Effects of acid and aluminum stress on seed germination and physiological characteristics of seedling growth in Sophora davidii

ABSTRACT

Sophora davidii, a vital forage species, predominantly thrives in the subtropical karst mountains of Southwest China. Its resilience to poor soil conditions and arid environments renders it an ideal pioneer species for ecological restoration in these regions. This study investigates the influence of acidic, aluminum-rich local soil on the germination and seedling growth physiology of S. davidii. Experiments were conducted under varying degrees of acidity and aluminum stress, employing three pH levels (3.5 to 5.5) and four aluminum concentrations (0.5 to 2.0 mmol·L⁻¹). The results showed that germination rate, germination index, and vigor index of S. davidii seeds were decreased but not significantly under slightly acidic conditions (pH 4.5–5.5), while strong acid (pH = 3.5) significantly inhibited the germination rate, germination index, and vigor index of white spurge seeds compared with the control group. Aluminum stress (≥0.5 mmol·L⁻¹) significantly inhibited the germination rate, germination index, and vigor index of S. davidii seed. Moreover, the seedlings’ root systems were sensitive to the changes of aluminum concentration, evident from significant root growth inhibition, characterized by root shortening and color deepening. Notably, under aluminum stress (pH = 4.3), the levels of malondialdehyde and proline in S. davidii escalated with increasing aluminum concentration, while antioxidant enzyme activities demonstrated an initial increase followed by a decline. The study underscores the pivotal role of cellular osmoregulatory substances and protective enzymes in combating aluminum toxicity in S. davidii, a key factor exacerbating growth inhibition in acidic environments. These findings offer preliminary theoretical insights for the practical agricultural utilization of S. davidii in challenging soil conditions.

KEYWORDS:

• Acidic soil
• aluminum toxicity
• antioxidant enzymes
• germination rate
• Sophora davidii

Introduction

Acidic soils, defined as those with a pH of 5.5 or lower,¹ encompass approximately 5.5% of China’s landmass, predominantly in its southern and southwestern regions.² Acid rain, a significant contributor to soil acidification, exacerbates the decline in soil pH upon infiltration into the ground.³ High acidity in soils can lead to direct plant damage through hydrogen ion toxicity, disrupting the stability of plant root protoplasmic membranes. This disruption manifests in reduced root systems, altered root morphology, and, in extreme cases, root tip death, ultimately culminating in plant mortality.^4,5

The primary challenges for plant growth in acidic soils include an abundance of hydrogen, aluminum (Al), manganese ions, and a deficiency of phosphorus, with Al being the most critical factor limiting plant growth in such environments.⁶ Al, the most prevalent metal in the Earth’s crust, becomes soluble and bioavailable in the form of Al³⁺ in soils with a pH below 5.5.^7,8 Even at micromolar concentrations, Al is readily absorbed by plant roots, resulting in reduced crop yields. Under acidic conditions, Al stress not only impairs root growth and hinders water and nutrient absorption but also disrupts the osmotic balance across plant cell membranes and enhances reactive oxygen species accumulation, thereby diminishing plant biomass.⁹ To combat these effects, plants develop adaptive mechanisms, such as secreting antioxidant enzymes and osmoregulatory substances, to mitigate reactive oxygen species damage and prevent Al ions from entering the cytoplasm, thereby enabling survival in Al-rich environments.^10,11

Sophora davidii, a perennial xerophytic shrub or small tree from the legume family, exhibits notable ecological traits including drought tolerance, poor soil adaptability, and soil erosion prevention.^12,13 Besides its ecological benefits, it serves as a forage, medicinal, and honey plant due to the rich nutrient composition of its flowers, leaves, and seeds, which include amino acids, vitamins, alkaloids, and minerals.¹⁴ Predominantly found in the karst terrains of Guizhou, Guangxi, Sichuan, and Hunan provinces in China, these regions are characterized by predominantly acidic soils.¹⁵ While existing research on S. davidii has focused on seed germination, plant chemical composition, and seedling physiological and biochemical responses under adverse conditions,^16–18 studies addressing the plant’s response to acid and Al stress in acidic soils are lacking. This study aims to analyze and discuss the germination, seedling morphology, and physiological characteristics of S. davidii under varying acid and Al stresses. The goal is to elucidate the morphological and physiological mechanisms enabling S.davidii to adapt to these stresses, thereby providing a theoretical foundation for its cultivation in acidic soils and contributing to the development of acid and Al-tolerant forage varieties.

Material and methods

Test materials and experimental design

To simulate acid stress, hydrochloric acid (HCl) was utilized, setting three distinct pH values: 3.5, 4.5, and 5.5. Distilled water served as the control, and treatments were labeled as pH-CK, pH-3.5, pH-4.5, and pH-5.5. Aluminum stress was simulated using aluminum chloride hexahydrate (AlCl₃·6 H₂O) at pH 4.3, with concentrations of 0 mmol·L⁻¹ (distilled water as control), 0.5 mmol·L⁻¹, 1.0 mmol·L⁻¹, 1.5 mmol·L⁻¹, and 2.0 mmol·L⁻¹, respectively, denoted as Al-CK, Al-0.5, Al-1.0, Al-1.5, and Al-2.0.

Experimental conditions and procedure

The experiment was conducted in the laboratory of the Department of Grassland Science, College of Animal Sciences, Guizhou University from January to March 2022. Seeds of S. davidii were selected based on uniform size and intactness, sterilized with 3% NaClO for 3–5 minutes, and rinsed 3–5 times with distilled water. To facilitate germination, seeds were treated with concentrated sulfuric acid for 15–20 minutes, followed by additional rinsing and air-drying. For each treatment, 50 seeds were placed in a 9 cm diameter petri dish on double-layer filter paper, moistened with 8 mL of the respective treatment solution. Each treatment was replicated thrice. The dishes were incubated at a constant temperature of 25°C with a 16/8-hour light/dark cycle, refreshing the filter paper and solution every 2 days. Seed germination was recorded daily, and on the seventh day, five seedlings from each treatment were selected for fresh weight measurement. Germination rate, potential, index, and vigor index were calculated. On the 21st day, five seedlings from each treatment were assessed for morphological indices, and their aboveground and root parts were analyzed for physiological indices.

Measurement indicators and methods

The seed germination index was determined as follows:

Germination rate (GR, %) = (Number of germinated seeds on the final day/Total number of tested seeds) × 100%

Germination potential (GP, %) = (Number of normally germinated seeds at day 4/Total number of tested seeds) × 100%

Germination index (GI) = Σ (Gt/Dt)

Vitality index (VI) = GI × Average fresh weight of seedlings on day 7

Where Gt is the number of seeds germinated on day t, and Dt is the corresponding day of germination.

Determination of seedling morphological indices

After 21 days of treatment, five seedlings were randomly selected from each group for measurement. Aboveground and root lengths were measured with a ruler. Fresh weights of the aboveground part and root were determined using a precision electronic balance, followed by oven drying at 105°C for 10 minutes and at 65°C until constant weight. Dry weights of the aboveground and root parts were then measured, and the root–shoot ratio (dry weight ratio of underground to aboveground parts) was calculated.

Determination of physiological indices of seedlings

On day 21, seedlings were dissected into shoot and root sections. A 0.1 g sample of the shoot was immediately weighed, placed in a 2 ml centrifuge tube, flash-frozen in liquid nitrogen, and stored at −80°C for physiological indicator analysis. Malondialdehyde (MDA), proline (Pro), soluble protein, and soluble sugar contents, as well as the activities of peroxidase (POD), superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) were determined using kits from Beijing Solarbio Science & Technology Co., Ltd., Beijing, China. Detailed assay procedures are provided in the Supplementary Material.

Data analysis

Data were recorded using EXCEL 2016, with figures generated in Origin2022. Displayed data represent mean ± standard error (n = 3). The SSPS23 software was employed for significant difference analysis, using one-way ANOVA and Duncan’s multiple range test for mean comparison at a significance level of p < .05.

Results

Effects of acid and Al stress on germination characteristics of S. davidii seeds

Increasing the Al concentration significantly reduced the germination rate, potential, index, and vigor of S. davidii seeds (p < .05) (Table 1). A notable difference in germination rate was observed between pH-3.5 and pH-CK (p < .05), indicating a decline in germination potential, index, and vigor with decreasing pH, reaching the lowest values at pH-3.5. These findings suggest that strong acid stress (pH = 3.5) and Al stress impede the germination of S. davidii seeds.

Table 1. Effects of acid and Al stress on seed germination of Sophora davidii.

Treatment	Germination rate	Germination power	Germination index	Vigor index
Al-CK	44.6 ± 5.0a	34.7 ± 5.9a	17.7 ± 2.0a	1.4 ± 0.2a
Al-0.5	36.0 ± 4.0b	21.3 ± 3.7b	11.5 ± 0.9b	0.7 ± 0.1b
Al-1.0	32.0 ± 4.0bc	20.7 ± 3.7b	12.2 ± 1.9b	0.7 ± 0.1b
Al-1.5	27.3 ± 1.2cd	18.7 ± 1.7b	10.3 ± 0.5b	0.6 ± 0.1b
Al-2.0	22.7 ± 2.3d	11.3 ± 0.7b	7.8 ± 0.4b	0.4 ± 0.1b
pH-CK	44.6 ± 5.0a	34.7 ± 5.9a	17.7 ± 2.0a	1.4 ± 0.2a
pH-5.5	42.7 ± 2.9a	35.3 ± 0.7a	18.9 ± 0.4a	1.4 ± 0.1a
pH-4.5	38.7 ± 2.4ab	31.3 ± 1.7a	15.9 ± 1.1ab	1.2 ± 0.1a
pH-3.5	32.7 ± 2.4b	23.3 ± 3.5a	12.8 ± 1.3b	0.8 ± 0.1b

The values in the table are expressed by mean ± standard error (n = 3); Different lowercase letters in the same column and the same treatment group indicate significant differences (p < 0.05).

Effects of acid and Al stress on growth characteristics of S. davidii seedlings

S. davidii seedlings exhibited varying responses to acid and Al stress during growth. An increase in Al concentration led to a reduction in seedling length. Significant decreases in the aboveground length were noted for Al-1.5 and Al-2.0 compared to Al-CK (p < .05), while other groups showed no significant differences (p > .05). Radicle length decreased substantially with increasing Al concentration (p < .05), reaching a minimum of 0.8 cm in Al-2.0. Under decreasing pH conditions, seedling length decreased, with the shortest length (2.2 cm) observed under pH-4.5. Radicle length also significantly diminished, with the smallest being 1.8 cm under pH-3.5 treatment.

As Figure 1 illustrates, increasing Al concentration darkened the root system color of the seedlings, eventually turning brown. Under acid stress, greater acidity resulted in darker root colors, turning brown at pH-3.5.

Figure 1. Seedlings of S. davidii under acid and aluminum stress for 21 days.

As per Table 2, Al concentration increases did not significantly affect shoot fresh and dry weights, but did significantly impact root fresh and dry weights (p < .05). The Al-2.0 group exhibited the greatest decrease in root fresh weight and dry weight, dropping by 61.5% and 50%, respectively. Different pH levels did not significantly affect shoot weights but did affect root fresh weight (p < .05). The smallest root fresh weight was observed in the pH-3.5 group, with a decrease of 36.3%. Root's dry weight showed no significant difference. The root–shoot ratio of S. davidii seedlings varied significantly under Al stress (p < .05), but not under acid treatment.

Table 2. Effects of acid and Al stress on morphological indexes of Sophora davidii seedlings.

Treatment	Seeding length/cm	Radicle length/cm	Fresh weight/mg		Dry weight/mg		Root-shoot ratio
			Shoot	Root	Shoot	Root
Al-CK	3.1 ± 0.06a	3.0 ± 0.23a	435.5 ± 7.7a	51.8 ± 3.1a	35.9 ± 1.6a	5.0±0.3a	0.14±0.01a
Al-0.5	3.0 ± 0.19ab	2.2 ± 0.13b	407.7 ± 15.7a	36.0 ± 0.5bc	34.5 ± 1.1a	4.1 ± 0.3ab	0.12 ± 0.01ab
Al-1.0	3.1 ± 0.23a	2.1 ± 0.20b	403.7 ± 18.2a	38.1 ± 3.8b	33.8 ± 1.5a	3.5 ± 0.4bc	0.10 ± 0.10b
Al-1.5	2.4 ± 0.03c	1.2 ± 0.07c	443.1 ± 22.2a	28.9 ± 1.4c	42.5 ± 2.4a	2.6 ± 0.3c	0.07 ± 0.01c
Al-2.0	2.6 ± 0.10bc	0.8 ± 0.09c	399.5 ± 11.9a	19.9 ± 0.9d	33.0 ± 0.7a	2.5 ± 0.2c	0.07 ± 0.00c
pH-CK	3.1 ± 0.06a	3.0 ± 0.23a	435.7 ± 7.7a	51.8 ± 3.1a	35.9 ± 0.3a	5.0 ± 0.3a	0.14 ± 0.01a
pH-5.5	2.8 ± 1.7a	2.3 ± 0.05b	447.8 ± 26.3a	42.5 ± 0.9b	34.8 ± 0.8a	3.9 ± 0.8a	0.11 ± 0.00a
pH-4.5	2.2 ± 0.06b	2.3 ± 0.11b	414.7 ± 20.8a	40.6 ± 1.0b	35.2 ± 2.5a	4.9 ± 2.5a	0.14 ± 0.01a
pH-3.5	2.3 ± 0.23b	1.8 ± 0.06c	388.6 ± 23.0a	33.0 ± 1.4c	35.1 ± 2.2a	4.5 ± 2.2a	0.13 ± 0.04a

The values in the table are expressed by mean ± standard error (n = 3); Different lowercase letters in the same column and the same treatment group indicate significant differences (p < .05).

Effects of acid and Al stress on MDA and osmoregulation substances in S. davidii seedlings

According to Figure 2, the MDA content in S. davidii seedlings increased with Al concentration. Al-1.0, Al-1.5, and Al-2.0 exhibited significant differences compared to Al-CK (p < .05), with the highest increase (49.9%) in Al-2.0. Under acid stress, the MDA content significantly decreased, with no notable differences among pH-5.5, pH-4.5, and pH-3.5.

Figure 2. Effect of acid and aluminum stress on the content of MDA and osmoregulatory substances in S. davidii seedlings. Different lowercase letters in the same treatment group indicate significant differences (p < .05).

The proline content in the seedlings increased with Al concentration, while soluble sugar and protein contents initially increased then decreased (Figure 2(b–d)). The greatest increase in the Pro content was observed in Al-2.0, rising by 38.2%. The highest and lowest values of soluble sugar and protein appeared in Al-1.0 and Al-2.0, respectively, compared to Al-CK. Under decreasing pH, osmoregulatory substances in S. davidii seedlings varied, with the Pro content showing a decrease then an increase (Figure 2(b)), with both soluble sugar and protein content significantly rising (p < .05). The largest differences were found in the pH-3.5 group, increasing by 205.9% and 36.5%, respectively (Figure 2(c,d)).

Effects of acid and Al stress on antioxidant enzyme activity in S. davidii seedlings

The activities of SOD, POD, CAT, and APX in S. davidii seedlings showed an initial increase followed by a decrease with rising Al concentration (Figure 3(a–d)). The peak increase in SOD activity occurred at Al-1.5 (Figure 3(a)), significantly higher than Al-CK (p < 0.05), with an increase of 95.3%. The peak activities of POD, CAT, and APX were at Al-0.5 (Figure 3(b–d)), all showing significant increases compared to Al-CK, with increases of 89.8%, 76.2%, and 45.7%, respectively. Under decreasing pH, SOD, and APX activities in S. davidii seedlings increased then decreased, peaking at pH-5.5 (Figure 3(a,d)), while POD and CAT activities showed a significant decreasing trend (p < .05), as depicted in Figure 3(b,c).

Figure 3. Effect of acid and aluminum stress on antioxidant enzyme activities in S. davidii seedlings. Different lowercase letters in the same treatment group indicate significant differences (p < .05).

Discussion

Seed germination and seedling growth of S. davidii under acid and Al stress

Seed germination, a critical phase in a plant’s lifecycle,¹⁹ is particularly susceptible to stress, providing a precise indicator of plant growth under adverse conditions. Key metrics for assessing seed germination and vigor include germination rate, potential, index, and vigor index.^20,21 This study found that both acid and Al stress significantly impeded S. davidii seed germination. Under strong acid conditions (pH = 3.5), there was a marked decline in germination rate, index, and vigor compared to the control, indicating a suppression of germination ability and seedling vigor in a highly acidic environment. Similar observations have been reported in other plants, such as Cinnamomum camphora L.²² and Pseudotsuga menziesi,²³ where lower pH levels adversely affected seed germination.

The germination of S. davidii was increasingly inhibited with rising Al concentrations. At 2.0 mmol·L⁻¹ Al, the inhibitory effect was most pronounced, with significant reductions in germination rate, potential, index, and vigor. This aligns with the findings by Choudhury & Sharma²⁴ on Cicer arietinum L., highlighting the substantial impact of even minimal Al levels on seed germination.

Acid and Al stress also affect the aboveground and belowground growth characteristics.²⁵ Strong acid (pH-3.5) likely hinders radicle length and root fresh weight in S. davidii by disrupting intracellular-free radicals and proton effects, leading to physiological dysfunction in the plant root system.^26,27 Al toxicity, known to significantly impact root elongation,²⁸ was observed to reduce root length in S. davidii, particularly at higher concentrations, corroborating the inhibitory effect of Al on root tip cell division.²⁹

Physiological characteristics of S. davidii seedlings under acid and Al stress

MDA, an indicator of membrane lipid peroxidation, reflects the extent of cellular damage.³⁰ In S. davidii seedlings, the MDA content increased with Al concentration, particularly at concentrations ≥1.0 mmol·L⁻¹, suggesting that the plasma membrane is sensitive to this level of Al concentration. High MDA production indicates increased lipid peroxidation and cell damage.³¹ Al toxicity, a primary growth inhibitor in acidic soils,³² exacerbated membrane damage in S. davidii seedlings under acidic conditions, as evidenced by higher MDA content under Al stress compared to acid stress alone.³³

Osmoregulatory substances like Pro, soluble sugars, and soluble proteins play crucial roles in maintaining cellular osmotic balance, membrane integrity, and enzyme protein stability.³⁴ Under acid stress, the soluble sugar and protein contents in S. davidii seedlings increased with decreasing pH, reaching their highest at pH = 3.5, suggesting a compensatory mechanism to counteract stress-induced damage.^35,36 Under Al stress, the Pro content rose with increasing Al concentration, indicating a strategy to mitigate Al toxicity.³⁷ At Al concentrations ≤1.5 mmol·L⁻¹, the increase in soluble sugars and proteins suggested an adaptive response to maintaining cellular osmotic potential. However, at higher Al concentrations (≥2.0 mmol·L⁻¹), the decline in these substances indicated a disturbance in the osmotic potential balance, adversely affecting seedling growth.³⁸

ROS accumulation under stress can lead to lipid peroxidation, damaging plant cell membranes.³⁹ S. davidii seedlings respond by enhancing antioxidant enzyme activity, such as SOD, POD, CAT, and APX, to mitigate oxidative stress.⁴⁰ With the decrease in pH, the activities of SOD, POD, CAT, and APX in the antioxidant enzyme system were inhibited and decreased, indicating that the acid-induced oxidative damage was greater than the scavenging effect of antioxidant enzymes, resulting in the peroxidation of membrane lipids in seedlings.⁴¹ Similarly, under Al stress, these enzyme activities initially increased but then decreased, with SOD playing a crucial role in removing reactive oxygen species.⁴² The decreased activities of POD, CAT, and APX at Al concentrations ≥1.5 mmol·L⁻¹ suggested a threshold of aluminum tolerance, beyond which cell membrane oxidation damage intensified. Therefore, the antioxidant enzyme system plays an important role in the resistance of seedlings to acid and aluminum stress.

Conclusion

The study demonstrated that acid and aluminum (Al) stress significantly inhibit the germination and growth of Sophora davidii, with the root system being primarily affected. The severity of toxicity escalates with increased acidity and Al concentration. Specifically, at a concentration of 2.0 mmol·L⁻¹ Al, S. davidii seedlings exhibited pronounced changes in root morphology – roots became thicker, shorter, and brown in color. Under Al stress (pH = 4.3), there was a notable increase in the malondialdehyde (MDA) content(p < .05), indicating substantial damage to the cytoplasmic membrane. In response to acid stress, S. davidii seedlings employed adaptive strategies to mitigate injury and bolster physiological functions. This was achieved through an increase in the content of soluble sugar and protein, facilitating normal cellular operations. Among the antioxidant enzymes, superoxide dismutase (SOD) activity emerged as the primary protective mechanism against acid and Al stress in S. davidii seedlings. Comparative analysis of seed germination, morphological, and physiological indices under acid and Al stress revealed that Al significantly contributes to the growth inhibition of S. davidii in acidic environments. This finding underscores the critical role of Al as a limiting factor in the survival and development of this species under such conditions. The insights gained from this study not only enhance our understanding of the physiological responses of S. davidii to environmental stressors but also provide valuable information for the cultivation and management of this species in challenging soil conditions.^,

Author contributions

The concept and design of the study were collaboratively undertaken by all authors. Sisi Long, Wenhui Xie, Wenwu Zhao, and Danyang Liu were responsible for material preparation, data collection, and analysis. The initial draft of the manuscript was composed by Sisi Long, while Lili Zhao and Puchang Wang contributed significantly to the manuscript revisions. All authors are actively engaged in discussing and refining previous versions of the manuscript. The final manuscript was read and approved by all contributing authors, ensuring a collective agreement on the content and findings presented.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The authors have determined that all of the data underlying this article has been displayed in the article. Competing Interests: The authors have no relevant financial or nonfinancial interests to disclose.

Additional information

Funding

The authors express their deep gratitude for the financial support received for this research from multiple sources. This study was funded in part by the Guizhou Provincial Science and Technology Program, under the grant numbers Qiankehe Support [2021] General 155, Qiankehe Platform Talent [2021] [No. 5636], and Qiankehe Foundation [2020] [1Z026]. Additionally, the National Natural Science Foundation of China provided substantial support through the grants [32260340 and 32060391].