample was measured at 520 nm.

Determination of malondialdehyde and ROS accumulation

Malondialdehyde (MDA) was measured according to the procedure described by Tan et al. (2019). The leaf sample (0.5 g fresh weight) was ground with five mL of 5% trichloroacetic acid and centrifuged for 10 min (8000 g, 4 °C). one mL of supernatant was mixed with one mL of 0.67% thiobarbituric acid (TBA). The mixture was then boiled in water for 30 min, the absorbance at 532 nm and 600 nm was measured respectively to calculate MDA content.

To measure H₂O₂, the methods described by Nawaz et al. (2018) was followed. Fresh leaves (100 mg) were ground in a mortar with 900 µL buffer, H₂O₂ content was recorded at a wavelength of 405 nm. The kit method (Nanjing Jiancheng Institute of Biological Engineering, Nanjing, China) was applied to detect O₂^•−−, Fresh leaves (100 mg) were centrifuged at 4000 g and 25 °C for 10 min. 50 µL of the supernatant was mixed with the four mL reagent solution. The mixture was bathed at 37 °C for 40 min and then two mL reagent solution was added. O${}_2^{•--}$ content was recorded at a wavelength of 550 nm.

According to the method of DAB staining (Orozco-Cardenas & Ryan, 1999), fresh leaves were soaked in DAB solution (1 mg mL⁻¹), the pH was adjusted to 3.8, vacuum was permeated for 30 min, shaker dark treatment was placed for 8 h, washed twice with 95% anhydrous ethanol, then was boiled in a water bath (20 min), and stored in 50% glycerin after cooling, stored at 4 °C. At least three leaves were selected for each treatment, and photographed with a stereomicroscope.

Extraction of RNA and analysis of related gene expression

Sequence search and primer design were carried out according to previous work (Gao et al., 2018). The list of primers is shown in Table S1. The total RNA of naked oats was extracted by TRIzol method. First-strand cDNA was synthesized using the PrimeScript™ RT reagent kit with the gDNA Eraser (Takara, Shiga, Japan). qRT-PCR was performed on a Bio-Rad CFX96 Real-Time PCR System (Bio-Rad, Hercules, CA, USA) using FastStart Essential DNA Green Master (Tiangen, Beijing, China). The procedure is as follows: 95 °C for 10 min, 1 cycle, 95 °C for 10 s, 60 °C for 30 s, 40 cycles. Finally, a melting curve was drawn to reconfirm the specificity of the primers by heating the product from 60 °C to 95 °C. The internal reference gene was Actin (KP257585.1) (Gao et al., 2018; Genty, Briantais & Baker, 1989). Three independent biological replications were performed for each experiment. The relative gene expression levels were calculated according to the 2^−ΔΔCt method and presented as fold changes.

Data analysis

All the measurements reported in this study are the means of three replicates. Vertical bars represent ± S.D. SPSS26.0 software was used for data statistics univariate analysis and minimum significant difference (LSD) test to compare the average values. We use prism 8.00 to organize pictures.

MT improves the growth of naked oat seedlings under lead stress

In the simulated experimental group (Control, Pb, MT₅₀, MT₁₀₀, MT₅₀ +Pb, MT₁₀₀ +Pb), on the 7th day (Fig. 1), the Pb solution decreased the plant height, fresh weight, dry weight and total chlorophyll content of naked oat seedlings. Under Pb stress, MT treatment improved seedlings growth with the plant height of seedlings increasing by 27.8% in MT₁₀₀ +Pb group, and 18.6% in MT₅₀ +Pb group compared with Pb group (Fig. 1A). The fresh weight of seedlings decreased by 47.2% after Pb treatment, which was significantly alleviated by the application of MT (Fig. 1B). Compared with the the Pb group, the dry weight of MT₅₀ +Pb and MT₁₀₀ +Pb groups increased by 20% and 33.3%, respectively (Fig. 1C). The chlorophyll content of the seedlings treated with 50 and 100 µM MT increased by 21.4% and 1.7% respectively compared with the control group, and the effect of 50 µM MT was more significant. Pb stress decreased the total chlorophyll content, but after MT treatment, the total chlorophyll content of naked oat seedlings increased considerably, with leaves also turning to green. Furthermore, the toxic symptoms were alleviated, with the total chlorophyll content increasing by 66.7% and 33.3% respectively compared with the Pb group. It can be observed that Pb inhibited the growth of naked oat seedlings, while the application of exogenous MT improved the tolerance of naked oat seedlings to Pb.

Exogenous application of MT enhances the activity of antioxidant enzymes in naked oat seedling cells

SOD, POD and CAT are important protective enzymes in plants, and they are all important indicators of plant resistance to stress. Pb stress changed the antioxidant enzyme activity of naked oat seedlings (Fig. 2), in which SOD, POD, CAT activity increased. After 50 and 100 µM MT pretreatment, SOD activity was significantly increased by 84.0% and 76.4%, respectively. Interestingly, the efficiency of MT to increase SOD activity decreased under Pb stress, but after MT₅₀ +Pb and MT₁₀₀ +Pb treatment, it increased 31.8% and 28.1% respectively compared with Pb group. Compared with the Pb stress group, the POD activity of the group treated with MT₅₀ +Pb and MT₁₀₀ +Pb increased by 50.7% and 62.0%, while the activity of CAT increased by 49.5% and 36.0%. The results showed that exogenous MT could enhance the activity of antioxidant enzymes in naked oats cells under Pb stress, so as to strengthen the antioxidant system of plants and reduce the toxicity of Pb to naked oats.

Changes of osmotic regulation substances in naked oat seedlings

After naked oat seedlings cells sensed the external Pb stress, plant soluble proteins and proline increased or decreased accordingly to maintain the redox balance and alleviate the Pb poisoning of plant cells (Fig. 3). MT₅₀ and MT₁₀₀ treatment did not cause significant changes in intracellular soluble protein content in the control group, and Pb stress decreased the soluble protein content, but after 50 µM or 100 µM MT pretreatment, the soluble protein content increased. Compared with the Pb group, the soluble protein content of MT₅₀ +Pb and MT₁₀₀ +Pb group increased by 99.3% and 96.9%, respectively (Fig. 3A). The content of proline increased by 31.5% and 24.2% in MT₅₀ and MT₁₀₀ treatments, respectively compared with the control group (Fig. 3B). Pb stress significantly increased proline content, and the effect of MT treatment was more significant. In MT₅₀ +Pb treatment group it increased by 25.4% while for MT₁₀₀ +Pb group the number was 19.8%. Therefore, MT might eliminate the increase of ROS in naked oat seedlings caused by Pb stress, stabilize the molecular structure of soluble proteins and enhance the tolerance of naked oats to heavy metal Pb stress. At the same time, the ROS of naked oat seedlings were elevated under Pb stress, and MT application could effectively alleviate the accumulation of ROS in naked oat seedlings.

Pb stress caused a significant increase in the contents of H₂O₂ and O${}_2^{•-}$ in young leaves of naked oats (Fig. 4). MT could significantly alleviate this phenomenon. Compared with the Pb group, the contents of H₂O₂ in MT₅₀ +Pb and MT₁₀₀ +Pb groups decreased by 30.3% and 25.7%, respectively, and the content of O${}_2^{•-}$ decreased by 25.7% and 24.7%, respectively. In the experiment, the content of MDA increased significantly under Pb stress, but MT treatment could reduce the content of MDA, and the content of MDA decreased by 42.9% and 34.4% in MT₅₀ +Pb and MT₁₀₀ +Pb groups, respectively (Fig. 4C). DAB histochemical staining of naked oat seedling leaves (Fig. 4D) showed that Pb stress induced oxidative stress of naked oats, and a large amount of H₂O₂ was accumulated in seedling leaves, which affected the leaf morphology. After Pb poisoning, the seedling leaves became smaller and narrower, and the DAB staining was aggravated. After MT treatment, the symptoms of the leaves treated with MT₅₀ +Pb and MT₁₀₀ +Pb were relieved, and the accumulation of H₂O₂ was reduced. The above results showed that after the Pb ion entered into naked oat seedlings, oxidative stress produced O₂^•−, H₂O₂ and other reactive oxygen species, which could be reduced by external application of MT. It is speculated that MT might be used as an antioxidant to help plants remove excessive ROS, lessen the degree of membrane lipid peroxidation in naked oat leaves, improve their tolerance under Pb stress, and improve the growth of naked oat seedlings.

Exogenous MT application increases the expression of genes related to oat seedlings

The expression of Lipoxygenase (LOX) and peroxygenase (POX) of naked oats changed significantly after exogenous application of MT (Figs. 5A and 5B). A total of 50 and 100 µM MT significantly increased the expression of LOX by 431.2% and 475.5%. Under Pb stress, the expression of LOX in MT₅₀ +Pb and MT₁₀₀ +Pb groups was also significantly increased by 214.3% and 262.3% (Fig. 5A). POX gene expression also increased significantly after Pb stress, and exogenous MT pretreatment increased 127.7% and 135.1% respectively (Fig. 5B). The results showed that MT treatment changed the expression level of lipid peroxidase in naked oat cells under Pb stress, which may protect plants from oxidative stress by up-regulating LOX and POX.

Asmap1 gene is a MAPK (mitogen activated protein kinase) protein in naked oats. After MT pretreatment, there was no significant change in MAPK cascade response in the Control group, after Pb treatment, Asmap1 gene expression increased significantly by 273.3% and 233.7% respectively (Fig. 5C) in MT₅₀+Pb and MT₁₀₀+Pb groups treated with MT, indicating that MT₅₀ can induce a stronger MAPK cascade response. It is suggested that MT can induce gene upregulation in MAPK cascade reaction and enhance the tolerance of naked oat seedlings to Pb stress.

Compared to the Pb group (Fig. 5D), the gene (NAC) expression of MT₅₀ +Pb and MT₁₀₀ +Pb groups increased significantly by 99.1% and 267.2% respectively. Compared to the Pb group, the gene (WRKY1) expression of MT₅₀ +Pb and MT₁₀₀ +Pb groups increased by 5.0% and 66.1%. It is speculated that exogenous MT may affect the expression of related TFs in naked oat seedlings in some way, and further enhance the tolerance of naked oat seedlings to Pb stress.