Boron alleviates the aluminum toxicity in buckwheat by regulating antioxidant defense system and maintaining osmotic balance

doi:10.21203/rs.3.rs-2617131/v1

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Boron alleviates the aluminum toxicity in buckwheat by regulating antioxidant defense system and maintaining osmotic balance

https://doi.org/10.21203/rs.3.rs-2617131/v1

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Aluminum (Al) pollution in acidic soils has become a serious environmental problem worldwide. Boron can help plants mitigate aluminum phytotoxicity in acidic soils, but its underlying mechanism in buckwheat remains completely unknown. In this study, the buckwheat germination and greenhouse pot experiments were carried out under the conditions of four Al concentrations (0, 5, 10, and 15 mM) and four boron concentrations (0, 10, 30, and 50 µM). This research aimed to investigate the mitigation effect of exogenous boron on the Al toxicity of buckwheat and reveal the mechanism from the aspects of growth characteristics, antioxidant enzyme system, and osmotic regulation. The results showed that boron supply reduced the accumulation of Al in buckwheat roots, increased the antioxidant enzyme activity and the content of osmotic adjustment substances in roots and leaves, reduced the Al damage to root and leaf cells, maintained the osmotic balance of the cells, improved the root activity and the content of leaf photosynthetic pigment, ultimately leading to a significant increase in buckwheat biomass. In conclusion, different levels of boron treatment increased the tolerance of buckwheat root and leave to Al toxicity, and had a promoting effect on the growth of buckwheat. Therefore, applying boron is a promising green remediation technique to improve crop tolerance in Al-contaminated acidic soil.

Acidic soil

Aluminum toxicity

Antioxidant enzyme

Boron

Buckwheat

Osmotic balance

Aluminum toxicity has become a critical constraint to plant growth in potentially arable acidic soils worldwide. Currently, more than 50% of the world's potentially arable lands are acidic, posing a serious threat to food safety and public health (Kochian et al., 2015; Wei et al., 2021). Normally, Al in the soil exists as insoluble phosphates, sulfates, silicates, and oxides that are less toxic to plant growth and development, but under acidic soil (pH ≤ 5) conditions, Al is released into the soil solution in the form of Al³⁺, which is toxic to the plant (Barcelo and Poschenrieder, 2002). The activity of Al³⁺, the main toxic Al species, increases by 1000 times for every unit decrease in soil pH (Kopittke and Blamey, 2016). Inhibited root growth and morphological abnormalities, such as swollen root tips and atrophy of root hair, are the early symptoms of Al toxicity in plants (Huang et al., 2014). Additionally, especially at the leaf tip, the top young leaves displayed chlorosis and burnt symptoms of Al toxicity (Yan et al., 2021). Al toxicity can further damage mitochondria and chloroplasts, affect nuclear function, and cause apoptosis (Vázquez et al., 1999; Panda and Matsumoto, 2007). Al also prevents cell division and elongation in roots, resulting in irregular cell arrangement and thickening of the cell wall, and then significantly impairs root absorption of water and nutrients, and ultimately reduces crop yield and quality (Ryan, 2018; Zhu et al., 2018; Yang et al., 2019; Yan et al., 2021). Studies have found that under Al stress, the yield of rapeseed decreased by 28.7% (Xiong et al., 2019), that of wheat decreased by 73.45% (Valle et al., 2009), and that of groundnut decreased by 39.58% (Liao et al., 2000). The relative crop yield losses due to Al phytotoxicity will increase from about 4% in 2010 to 24% in 2050 if appropriate measures are not taken (Zhu et al., 2020). Therefore, Al phytotoxicity is considered to be a key factor limiting crop yield in acidic soils. Given the growing population and extreme land degradation, enhancing the crop productivity on these Al-toxic soils is especially necessary to ensure sustained global food security. Ameliorating the Al phytotoxicity in acid soils has attracted extensive attention from scientists, and lime and fertilizers have been widely used to mitigate Al toxicity in acidic soils by neutralizing soil acidity (Fontoura et al., 2019; Zhang et al., 2019; Shi et al., 2019).

Boron is an essential nutrient for plant growth and development and plays a vital role in the cellular structure and metabolic activities of plants (O'Neill et al., 2001). Boron is present in the soil mainly in the form of uncharged boric acid [B(OH)₃] and is absorbed by plants in the form of molecules with a structure similar to Al(OH)₃ (Blevins and Lukaszewski, 1998). Boron can alleviate Al toxicity by changing the characteristics of the cell wall. The synthesis and degradation of homogalacturonan (HG) participate in the structure and function of the cell wall, and the addition of boron can reduce the disordered distribution of HG in pectin caused by Al toxicity and enhance the plant tolerance to Al (Riaz et al., 2018a). Studies in the sunflower have found that boron can promote the anabolic effects of ascorbic acid and glutathione, and reduce the oxidative damage caused by Al toxicity (Ruiz et al., 2006). While boron can also reduce the absorption of Al in rapeseed roots and regulate the activity of antioxidant enzymes to alleviate the inhibitory effect of Al on root elongation (Yan et al., 2018c). It has been reported in trifoliate orange that boron can lessen oxidative stress caused by Al toxicity and decrease plasma membrane damage by regulating the antioxidant defense system and reducing the uptake of Al in the roots (Riaz et al., 2018b). Pirzadah et al. (2019) discovered that the antioxidant system is essential to protect plants from the initial deleterious effects of Al. Similarly, it has been proven that exogenous P, fluorine, and Ca mitigate Al toxicity in plants by enhancing the overall antioxidant capacity (Guo et al., 2006; Dawood, et al., 2022; Karimaei et al., 2022). Therefore, it is feasible to take certain measures to alleviate Al toxicity by regulating the activity of antioxidant enzymes in plants.

Buckwheat (Fagopyrum esculentum Moench L.) belongs to the Fagopyrum of Polygonaceae and is widely grown around the world (Fang et al., 2018). Buckwheat is an important underutilized crop, also known as an Al-tolerant and Al-accumulating species. (Ma et al., 1997; Pirzadah et al., 2019; Salazar-Chavarría et al., 2020). Buckwheat grows well in acidic soils through different treatment strategies for Al toxicity, the roots take up Al through the form of Al³⁺, which complexes with oxalate in a 1:3 ratio to form a non-phytotoxic form of Al, and it is also able to accumulate a large amount of Al in its leaves (Ma et al., 1997, 1998). In recent years, the rate of soil acidification has increased significantly worldwide due to various anthropogenic activities, including acid deposition, excessive application of ammonium-base fertilizers, and crop removal (Guo et al., 2010; Zhu et al., 2018; Cecchini et al., 2019; Cho et al., 2019; Meng et al.,2019). Although buckwheat has the unique advantage in tolerance to Al toxicity, a large amount of active Al³⁺ in acidic soil severely inhibits the growth and yield of buckwheat. Therefore, it is of great significance for the sustainable production of buckwheat to study the response of buckwheat to soil acid Al toxicity and to take corresponding mitigation measures.

The seeds absorb water continuously during the germination process, and Al³⁺ can enter the interior of the seeds with water as the medium, which has a series of influences on the buds’ activity after the seeds germinate. However, the current research on boron to alleviate Al toxicity is mostly focused on the seedling stage, and there is a lack of research on the physiological performance of buds in the germination stage. At the same time, most of the researchers carried out studies on plant tolerance to Al toxicity through hydroponic experiments, there are relatively few reports on plant response to Al³⁺ toxicity in acidic soil. Keeping in view of the above facts, this research explored the effects of exogenous boron on the growth, physiological and biochemical characteristics of buckwheat buds under different degrees of Al stress conditions, while the pot experiment of buckwheat was carried out to explore the mitigation effect of exogenous boron on the growth of buckwheat seedlings in acidic soil polluted by different degrees of Al³⁺ conditions. By studying the mechanism of boron alleviating buckwheat Al toxicity in two growth stages to reveal the possible advantageous role of boron on buckwheat germination, the growth, physiology, and antioxidant system of buckwheat in Al-contaminated soil. The study results will be used to develop technical recommendations to mitigate Al phytotoxicity and to ensure safe production in Al-contaminated acidic soils using boron fertilizer.

2.1 Plant material

Xinong 9940, a Tartary buckwheat variety, provided by Northwest A&F University, Yangling, China. First of all, we selected 2000 buckwheat seeds with the same size, complete shape, and well-developed shape. The seeds were soaked in 1% sodium hypochlorite for 20 min and then rinsed with distilled water five times, and then the sterilized seeds were used for this experiment.

2.2 Experimental design

2.2.1 Experimental treatment

The Al³⁺ solution in the experiment was prepared from AlCl₃ 6H₂O and distilled water, and the B³⁺ solution was prepared from H₃BO₃ and distilled water. There were 16 treatments in this experiment, each treatment was repeated three times, the pH of each treatment group was 4.5, and the boron and Al concentrations used in the experiment were obtained from extensive pretests. To facilitate the understanding of the study results, these treatments are shown in Table 1.

Table.1 Concentration of aluminum ions and boron ions in each experiment treatment

Treatment	Number	Al³⁺ (mmol L^− 1)	B³⁺ (µmol L^− 1)
T1	A0B0	0	0
T2	A0B10	0	10
T3	A0B30	0	30
T4	A0B50	0	50
T5	A1B0	5	0
T6	A1B10	5	10
T7	A1B30	5	30
T8	A1B50	5	50
T9	A2B0	10	0
T10	A2B10	10	10
T11	A2B30	10	30
T12	A2B50	10	50
T13	A3B0	15	0
T14	A3B10	15	10
T15	A3B30	15	30
T16	A3B50	15	50

2.2.2 Seed germination experiment

In the Petri dish germination experiment, 30 sterilized seeds were spread in a Petri dish with two layers of filter paper, and 10 mL of treatment solution was added to each petri dish and incubated in an artificial climate chamber at variable temperature (25℃, 16 h, light/18℃, 8 h, dark). The filter papers in the Petri dishes were changed every two days to ensure that the concentration of the treatment solution in the Petri dishes remained constant. The measurement of each index was performed on the 7th day.

2.2.3 Seedling growth experiment

The pot experiment was designed to further investigate the mitigation effect of boron on the growth of buckwheat seedlings under Al stress, and it was carried out in a greenhouse under natural sunlight conditions at Northwest A&F University. In the pot experiment, buckwheat seedlings were grown in plastic pots (20 × 15 cm) filled with a mixture of nutrient soil and vermiculite (3:1, v/v), each pot was filled with one kilogram of mixed soil, and then 30 sterilized seeds are sown in each pot. Finally, the seeds were covered with soil and watered with the 200 mL treatment solution (Al3 + solution). After that, the 50 mL treatment solution (B3 + solution) was poured every day, and the same volume of distilled water was poured as a control. After 14 days, relevant physiological and biochemical indicators of buckwheat seedlings were measured.

2.3 Determination of indicators

2.3.1 Morphological traits and biomass

In the petri dish germination experiment, after the seeds were germinated, the length of the roots was measured with vernier calipers and the fresh weight of buds was measured with a one-thousandth balance. In the pot experiment, the plant height and root length were measured with a straightedge, and the fresh weight of seedlings was measured with a one-thousandth balance. The buds and seedlings were killed at 105℃ for 30 min, dried at 70℃ to constant weight, and then the dry weight was measured on a one-thousandth balance.

2.3.2 Antioxidant enzyme activity, malondialdehyde, and soluble protein content

Superoxide dismutase (SOD), peroxidase (POD), catalase (CAT) activities, malondialdehyde (MDA), and soluble protein content were determined by the commercially available kits, BC0170, BC0090, BC0200, BC0025, and BC0030 (Beijing Solarbio Technology Co., Ltd., China), and experiments were performed according to the manufacturer's guidelines.

2.3.3 Osmotic adjustment substances

The concentration of Proline was estimated using L-proline as a standard according to Bilal’s method (1973), the soluble sugars were analyzed spectrophotometrically at 490 nm with some modifications in Dubois’s protocol (2002).

2.3.4 Photosynthetic pigment content

The method of Harmut (1987) was followed to measure photosynthetic pigment content. Fresh leaf samples (0.2 g) were cut into small pieces and soaked in anhydrous ethanol (10 mL) and kept in the dark, for 24 h. Afterward, the filtrates were run in a spectrophotometer to measure absorbance at 645 and 663 nm. Finally, the pigment concentrations were calculated by using the following formula:

Chlorophyll a (mg g^− 1 FW) = [12.7(OD₆₆₃) − 2.69(OD₆₄₅)] × V/ (1000 × W) (1)

Chlorophyll b (mg g^− 1 FW) = [22.9(OD₆₄₅) − 4.68(OD₆₆₃)] × V/ (1000 × W) (2)

2.3.5 Root activity

0.5 g of roots were immersed in 0.4% triphenyltetrazolium chloride solution and 5 mL of 0.1 mM phosphate buffer (pH 7.0), incubated at 37℃ for 2 h and then removed and 1 M H₂SO₄ was added to terminate the reaction, then the roots were immersed in 10 mL of methanol and placed in a constant temperature incubator at 37℃, and the absorbance of methanol was recorded at 485 nm with a spectrophotometer when the roots turned white (Islam et al., 2007).

2.3.6 Hematoxylin staining

Hematoxylin staining was used to detect the distribution and concentration of Al in the roots. Hematoxylin staining was performed according to Polle’s method (1978). Roots treated with B and Al were washed in distilled water for 15 min to remove the residual Al from the root tip surface and then stained in a hematoxylin staining solution for 15 min. The stained roots were placed in distilled water for 15 min until there was no more color in the soaking solution. The staining of the root tips was observed and photographed with a camera.

2.4 Data statistics and analysis

Experiments were conducted in replicates (N = 3) and the data were expressed as mean ± standard error. The data were examined with one-way analysis of variance (ANOVA), and the mean values were compared using Duncan’s multiple range test (P < 0.05). All data were statistically analyzed using SPSS 26.0 (SPSS, Chicago, IL, USA) for each treatment, and data were plotted by Origin pro.2021 (Origin Lab Corporation, USA) software.

3.1 Effect of boron on seed germination of buckwheat under Al stress

3.1.1 Root length

It was observed that under the boron deficient conditions, compared with the control, the main roots of buckwheat after germination gradually became shorter with the increase of Al³⁺ concentration, the number of lateral roots exhibited a decreasing trend, and became brittle and easily broken, while the roots also showed a slight browning (Fig. 1). Under the Al-stressed treatments, the number and length of lateral roots increased along with the main root system becoming longer after the boron treatments. In the absence of boron, the root length gradually decreased with the increase of Al³⁺ concentration. When the Al³⁺ concentration was 15 mmoL^− 1, the inhibitory effect of Al³⁺ on root length was most obvious and root length decreased by 82.73% compared with the control (Fig. 1). Regardless of the presence or absence of Al³⁺, the boron treatments promoted root growth after germination of buckwheat. When the Al³⁺ concentration was 0, 5, and 15 mmoL^− 1, the promoted effect on root length was the most obvious under the B50 treatment, compared with the control, the root length increased by 44.53%, 37.78%, 21.28%, respectively (Fig. 1). When the Al³⁺ concentration was 10 mmoL^− 1, the root length reached the maximum value under the B30 treatment, and the root length increased by 93.42% compared with the control.

3.1.2 Fresh weight and dry weight

The results showed that the bud fresh and dry weights exhibited a decreasing trend with the increase of Al³⁺ concentration. Under the three Al-stressed treatments, the fresh weight decreased by 51.27%, 64.34%, and 84.95%, and the dry weight reduced by 57.02%, 68.79%, and 91.84%, respectively, compared with the control. The Boron treatment significantly alleviated the inhibitory effect of Al³⁺ on the fresh and dry weights. Under the three Al-stressed conditions, the B50 treatment promoted the fresh weight most significantly, the fresh weight increased by 64.24%, 73.64%, and 97.65%, respectively, compared with the control (Table 2). When the Al³⁺ concentration was 5, 10 mmoL^− 1, the B50 treatment had a positive effect on the dry weight, which increased by 44.41% and 43.46%, respectively, compared to the treatment without the addition of boron. At an Al³⁺ concentration of 15 mmoL^− 1, the dry weight under the B30 treatment was the maximum value, which increased by 39.71% compared to the control (Table 2).

Table 2

Effect of boron on fresh and dry weights of buckwheat seed buds under Al stress
Indicator	Treatment	Al concentration
Indicator	Treatment	0 mmoL^− 1	5mmoL^− 1	10mmoL^− 1	15mmoL^− 1
Fresh weight of buds (mg)	B0 B10 B30 B50	112.9 ± 7.8 b 118.4 ± 14.2 b 156.7 ± 27.9 a 114.6 ± 19.0 b	55.0 ± 8.0 c 70.8 ± 5.9 b 78.4 ± 6.0 b 90.4 ± 6.9 a	40.3 ± 6.1 c 46.3 ± 3.8 c 58.6 ± 5.2 b 69.9 ± 3.3 a	17.0 ± 3.7 c 23.6 ± 4.6 bc 27.0 ± 7.9 ab 33.6 ± 6.1 a
Dry weight of buds (mg)	B0 B10 B30 B50	16.7 ± 2.2 a 16.4 ± 1.6 a 16.5 ± 1.9 a 18.0 ± 3.7 a	7.2 ± 1.3 b 7.2 ± 1.0 b 9.2 ± 2.3 ab 10.3 ± 1.3 a	5.2 ± 2.4 b 5.4 ± 0.9 ab 6.0 ± 0.6 ab 7.5 ± 1.6 a	1.4 ± 0.3 b 1.6 ± 0.3 ab 1.9 ± 0.2 a 1.8 ± 0.2 a

Each value is demonstrating the mean of three repeats of every treatment. The similar letters inside a column specify that there was no significant difference at a 95% probability level at the p < 0.05 level, correspondingly

3.1.3 Antioxidant enzyme activity

The results exhibited that with the increase of Al³⁺ concentration, the SOD activity showed a decreasing trend, while the POD and CAT activities showed the opposite trend after the seeds germination (Fig. 2A-C). When the Al³⁺ concentration was 15 mmoL^− 1, the SOD activity reached the minimum value, the POD and CAT activities reached the maximum value, and the SOD activity decreased by 47.21%, the POD activity increased by 75.34% and the CAT activity increased by 120.05%, compared with the control. Boron could modulate the antioxidant enzyme activity of buckwheat under the Al stress. Under the three Al-stressed conditions, SOD activity increased continuously with the increase of boron concentration and reached the maximum value under the B50 treatment. In contrast to the SOD activity, CAT activity reached the minimum value under the B50 treatment, the B50 treatment increased SOD activity by 49.73%, 53.07%, and 60.79%, and decreased CAT activity by 27.64%, 33.51%, and 33.87% compared with the control, respectively (Fig. 2A, C). The POD activity reached the maximum value under the B30 treatment when the Al³⁺ concentration was 5 mmoL^− 1 and 10 mmoL^− 1, and POD activity increased by 121.91% and 48.91%, respectively, compared with the control. When the concentration of Al3 + was 15 mmoL-1, the POD activity reached the maximum value under the B50 treatment and increased by 19.33% (Fig. 2B)

3.1.4 MDA and soluble protein content

It was observed that the MDA and soluble protein content were low and stable under the no Al stress. Al toxicity led to the increase of MDA and soluble protein content, compared with the control, the three Al-stressed treatments increased the MDA content by 54.67%, 121.57%, 207.69%, and increased the soluble protein content by 10.3%, 39%, 40.55%, respectively. The MDA and soluble protein content showed a decreasing trend with the increase of boron concentration under Al stress. The MDA content was lower under the B50 treatment and reduced by 46.39%, 44.15%, and 25.26%, respectively, compared with the control under the three Al-stressed treatments (Fig. 3A). When the Al³⁺ concentration was 10 mmoL^− 1 and 15 mmoL^− 1, the soluble protein content reached the lowest value under the B50 treatment and decreased by 31.3% and 26.5%, respectively, compared with the control (Fig. 3B).

3.2 Effect of boron on the growth of buckwheat seedlings under Al stress

3.2.1 Plant height and root length

Different concentrations of B³⁺ and Al³⁺ had a significant regulatory effect on the height and root length of buckwheat seedlings (Fig. 4). The plant height and root length were negatively correlated with the increase of Al³⁺ concentration, compared with the control, the three Al-stressed treatments reduced the plant height by 15.29%, 21.93%, 31.59%, and decreased the root length by 29.27%, 44.93%, and 55.09%, respectively (Fig. 4A, B). The phenotypic improvements of Boron-treated buckwheat plants were observed in the picture (Fig. 4). Under the Al-stressed treatments, the boron treatment significantly increased the plant height and root length. In terms of the plant height, the B30 treatment performed the best under the three Al-stressed treatments, the plant height increased by 17.1%, 20.1%, and 27.94%, respectively (Fig. 4A). In terms of the root length, when the Al³⁺ concentration was 0 mmoL^− 1 and 10 mmoL^− 1, the root length reached the maximum value under the B50 treatment, and increased by 32.12% and 41.5%, respectively, compared with the control. When the Al³⁺ concentration was 5 mmoL^− 1 and 15 mmoL-1, the root length reached the maximum value under the B30 treatment, and increased by 23.78% and 54.16%, respectively, compared with the control (Fig. 4B).

Figure 4 Effect of boron on the growth of buckwheat seedlings under Al toxicity

The left side figure shows the growth of buckwheat seedlings treated with different B and Al, and the right side shows the effects of boron on plant height and root length in buckwheat seedlings under Al stress. Note: ANOVA followed by Tukey tests determined significant differences (P < 0.05) between different B and Al treatments. Bars are means of three replicates ± SD. Different letters in each sub-figure represent significant differences at (P < 0.05).

3.2.2 Fresh and dry weights of seedlings

As shown in Table 3, the fresh and dry weights of buckwheat seedlings were significantly inhibited with the increase of Al³⁺ concentration. Compared with those in the control seedlings, the three Al-stressed treatments reduced the fresh weight by 16.14%, 35.49%, 40.01%, and decreased the dry weight by 20.99%, 28.48%, 52.35%, respectively (Table 3). The addition of boron significantly alleviated the inhibitory effect of Al toxicity on the fresh and dry weights of buckwheat plants. When the Al³⁺ concentration was 5 mmoL^− 1 and 15 mmoL^− 1, the fresh weight increased with the increase of boron concentration, and reached the maximum value under the B50 treatment, compared with the control, the B50 treatment increased the fresh weight by 23.6%, and 40.35%, respectively (Table 3). As for the dry weight of seedlings, when the Al³⁺ concentration was 5 mmoL^− 1, the dry weight reached the maximum value under the B50 treatment and increased by 32.3% compared with the control. The dry weight reached the maximum value under the B30 treatment when the Al³⁺ concentration was 10 mmoL^− 1 and 15 mmoL-1, and the B30 treatment increased the dry weight by 17.66% and 44.06%, respectively, compared with the control (Table 3).

Table 3

Effect of B on fresh weight, and dry weight in buckwheat seedlings under Al toxicity
Indicator	Treatment	Al concentration
Indicator	Treatment	0 mmoL^− 1	5mmoL^− 1	10mmoL^− 1	15mmoL^− 1
Fresh weight of seedlings (mg)	B0 B10 B30 B50	447.3 ± 15.9 b 464.0 ± 20.8 b 466.2 ± 24.7 b 509.8 ± 26.9 a	375.1 ± 24.5 b 401.9 ± 16.8 b 437.4 ± 28.3 a 463.6 ± 23.3 a	288.5 ± 13.2 b 293.9 ± 46.3 b 371.2 ± 24.8 a 338.7 ± 27.7 a	268.3 ± 39.1 c 288.9 ± 28.6 c 332.9 ± 13.6 b 376.6 ± 40.0 a
Dry weight of seedlings (mg)	B0 B10 B30 B50	23.0 ± 1.7 b 26.3 ± 0.9 a 27.1 ± 2.2 a 27.5 ± 2.3 a	18.1 ± 1.1 b 19.6 ± 0.9 b 23.6 ± 2.7 a 24.0 ± 2.5 a	16.4 ± 1.7 b 15.8 ± 1.4 b 19.3 ± 1.3 a 19.0 ± 1.3 a	10.9 ± 1.3 c 13.7 ± 1.3 b 15.8 ± 0.8 a 14.9 ± 1.3 ab

3.2.3 Antioxidant enzyme activity of seedlings

As can be seen in Fig. 5, with the increase of Al³⁺ concentration, the SOD activity showed a decreasing trend, and the POD and CAT activities increased continuously. When the Al³⁺ concentration was 15 mmoL^− 1, the SOD activity reached the minimum value, and the POD and CAT activities reached the maximum value, compared with the control, the SOD activity decreased by 31.37%, the POD activity increased by 71.37%, and the CAT activity improved by 101% (Fig. 5A-C). On the other hand, the boron treatment increased the SOD activity under the Al-stressed treatment compared to their corresponding exclusively Al-stressed counterparts (Fig. 3A, G). The SOD activity reached the maximum value under the B50 treatment and enhanced by 83.37%, 122.54%, and 67.6% under the three Al-stressed treatments, respectively, compared to the control (Fig. 5A). While the boron treatment reduced the POD and CAT activities under Al stress, and the POD and CAT activities reached the minimum value under the B50 treatment. Under the three Al-stressed treatments, compared with the control, the B50 treatment decreased the POD activity by 22.96%, 38.59%, and 30.33%, and decreased the CAT activity by 29.49%, 22.04%, and 35.21%, respectively (Fig. 5B, C).

3.2.4 MDA content of seedlings

As compared to those in the control seedlings, substantial increments of the MDA content were

observed in Al-stressed treatments in a concentration-dependent fashion. The MDA content reached its maximum value when the Al³⁺ concentration was 15 mmoL^− 1, and increased by 71.35%, 146.76%, and 299.48% under the three Al-stressed treatments, respectively, compared with the no Al stress condition (Fig. 6). In contrast to the effect of Al³⁺, the MDA content under Al stress showed a decreasing trend with the increase of boron concentration. Under the three Al-stressed treatments, MDA content was minimized under the B50 treatment and reduced by 43.18%, 42.03%, and 52.57%, respectively, compared to their corresponding exclusively Al-stressed counterparts (Fig. 6).

3.2.5 Osmotic adjustment substances

The result showed that the soluble sugar content increased with the increase of Al³⁺ concentration. When the Al³⁺ concentration was 15 mmoL^− 1, the soluble sugar content reached its maximum value, which increased by 89.67% compared with the control (Fig. 7A). Under the three Al-stressed treatments, the boron treatment increased the soluble sugar content, the soluble sugar content reached the maximum value under the B50 treatment, which increased by 50.88%,49.2%, and 10.19%, respectively, compared with the control (Fig. 7A). The soluble protein content was significantly higher than the control and increased by 16.73%, 27.32%, and 40.58%, respectively, under the three Al stress conditions (Fig. 7B). In contrast to the Al treatment, the boron treatment decreased the soluble protein content, When the Al³⁺ concentration was 5 mmoL^− 1 and 15 mmoL^− 1, the soluble protein content reached the minimum value under the B50 treatment and decreased by 18.38% and 30.66%, respectively, compared with the control (Fig. 7B). The proline content also increased significantly with the increase of Al³⁺ concentration, which increased by 23.4%, 64.43%, and 92.8%, respectively, compared with the control (Fig. 7C). The boron treatment also increased the proline content under the three Al-stressed treatments, when the concentration of Al³⁺ was 5 mmoL^− 1 and 15 mmoL^− 1, the proline content reached the maximum value under the B50 treatment, compared with the control, increased by 52.12% and 39%, respectively (Fig. 7C).

3.2.6 Photosynthetic pigments

The total chlorophyll and chlorophyll a content reached the lowest value when the Al³⁺ concentration was 15 mmoL^− 1, and the chlorophyll b content reached the minimum value at an Al³⁺ concentration of 5 mmoL^− 1, which decreased by 34.18%, 44.39%, and 35.91%, respectively, compared with the control (Fig. 8A-C). In contrast to the effect of Al³⁺, the boron treatment promoted photosynthetic pigment concentration, and different boron concentrations had various promoted effects. Both total chlorophyll and chlorophyll a content reached the maximum value under the B50 treatment when Al³⁺ concentration was 10 mmoL^− 1 and 15 mmoL^− 1, and the B50 treatment increased the total chlorophyll by 18.56%, 43.42% and increased the chlorophyll a by 21%, 56.79%, respectively, compared with the control (Fig. 8A, C). When the Al³⁺ concentration was 5 mmoL^− 1 and 10 mmoL^− 1, the B50 treatment had the most obvious promoted effect on chlorophyll b, which increased by 15.28% and 29.78%, respectively, compared with the control (Fig. 8B).

3.2.7 Root activity and hematoxylin staining

Under the three Al-stressed treatments, the root activity of buckwheat seedlings showed a decreasing trend with the increase of Al³⁺ concentration, which decreased by 47.17%, 63.44%, and 78.71%, respectively. However, the boron treatment significantly improved the root activity (Fig. 9). Under the three Al³⁺ concentration conditions, the root activity reached the maximum value under the B50 treatment, compared to their corresponding exclusively Al-stressed counterparts, the root activity increased by 89.62%, 124.38%, and 142.29%, respectively (Fig. 9). This demonstrated that boron can enhance the tolerance of buckwheat to Al toxicity by improving the root activity. The hematoxylin staining method is considered to be the most direct and convenient method to identify the Al³⁺ content of plants. In the absence of Al³⁺, the root tips showed a clear bright white color. Under Al stress, the root tips of buckwheat seedlings appeared purple, and the purple color became more obvious with the increase of Al³⁺ concentration (Fig. 9). These phenomena indicated that the Al³⁺ accumulation in seedling roots was also gradually increasing. After the boron treatment, the root tips showed light purple color, and with the increase of boron concentration, the root tip color gradually became lighter, which indicated that the boron treatment reduced the Al³⁺ accumulation in the seedling roots.

4.1 Effects of boron on growth characteristics under Al stress

As soil pH decreases, aluminum is converted to toxic Al³⁺, which is easily absorbed by plants and interferes with normal plant growth mechanisms (Kochian, 1995; Ryan et al., 2001). Root elongation inhibition is the main symptom of Al poisoning (Ma and Furukawa, 2003). Similar results were obtained in this experiment, Al toxicity severely affected the root length of buckwheat (Fig. 1, 4B), plant height (Fig. 4A), and the fresh and dry weights (Table 2, 3). It has been shown that boron could alleviate the inhibitory effect of Al toxicity on plant growth, which has been reported in crops such as mung bean (Yang and Zhang, 1998), soybean (Yang et al., 2004), oilseed rape (Yan et al., 2018c), and pea (Yu et al., 2009; Li et al., 2017; Yang et al., 2022). This study found that boron can not only promote the germination growth of buckwheat seeds under Al stress but also relieve the inhibition of Al toxicity on the growth of seedlings and promote the growth of seedlings. It may be that the supply of exogenous boron reduced the Al³⁺ content in the root system, resulting in less damage to the root system, which was able to ensure that the root system of the plant could carry out normal nutrient and water uptake and maintain the normal plant growth and development.

4.2 Effects of Boron on antioxidant enzyme system under Al stress

In plants, ROS (reactive oxygen species) plays a pivotal role in programmed cell death under abiotic stress, while Al toxicity could lead to physiological metabolic processes damages such as impaired membrane lipids by over-production of ROS (Guo et al., 2004). Plasma membrane carboxylic and phosphate groups are easily attached to by Al ions, which can cause lipid peroxidation and the creation of MDA. (Cakmak and Horst, 1991). In a study of date palm seedlings under Al stress, the boron treatment reduced MDA and superoxide production levels (Bilal et al., 2022). Our results show that whether the germinated buckwheat or the buckwheat in the seedling stage, Al stress significantly increased MDA content in roots as well as leaves (Fig. 3, 6), but under the three Al-stressed treatments, boron can significantly reduce the MDA content (Fig. 3, 6), this may be due to the relationship between boron regulation of the antioxidant enzyme system in plants to scavenge ROS in plants and the reduction of damage to the membrane system.

Plants have evolved enzymatic and non-enzymatic protection mechanisms that efficaciously scavenge ROS. The SOD, POD, and CAT are the key antioxidant enzymes in the enzymatic detoxification system. In this study, we measured the activities of various antioxidant enzymes and found that Al toxicity significantly reduced SOD activity while marked enhanced the POD and CAT activities, which is similar to the results of previous studies (Guo et al., 2006; Huang et al., 2009). The SOD is the first line of defense program for various enzymatic systems to scavenge ROS (Sanchez-Casas and Klessig, 1994). Here, the appreciable improvement in SOD activity by boron application under Al exposure (Fig. 2A, 5A). It can be confirmed that the supply of boron is related to the reduction of the accumulation of free radicals in cells and the damage to the membrane system. POD and CAT as the main enzymes for scavenging ROS (Sanchez-Casas and Klessig, 1994), but POD and CAT activities significantly decreased with the addition of exogenous boron, which may be the addition of boron reduced the aluminum-induced H₂O₂ production, there was no need for POD and CAT to break down H₂O₂ to H₂O and O₂, thus POD and CAT can maintain lower activity. These suggested that boron can reduce MDA content, POD, and CAT activity by reducing lipid peroxidation of the cell membrane and maintaining the stability of cell membrane structure, and enhancing the ability to remove O₂⁻ (Yan et al., 2017).

4.3 Effects of boron on osmotic adjustment substances under Al stress

Numerous studies showed that plants could accumulate low molecular compounds to tolerate biotic or abiotic stress. In addition, proline accumulation may enhance enzyme activity and help stabilize protein integrity, while proline is considered a non-enzymatic antioxidant against stress by signaling and regulating metabolic activity ( Ashraf and Foolad, 2007; Lipiec et al., 2013). In our study, the proline content exhibited an increasing trend with the increase of Al³⁺ concentration (Fig. 7C). Under Al stress, the increase of proline showed a correlation between ROS production and ROS scavenging. The addition of external boron also improved the proline content under Al stress. Therefore, under the Al-stressed condition, boron could remove free radicals produced during oxidative stress and increase the plant tolerance to Al toxicity. In terms of soluble protein and soluble sugar content, Al stress significantly increased their content, which may be due to Al toxicity severely disrupting the osmotic balance of buckwheat, resulting in the buckwheat plant initiating an emergency osmoregulatory mechanism to break down a large number of macromolecular carbohydrates and proteins to increase the soluble sugar and soluble protein content, in response to the aluminum-induced osmotic imbalance. Similarly, soluble protein in barley seedlings tended to increase under Al stress (Zhang et al., 2013). Therefore, induction of protein synthesis has emerged as a possible mechanism against Al toxicity. However, under Al stress, the addition of different concentrations of boron resulted in the decrease of soluble protein content in both roots and leaves of buckwheat seedlings (Fig. 7B, 3B), which showed that boron could relieve Al toxicity to drop the soluble protein content by improving the decomposition of enzyme activity (Zhang et al., 2013). Previous studies have also demonstrated that boron treatment improved the osmotic regulation ability of the trifoliate orange by decreasing the soluble protein content (Yan et al., 2018b). However, the change of soluble sugar was opposite to that of soluble protein, the soluble sugar content showed an increasing trend after the addition of exogenous boron (Fig. 7A). These changes may be due to boron changing the content of osmotic adjustment substances by regulating the biosynthesis and metabolism of amino acids and carbohydrates in plants (Yan et al., 2019). Plant leaves, under the boron treatment, regulate the cellular osmotic potential and maintain the intercellular water balance through the accumulation of soluble sugar, soluble protein, and proline. In addition to the decrease in soluble protein content, the content of soluble sugar and proline tended to increase in our experiment, and it can be concluded that exogenous boron can alleviate the aluminum-induced damage to the leaf membrane system by changing the osmotic adjustment substances.

4.4 Effects of boron on chlorophyll content and root activity under Al stress

Chlorophyll is an important pigment involved in photosynthesis in plants, when plants are exposed to adverse environments for a long time, the chlorophyll content and photosynthetic rate will decrease, the determination of chlorophyll content is also commonly used in the study as an indicator to detect the growth status of plants under various stress conditions (Zhang and Liu, 2019). This experiment found that the buckwheat chlorophyll a and chlorophyll b contents decreased with the increase of Al^{3 +} concentration (Fig. 8A, B), which is consistent with previous studies (Jiang et al., 2009). It may be because the increase of aluminum-induced MDA content accelerated the rupture of chloroplast membrane and chloroplast decomposition, while Al toxicity caused the inhibition of chlorophyll synthesis, which eventually led to a significant decrease in total chlorophyll content. However, both chlorophyll a and chlorophyll b contents in the leaves showed an increasing trend after the addition of boron under the three Al-stressed conditions. The reason may be that the addition of exogenous boron inhibited the absorption of Al³⁺ by roots, reduced the content of active Al³⁺ transported from plant roots to leaves, and alleviated the damage of Al³⁺ to chloroplast membranes (Yan et al., 2018a).

Root activity refers to the ability of roots to absorb water and nutrients from the soil, and it plays an important role in the assimilation, synthesis, and transformation of several substances (Bai et al., 1994). Under Al stress, the root activity of buckwheat seedlings showed a significant positive correlation with the boron concentration, which may be related to the reduction of aluminum-induced root damage by boron application (Fig. 9). The results of hematoxylin staining showed that the roots became darker with the increase of Al³⁺ concentration, indicating that the addition of boron induced the root to reduce the absorption of Al, and further demonstrating that the addition of boron could protect the roots from Al toxicity by reducing the Al content in the roots (Yu et al., 2009; Li et al., 2017). There are three possible reasons for the decrease in Al content after boron treatment. First, boron might be shielding the sites of Al binding with boron application thus reducing the Al augmentation in the root system. Second, the cross-linking of boron with Rhamnogalacturonan II (RG-II) forms a stable cell wall structure, reducing the cell wall pore size and thus reducing the pathway for Al to enter sensitive tissues. Finally, sufficient B supply promotes the immobilization of Al, resulting in less Al in the symplast (Yang and Zhang, 1998; Tang et al., 2011; Li and Yu, 2013).

The results showed that Al toxicity in acidic soils could cause damage to the buckwheat root system, inhibit root elongation, reduce root activity, reduce the leaf photosynthetic pigment content, and eventually lead to a reduction in buckwheat biomass. Boron treatment with different concentrations could reduce the Al³⁺ concentration in the root, regulate the antioxidant enzymes activity and the osmotic adjustment substances content, reduce the membrane lipid peroxidation and MDA content, reduce the damage to the root system, improve the root activity, and promote the growth and development of the root system and the synthesis of photosynthetic pigments in the leaves, thereby enhance the tolerance of buckwheat to Al toxicity, and the biomass of the plant can be significantly increased. The results obtained from this study will provide important references for the alleviation of Al phytotoxicity in acidic soils using boron fertilizer, especially under the intensive agricultural systems.

Authors’ contribution

Jinfeng Gao conceived and designed experiments; Jiahao Ge performed experiments, analyzed data, and wrote manuscript; Qi Wang provided insightful comments on an early draft of this article; Xiaoying Xu, Xinhui Lei, Jincai Tao, Feng Gao, Xiaoli Gao, and Pu Yang assisted the whole experiment process.

Funding This study was financially supported by the National Natural Science Foundation of China (31671631), the Science and Technology Key Research & Development Project of Shaanxi Province (2022NY-178), and the Technical System of Minor Grain Crops Industry in Shaanxi Province (NYKJ-2021-YL(XN)40).

Data availability The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.

Ethical approval There are no ethical issues in this article.

Consent to participate All the authors agree to participate in this paper.

Consent for publication All the authors agree to the publication of this paper.

Competing interest The authors declare no competing interests.

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Boron alleviates the aluminum toxicity in buckwheat by regulating antioxidant defense system and maintaining osmotic balance

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Abstract

Figures

1. Introduction

2. Materials And Methods

2.1 Plant material

2.2 Experimental design

2.2.1 Experimental treatment

2.2.2 Seed germination experiment

2.2.3 Seedling growth experiment

2.3 Determination of indicators

2.3.1 Morphological traits and biomass

2.3.2 Antioxidant enzyme activity, malondialdehyde, and soluble protein content

2.3.3 Osmotic adjustment substances

2.3.4 Photosynthetic pigment content

2.3.5 Root activity

2.3.6 Hematoxylin staining

2.4 Data statistics and analysis

3 Results

3.1 Effect of boron on seed germination of buckwheat under Al stress

3.1.1 Root length

3.1.2 Fresh weight and dry weight

3.1.3 Antioxidant enzyme activity

3.1.4 MDA and soluble protein content

3.2 Effect of boron on the growth of buckwheat seedlings under Al stress

3.2.1 Plant height and root length

3.2.2 Fresh and dry weights of seedlings

3.2.3 Antioxidant enzyme activity of seedlings

3.2.4 MDA content of seedlings

3.2.5 Osmotic adjustment substances

3.2.6 Photosynthetic pigments

3.2.7 Root activity and hematoxylin staining

4. Discussion

4.1 Effects of boron on growth characteristics under Al stress

4.2 Effects of Boron on antioxidant enzyme system under Al stress

4.3 Effects of boron on osmotic adjustment substances under Al stress

4.4 Effects of boron on chlorophyll content and root activity under Al stress

5. Conclusion

Declarations

References

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