Enhancing lettuce resilience to cadmium stress: Insights from raw vs. cystamine-modified biochar

doi:10.21203/rs.3.rs-5324832/v1

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Research Article

Enhancing lettuce resilience to cadmium stress: Insights from raw vs. cystamine-modified biochar

https://doi.org/10.21203/rs.3.rs-5324832/v1

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Aims and Methods

Understanding the interactions among biochar, plants, soils, and microbial communities is essential for developing effective, eco-friendly soil remediation agents. This study investigates the mechanisms by which cystamine-modified biochar (Cys-BC) alleviates cadmium (Cd) toxicity in lettuce, comparing its effects to those of raw biochar across key parameters: plant growth, antioxidant enzyme activities, available Cd in root-sheet-soil, and shifts in microbial communities.

Results

Cys-BC significantly enhances biomass, increasing above-ground growth by 40.54–44.95% and root biomass by 37.54–47.44% compared to Cd-stressed controls. Photosynthetic parameters, including chlorophyll a content and net photosynthetic rate, improve by up to 91.02% and 37.93%, respectively. Cys-BC mitigates oxidative stress, enhancing antioxidant activities by 73.83–99.39%. Additionally, it reduces available soil Cd levels while promoting microbial diversity, as evidenced by increases in Shannon, Chao1, and ACE indices of 12.81%, 14.48%, and 17.15%, respectively.

Conclusions

Cys-BC enhances glutathione reductase activity and increase cysteine levels within the ascorbate-glutathione cycle, functioning through increased soil cation exchange for Cd passivation rather than through pH modifications. Significant shifts in microbial communities, particularly the increases in Deltaproteobacteria and Nitrospira, underscore their roles in sulfur and nitrogen metabolism. These findings provide new insights into how modified biochar, specifically Cys-BC, can effectively address Cd contamination, highlighting its potential for broader applications in soil remediation.

Cd Stress

Modified biochar

Antioxidant response

Soil properties

Microbial communities

Mitigation mechanisms

Cadmium (Cd), a non-essential and toxic heavy metal, is a significant pollutant in agricultural soils, posing serious threats to food safety, human health, and crop productivity (Mubeen et al. 2023). Long-term exposure to Cd-contaminated soils necessitates the urgent development of sustainable and effective remediation technologies (Rai et al. 2023). Recent studies have highlighted various innovative approaches, including microbial remediation, which utilizes beneficial microbes to transform heavy metals into less toxic forms, and phytoremediation, where plants absorb and stabilize contaminants (Vasilachi et al. 2023). Enhanced bioremediation and in-situ remediation techniques are also gaining traction due to their environmental friendliness and effectiveness (Haider et al. 2024). Among these methods, biochar, a carbon-rich material derived from the pyrolysis of biomass, has garnered considerable attention for its high effectiveness in immobilizing heavy metals (Zong et al. 2021; Amalina et al. 2022; Yadav and Ramakrishna W 2023). Biochar possesses properties such as a high surface area, a porous structure, and the ability to sequester carbon, making it a valuable partner in agriculture (Rahim et al. 2022).

However, raw biochar exhibits irregular morphology, an unstable structure, and non-specific adsorption properties, which may limit its practical application across diverse soil conditions and varying levels of Cd contamination (Cui et al. 2020). Recent advances in biochar research have demonstrated that functionalizing biochar with materials such as phosphorus, iron (Fe), manganese (Mn), magnesium (Mg), or nanoparticles can significantly enhance its Cd adsorption capacity, thereby reducing metal bioavailability and toxicity (Usman et al. 2020; Hafeez et al. 2022). These modified biochars have shown promising results in improving plant growth, nutrient uptake, and microbial activity in Cd-contaminated soils (Bilias et al. 2021; Gao et al. 2022; Hamid et al. 2022; Amin et al. 2023; Han et al. 2023). However, the underlying mechanisms by which the chemical modifications of biochar influence plant cellular processes and soil microbial dynamics remain poorly understood (Mukherjee et al. 2022).

In this study, we investigated the comparative effects of raw biochar (BC) and cystamine-modified biochar (Cys-BC) on the mitigation of Cd stress in lettuce (Lactuca sativa). Cys-BC, which incorporates sulfur and nitrogen functional groups, demonstrated superior Cd adsorption and selectivity compared to unmodified biochar, attributed to enhanced electrostatic interactions and surface complexation (Tan et al. 2021). We assessed various plant growth parameters, antioxidant enzyme activities, Cd mobility in soils and plant, and changes in soil biological properties to gain a deeper understanding of how biochar surface modifications interact with plant roots and soil microbial communities.

Our findings demonstrate that Cys-BC not only enhances plant resilience to Cd stress by reducing Cd uptake and improving antioxidant defenses but also promotes beneficial shifts in soil microbial community structure. This comprehensive approach offers novel insights into the synergistic effects of biochar modifications on plant-soil-microbe interactions, providing a foundation for developing tailored biochar-based remediation strategies. Future research will focus on optimizing biochar formulations to align with specific soil types and crop systems, thereby advancing agricultural productivity and environmental sustainability in regions affected by heavy metal contamination.

Materials

Lettuces of the variety were sourced from Rijk Zwaan Agricultural Service Co., Ltd. (Qingdao, China). Rice husk biochar, pyrolyzed at 600°C, was obtained from Liao Ning Golden Future Agriculture Technology Co., Ltd. (Xiuyan, China).

The raw biochar was modified using cystamine dihydrochloride and glutaraldehyde as ligands, as proposed in our previous studies (Chen et al. 2019), 5 g of rice husk biochar were dispersed in 25 mL of a 2% glutaraldehyde solution and stirred at 160 rpm for 4 hours at room temperature. Subsequently, 0.75 g of cystamine was added to the mixture solution, and the solution were stirred at 160 rpm for an additional 12 hours at room temperature. The resulting cystamine-modified biochar (Cys-BC) was separated using a G3 sand core funnel and washed with anhydrous ethanol and distilled water until a neutral pH was achieved. Finally, the Cys-BC was dried under vacuum at 60°C to a constant weight and stored.

Soil and pot treatments

Soil Cadmium Content Setting

The maximum permissible concentration of Cd in agricultural soil, according to China’s Soil Environmental Quality Standard (GB15618-2008), is 1.0 mg kg^− 1. To simulate various levels of contamination, Cd solutions were added to the soil at concentrations of 1, 3, and 5 mg kg^− 1. The soil was then equilibrated for 30 days to ensure a homogeneous distribution of Cd.

Potting Test

Polyethylene pots with dimensions of 120 mm (top diameter) × 110 mm (bottom diameter) × 95 mm (height) were filled with 600 g of Cd-treated soil. Soil moisture was maintained at approximately 60% of its water-holding capacity by adding deionized water every other day. The pots were equilibrated for 30 days. Once the lettuce seedlings developed 5 to 6 leaves, they were transplanted into the pots at 28 days. Each treatment was replicated 25 times, and the plants were irrigated with a nutrient solution prepared from deionized water.

Biochar Treatment design

To assess the effect of biochar on Cd uptake, four concentrations of biochar (0.3%, 0.6%, 1.2%, and 1.8%) along with cysteine-modified biochar (Cys-BC) were incorporated into the soil. The optimal biochar addition investigations and soil treatment designs are detailed in Table S1 and Table S2, respectively.

Measurement of lettuce growth parameters

Lettuce growth parameters were assessed over a period of 35 days, with samples collected at 7-day intervals. After this duration, five plants from each treatment group, selected based on uniform growth, were evaluated. Key growth metrics, including plant height, stem diameter, leaf length, and leaf width, were measured with precision.

The plants were then washed, and their fresh weight was recorded. Root system characteristics were analyzed using an Epson Expression 10000XL root scanner, which provided data on root length, surface area, volume, and tip count. The determination of dry weight involved a two-stage drying process: an initial drying at 105°C for 30 minutes, followed by prolonged drying at 75°C until a stable weight was achieved.

Measurement of physiological indices in lettuce

A comprehensive array of analytical techniques was utilized to quantify essential photosynthetic and antioxidative indices in lettuce. Photosynthetic efficiency was measured using the LI-6400 portable photosynthesis system (LI-COR, USA) to determine the net photosynthetic rate. Photosynthetic pigment concentrations were determined using UV spectrophotometry.

Antioxidant enzyme activities (SOD, POD, CAT, GR) were assessed using assay kits from Keming Biotechnology Co. For sample pre-treatment, 0.1 g of lettuce tissue was homogenized and centrifuged at 8000 g for 10 minutes at 4°C, and the resulting supernatant was analyzed. Markers of oxidative stress (MDA, H₂O₂, GSH) were quantified using the corresponding assay kits.

Cysteine Content: The cysteine content is measured by homogenizing plant tissue in chilled perchloric acid, followed by centrifugation. The concentration of cysteine in the supernatant is then determined at 560 nm using acidic ninhydrin.

Measurement of cadmium content in lettuce

The above- ground parts and below-ground parts of the lettuce were collected, ground, and passed through a 0.5 mm sieve. A 0.3 g sample was transferred into an ablation tank containing 5 mL of HNO₃ and left to sit overnight. Subsequently, 2 mL of H₂O₂ was added, and the ablation tank underwent microwave digestion (Mars 6, CEM, USA) with the following settings:

The temperature was increased to 120°C over a period of 10 minutes, held for 5 minutes, then raised to 180°C in 6 minutes and maintained for 40 minutes. The cadmium concentration was subsequently measured using an inductively coupled plasma mass spectrometer (ICP-MS) (NexION 300X, Perkin Elmer, USA).

Measurement of cadmium in soil

Soil samples were collected, air-dried, and sieved to achieve a uniform particle size of less than 2 mm. Weigh 2 g of the sieved soil and place it into a 50 mL centrifuge tube. Add 20 mL of the DTPA extracting solution (0.005 M DTPA, 0.01 M CaCl₂, and 0.1 M triethanolamine, pH 7.3). The mixture is shaken at 250 rpm for 2 hours at room temperature. After shaking, centrifuge the tubes at 3000 rpm for 10 minutes. Decant the supernatant into a clean container and filter it through filter paper. Analyze the filtered extract using inductively coupled plasma mass spectrometry (ICP-MS). The determination of cadmium (Cd) fractions in the soil was conducted using the BCR three-step sequential extraction method, followed by ICP-MS detection.

Assessment of soil microbial diversity

In this study, four treatments were selected 35 days after the experiment described in Section 2.2: (1) CK: no cadmium or biochar added; (2) T_0 − 5: cadmium added at 5 mg kg^− 1 without biochar; (3) C_1.2−5: 1.2% raw biochar added with cadmium at 5 mg kg^− 1; and (4) T_1.2−5: 1.2% modified biochar added with cadmium at 5 mg kg^− 1. Each treatment was replicated three times, resulting in a total of 12 samples.

DNA extraction was conducted using the CTAB method, and the purity and concentration of the extracted DNA were verified through agarose gel electrophoresis. Bacterial 16S rRNA was amplified using primers specific to the V4 + V5 region (F: 5'-GTGCCAGCMGCCGCGGTAA-3'; R: 5'-CCGTCAATTCCTTTGAGTTT-3'), while fungal rRNA was amplified using primers for the ITS1-5F region (F: 5'-GGAAGTAAAAGTCGTAACAAGG-3'; R: 5'-GCTGCGTTCTTCATCGATGC-3'). The PCR products were purified using the Thermo Scientific Gel Extraction Kit, and sequencing libraries were constructed. Following quality control, sequencing was performed on the Thermo Fisher Ion S5™ XL platform. Raw reads were filtered to eliminate chimeras, resulting in high-quality reads. These reads were utilized for operational taxonomic unit (OTU) clustering and species classification. Diversity indices and inter-group differences were assessed, followed by taxonomic analysis at various classification levels.

Data analysis

To compare the differences among various treatment groups, an Analysis of Variance (ANOVA) was performed, followed by Duncan’s multiple range test to identify significant differences between the means. These statistical analyses were conducted using SPSS software version 20.0 (SPSS-IBM, Chicago, IL, USA). Differences were considered statistically significant at P < 0.05. Additionally, all data visualizations were created and analyzed using OriginPro 2016 and GraphPad Prism 7 software.

Effects of various biochar treatments on lettuce growth

The effects of cadmium (Cd) stress, in conjunction with raw biochar (BC) and cystamine-modified biochar (Cys-BC) treatments, on lettuce growth are presented in Table 1. Cd exposure significantly inhibits lettuce growth, particularly at the highest concentration of 5 mg kg^− 1. This inhibition is evident through reductions in plant height, leaf length, and fresh weight. Under high Cd stress (T_0 − 5), fresh weight decreases by 51.77% compared to the control group (CK), which is not exposed to Cd. However, treatments raw BC (C_1.2−b) and Cys-BC (T_1.2−b) mitigate these adverse effects, resulting in increases in plant fresh weight of 18.99% and 43.07%, respectively, compared to the T_0 − 5 treatment.

Table 1

Effects of different biochar treatments on lettuce growth parameters.
Treatment	Plant height (cm)	Stem diameter (mm)	Leaf number	Leaf width (cm)	Leaf length (cm)	Fresh weight (g·plant^− 1)
CK	23.80 ± 0.24a	9.85 ± 0.24A	13.40 ± 0.80a	9.80 ± 0.22A	12.7 ± 0.28a	143.28 ± 1.04A
T_0 − 1	16.81 ± 0.47e	5.58 ± 0.18C	11.20 ± 0.75b	6.72 ± 0.56C	9.52 ± 0.54d	88.28 ± 0.84F
T_0 − 3	16.09 ± 0.19ef	5.34 ± 0.04C	10.80 ± 0.40b	6.22 ± 0.10C	8.31 ± 0.29e	79.71 ± 1.19H
T_0 − 5	15.78 ± 0.10f	5.09 ± 0.17C	10.40 ± 0.49b	5.85 ± 0.3C	7.07 ± 0.24f	69.93 ± 1.41I
C_1.2−1	22.36 ± 0.34cd	9.06 ± 0.27B	12.80 ± 0.4a	8.88 ± 0.35B	11.85 ± 0.65abc	108.61 ± 1.12D
C_1.2−3	22.77 ± 0.57bc	9.07 ± 0.34B	12.60 ± 0.49a	8.84 ± 0.51B	11.35 ± 0.48bc	97.67 ± 0.71E
C_1.2−5	21.65 ± 0.50d	9.06 ± 0.56B	12.80 ± 0.4a	8.53 ± 0.27B	10.97 ± 0.33c	83.21 ± 1.61G
T_1.2−1	23.48 ± 0.42ab	9.55 ± 0.49AB	13.40 ± 0.49a	9.08 ± 0.61AB	11.92 ± 0.47abc	123.19 ± 0.96B
T_1.2−3	22.58 ± 0.42c	9.28 ± 0.24AB	13.00 ± 0.63a	9.31 ± 0.52AB	12.23 ± 0.28ab	111.64 ± 1.5C
T_1.2−5	22.60 ± 0.31c	8.96 ± 0.2B	13.00 ± 0.63	9.24 ± 0.55AB	11.25 ± 0.86bc	100.05 ± 1.18E
CK group represents the control group without Cd or biochar added to the soil. The T/C_a−b treatments refer to the use of cystamine-modified (T) and raw (C) biochar, with "a" representing the biochar addition level (0 ~ 1.2%), and "b" representing the concentration of added Cd (1, 3, 5 mg kg^− 1) in the soil.

Effects of various biochar treatments on root morphology

Scanning images of the lettuce root system under various biochar treatments (Fig. S1) reveal significant reductions in lateral root development under different levels of Cd stress. Table 2 presents a quantitative analysis of root morphology, including total root length, surface area, volume, and tip count. In the T_0 − 5 treatment, total root length decreases by 48.72% compared to the control (CK) during early growth. However, both raw BC and Cys-BC treatments result in notable improvements. The Cys-BC treatments significantly increase root surface area and volume by 1.06 and 2.32 times, respectively, compared to the T_0 − 5 treatment. With prolonged biochar application, all root parameters further improve, particularly in the Cys-BC treatments, where total root length, surface area, and volume are 1.10, 1.09, and 1.07 times higher, respectively, compared to the T_0 − 5 treatments.

Table 2

Quantitative analysis of root morphology in lecture under different biochar treatments.
Days	Treatment	Root length (cm)	Root surface area (cm²)	Total root volume (cm³)	Root diameter (mm)	Tips count
14	CK	123.28 ± 5.47g	48.42 ± 2.46fg	1.541 ± 0.03de	1.035 ± 0.11abc	376.33 ± 28.99a
	T_0 − 5	63.32 ± 3.21h	25.07 ± 1.35h	0.787 ± 0.13e	0.858 ± 0.146bc	159.53 ± 23.71h
	C_1.2−5	115.37 ± 7.91g	35.06 ± 1.84gh	0.848 ± 0.13e	0.967 ± 0.163abc	275.32 ± 12.53g
	T_1.2−5	138.29 ± 4.09fg	45.72 ± 3.54fgh	1.203 ± 0.44de	1.052 ± 0.127abc	398.67 ± 58.62ef
21	CK	186.01 ± 8.45de	62.10 ± 4.29ef	1.650 ± 0.66de	1.083 ± 0.147abc	504.53 ± 34.25d
	T_1.2−5	167.73 ± 10.10ef	44.17 ± 2.78fgh	0.925 ± 0.46e	0.838 ± 0.164c	467.52 ± 42.53e
	C_1.2−5	202.26 ± 13.54de	73.49 ± 5.98de	2.104 ± 0.88cd	1.145 ± 0.131abc	427.53 ± 62.53e
	T_1.2−5	214.19 ± 17.93d	90.95 ± 4.35cd	3.074 ± 0.50bc	1.352 ± 0.154a	566.32 ± 32.53cd
28	CK	290.54 ± 15.56c	112.11 ± 10.87bc	3.442 ± 0.57b	1.228 ± 0.112abc	725.62 ± 42.53ab
	T_0 − 5	222.25 ± 10.35d	91.525 ± 8.32cd	2.999 ± 0.49bc	1.311 ± 0.224ab	532.43 ± 62.53d
	C_1.2−5	361.00 ± 19.47b	123.61 ± 14.77b	3.368 ± 0.44b	1.090 ± 0.151abc	640.65 ± 52.53b
	T_1.2−5	467.37 ± 25.63a	191.04 ± 12.60a	6.214 ± 1.45a	1.301 ± 0.197a	828.41 ± 62.53a

Effects of various biochar treatments on photosynthesis

Table 3 illustrates a negative correlation between increasing Cd concentrations and chlorophyll content in lettuce leaves. Chlorophyll-a levels decrease by 17.53–46.75%, while chlorophyll-b levels decline by 19.67–54.10%. These reductions reflect the plant's adaptive response to Cd stress, as it modulates chlorophyll composition to mitigate the toxic effects of Cd. The significant decline in chlorophyll-b indicates a strategic adaptation aimed at maintaining photosynthetic efficiency under Cd stress. Both raw and Cys-BC treatments contribute to restoring or maintaining the standard chlorophyll a/b ratio during Cd-induced stress. The C_1.2−3 treatment increases chlorophyll a and b levels by 13.86% and 16.67%, respectively, compared to the T_0 − 3 control. More pronounced improvements are observed in the T_1.2−5 treatment, with chlorophyll-a increasing by 51.22% and chlorophyll-b by 67.86%, relative to the T_0 − 5 control.

Table 3

Effects of different biochar treatments on chlorophyll pigment content in lettuce.
Treatments	Chlorophyll a content (mg g^− 1 FW)	Chlorophyll b content (mg g^− 1 FW)	Chl a/ Chl b
CK	1.54 ± 0.14a	0.61 ± 0.12A	2.52 ± 0.11bc
T_0 − 1	1.27 ± 0.08g	0.49 ± 0.02EF	2.59 ± 0.01d
T_0 − 3	1.01 ± 0.07h	0.36 ± 0.11FG	2.81 ± 0.03e
T_0 − 5	0.82 ± 0.04i	0.28 ± 0.04G	2.93 ± 0.07e
C_1.2−1	1.35 ± 0.08c	0.53 ± 0.05CD	2.55 ± 0.10bc
C_1.2−3	1.15 ± 0.04e	0.42 ± 0.02EF	2.74 ± 0.09cd
C_1.2−5	0.93 ± 0.02f	0.32 ± 0.01G	2.91 ± 0.14a
T_1.2−1	1.51 ± 0.12b	0.60 ± 0.01AB	2.52 ± 0.04bc
T_1.2−3	1.47 ± 0.10c	0.57 ± 0.02BC	2.58 ± 0.07bc
T_1.2−5	1.24 ± 0.08d	0.47 ± 0.01DE	2.64 ± 0.12ab

The effects of different biochars on the photosynthetic parameters of the lecture are presented in the supplementary materials. Fig. S2 and S3 illustrate the effectiveness of biochar treatments in alleviating the negative impacts of Cd stress on lettuce photosynthesis, with Cys-BC exhibiting superior performance.

Effect of various biochar treatments on oxidative stress markers

Malondialdehyde (MDA)

Figure 1a shows that MDA levels significantly increase under Cd stress (T_0 − 5). In the absence of biochar, MDA concentrations rise to 2.17 and 3.02 times in above- and below-ground parts, compared to the control (CK). However, the introduction of biochar treatments-specifically, raw biochar (C_1.2−b) and Cys-BC (T_1.2−b)-markedly reduces MDA levels. In below-ground parts, MDA levels are reduced by 31.25% and 55.28%, respectively, and in above-ground parts by 25.24% and 43.57%, demonstrating the potential of Cys-BC in mitigating oxidative stress.

Hydrogen peroxide (H₂O₂)

Figure 1b indicates that H₂O₂ levels are 2.18 and 2.33 times higher under Cd stress compared to CK in above- and below-ground parts, respectively. The T_1.2−b treatment reduces H₂O₂ levels by 46.45% in above-ground parts and 56.62% in below-ground parts. This highlights the efficacy of Cys-BC in reducing oxidative damage induced by Cd stress.

Glutathione (GSH)

Figure 1c shows a decrease in GSH content under Cd stress, with levels 2.38-fold and 2.02-fold lower than CK in above- and below-ground parts, respectively. T_1.2−b treatment increases GSH levels by 1.30, 2.63, and 1.74 times in below-ground parts, compared to CK, T_0 − b, and C_1.2−b treatments, respectively. This increase suggests that Cys-BC plays a vital role in enhancing the plant’s antioxidant defense system under Cd stress.

Cysteine (Cys)

Figure 1d details that Cd stress induces a substantial increase in cysteine content, with levels in below-ground parts rising 3.08-fold compared to CK. The T_1.2−b treatment further amplifies Cys content, with increases of 4.71, 1.09, and 1.32 times in above-ground parts and 13.86, 1.42, and 2.31 times in below-ground parts, compared to CK, T_0 − b, and C_1.2−b treatments. This indicates that Cys-BC contributes to boosting cysteine production, a key player in plant defense against Cd toxicity.

Effects of various biochar treatments on antioxidant enzyme activity

Superoxide Dismutase (SOD)

As illustrated in Fig. 2a, Cd stress (T_0 − 5) results in a significant reduction in SOD activity, with decreases of 55.17% in above-ground parts and 61.77% in below-ground parts, compared to control (CK). In contrast, the Cys-BC treatment (T_1.2−5) leads to substantial increases in SOD activity, with an increase of 73.83% in above-ground parts and 99.39% in below-ground parts. These findings highlight the beneficial effect of Cys-BC in enhancing the enzymatic defense against oxidative stress.

Peroxidase (POD)

Figure 2b illustrates a reduction in POD activity of 47.77% and 47.20% in the above-ground and below-ground parts under Cd stress (T_0 − 5). The application of raw biochar treatment (C_1.2−5) results in a slight improvement in POD activity, with increases of 35.60% in the above-ground and 23.79% in the below-ground parts. Notably, Cys-BC (T_1.2−b) demonstrates the most significant enhancement, with POD activity increased by 64.37% in the above-ground and 70.42% in the below-ground parts, highlighting its role in enhancing the plant’s antioxidant capacity.

Catalase (CAT) Activity: Under Cd stress, CAT activity decreases by 56.15% in above-ground tissues and 47.50% in below-ground tissues, as seen in Fig. 2c. Raw biochar (C_1.2−5) enhances CAT activity, resulting in increases of 58.56% in above-ground tissues and 12.12% in below-ground tissues. However, Cys-BC (T_1.2−5) demonstrate the most significant, increasing CAT activity by 86.30% in above-ground tissues and 71.52% in below-ground tissues. This underscores its effectiveness in mitigating oxidative damage through enhanced CAT activity.

Glutathione reductase (GR)

Figure 2d illustrates that Cd stress has a more pronounced effect on GR activity in below-ground tissues, resulting in reductions of 13.49% in above-ground parts and 45.31% in below-ground parts compared to CK. Raw biochar (C_1.2−5) moderately enhances GR activity, increasing it by 31.62% in above-ground parts and 15.13% in below-ground parts. The Cys-BC treatment (T_1.2−5) shows the most substantial improvement, with GR activity rising by 67.18% in above-ground parts and 50.39% in below-ground parts. This further supports the capacity of Cys-BC to strengthen the plant's enzymatic defense mechanisms.

Effects of various biochar treatments on cadmium transfer rate in lettuce

Comparing Fig. 3a and 3b reveals that Cd accumulates predominantly in the roots of lettuce, exhibiting a higher transfer rate from soil to roots than from roots to shoots. The Cys-BC treatment is particularly effective in reducing Cd transfer rates, as evidenced by the lower slopes and intercepts in Fig. 3c and 3d compared to those observed with. The root-to-shoot Cd translocation slope ratio under the Cys-BC treatment is 3.03, significantly higher than the 2.63 ratio observed with raw biochar. This indicates that Cys-BC effectively limits Cd movement to the edible parts of the plant, thereby enhancing food safety.

Effects of various biochar treatments on soil properties

Figure 4a and 4b illustrate how the addition of raw BC and Cys-BC alters soil Cd fractions. Both treatments lead to a significant reduction in the proportions of acid-extractable, oxidizable, and reducible Cd forms, while markedly increasing the residual Cd fraction. The residual Cd content rises from 15% ± 0.8% in the non-biochar treatments to 44% ± 0.5% with raw BC treatments, and further increases to 67% ± 1% with Cys-BC treatments. These findings indicate that Cys-BC is more effective than raw BC in converting active Cd into stable forms, achieving a 50% reduction in acid-extractable and reducible Cd compared to raw BC.

Figure 4c and 4d illustrate the effects on soil pH and cation exchange capacity (CEC). Both raw BC and Cys-BC significantly enhance soil pH. Raw BC raises soil pH by 0.25 to 0.79 units, while Cys-BC increases it by 0.10 to 0.32 units. Furthermore, both treatments improve soil CEC. The application of raw BC results in an increase in CEC of 3.69 and 5.23 mol kg^− 1 at dosages of 1.2% and 1.8%, respectively. Cys-BC demonstrates an even greater effect, with increases of 5.79 and 6.37 mol kg^− 1 at the same dosages.

Table 4 presents the linear regression models that correlate soil pH and CEC with DTPA-extractable Cd. In treatments with raw BC, soil pH has a substantial influence on DTPA-extractable Cd (P = 0.001–0.003), as indicated by a high correlation coefficient (R² = 0.995–0.999). In contrast, in treatments with Cys-BC treatments, the influence of soil pH on available Cd is less significant (P = 0.073–0.135), with a lower correlation (R²=0.747–0.859). While soil CEC correlates with DTPA-extractable Cd in both treatments, the correlation is stronger in Cys-BC treatments (R² = 0.9865–0.9972, P = 0.0014–0.0068). These results suggest that raw BC primarily reduces Cd availability by increasing soil pH, whereas Cys-BC exerts its effect mainly by enhancing soil CEC. The improved CEC in Cys-BC treatments may be attributed to the structural properties of Cys-BC, particularly the abundance of functional groups such as amino, thiol, aldehyde, and hydroxyl groups on its surface, which enhance its cation exchange capacity.

Table 4

Linear regression models correlating soil pH and CEC with DTPA-extractable Cd.
Soil	Treatments	Cd levels (mg kg^− 1)	Fitting equation	Correlation (R²)	P value
pH	Raw BC	1	Y = -0.102*X + 0.991	0.999	0.001**
		3	Y = -0.316*X + 3.093	0.997	0.001**
		5	Y = -0.499*X + 5.052	0.995	0.003**
	Cys-BC	1	Y = -0.359*X + 2.814	0.859	0.073*
		3	Y = -1.379*X + 10.640	0.851	0.077*
		5	Y = -1.808*X + 14.300	0.747	0.136
CEC	Raw BC	1	Y = -0.021*X + 0.4777	0.964	0.018*
		3	Y = -0.066*X + 1.497	0.946	0.027*
		5	Y = -0.103*X + 2.533	0.942	0.030*
	Cys-BC	1	Y = -0.035*X + 0.6342	0.987	0.007**
		3	Y = -0.137*X + 2.285	0.994	0.003**
		5	Y = -0.191*X + 3.505	0.997	0.001**

Effects of various biochar treatments on microbial diversity

Table 5 demonstrates that the goods coverage indices for all samples exceed 98%, confirming sufficient sequencing depth. The Shannon diversity index decreases by 8.83% for bacteria and 15.81% for fungi in the T_0 − 5 treatment compared to the control (CK) group. Similarly, the Chao1 indices indicate reductions of 8.71% for bacteria and 13.07% for fungi in T_0 − 5 relative to CK. The ACE indices also decline by 8.69% for bacteria and 13.14% for fungi. In contrast, the application of Cys-BC significantly mitigates the adverse of cadmium on soil microbial diversity, resulting in increases in the Shannon, Chao1, and ACE indices of 12.81%, 14.48%, and 17.15%, respectively.

Table 5

Alpha indices statistics for bacteria and fungi in soil sample groups
Soil microbe	Treatments	Number of OTUs	Diversity index		Richness estimator		Goods coverage	Phylogenetic diversity
Soil microbe	Treatments	Number of OTUs	Shannon	Simpson	Chao1	ACE	Goods coverage	Phylogenetic diversity
Bacteria	CK	2432	9.390	0.996	3078.653	3072.826	0.982	165.653
	T_0 − 5	2268	8.561	0.995	2842.903	2805.170	0.981	162.321
	C_1.2−5	2607	9.566	0.996	3121.403	3096.870	0.981	175.993
	T_1.2−5	2686	9.658	0.997	3254.430	3286.196	0.982	182.432
Fungi	CK	987	7.222	0.988	1134.655	1073.938	0.997	306.808
	T_0 − 5	968	6.080	0.998	986.443	932.886	0.994	312.511
	C_1.2−5	1022	7.346	0.982	1109.757	1121.158	0.997	311.814
	T_1.2−5	1148	7.719	0.969	1276.26	1289.536	0.997	354.215

Figure 5a illustrates significant variability in bacterial species abundance between raw BC and CK. The predominant bacterial classes identified include Gammaproteobacteria, Deltaproteobacteria, Planctomycetacia, and Bacilli. Both Gammaproteobacteria and Deltaproteobacteria are instrumental in mitigating Cd toxicity through the degradation of harmful substances and the facilitation of heavy metal transformation (Nawaz et al. 2024). Planctomycetacia plays a vital role in the cycling of nutrients, particularly nitrogen and carbon. Bacilli play a crucial role in plant resistance by producing antioxidants and bioactive compounds.

In contrast, Fig. 5b highlights the significant variability in bacterial species abundance between Cys-BC and CK. The primary bacterial classes observed include Deltaproteobacteria, Nitrospira, and Chloroflexia. Deltaproteobacteria play a crucial role in sulfur and organic matter metabolism, potentially reducing the bioavailability and toxicity of heavy metals such as cadmium. Nitrospira are essential to the nitrogen cycle, particularly in the process of nitrite oxidation, which helps maintain soil nutrient balance. Chloroflexia significantly contributes to photosynthesis and the degradation of organic matter, thereby promoting soil health by facilitating the breakdown of organic materials.

Figure 5c and 5d demonstrate significant variability in fungal species abundance across different treatments. The key fungal classes identified include Mucoromycota-Mucoromycete, Basidiomycota, Tremellomycetes, and Leotiomycetes. Mucoromycota play a crucial role in organic matter decomposition and nutrient cycling, with Mucoromycete potentially forming symbiotic relationships with plants to enhance nutrient uptake and improve tolerance to heavy metals. Basidiomycota includes several important decomposers capable of breaking down complex organic matter, such as lignin and cellulose, thereby enhancing soil quality under Cd stress through enhanced biodegradation (Kumar et al. 2023). Tremellomycetes are often associated with saprophytic and symbiotic relationships that facilitate organic matter decomposition and nutrient release, while they may also degrade organic pollutants by producing extracellular enzymes in response to Cd stress. Finally, Leotiomycetes are primarily involved in humus formation and the maintenance of soil health, supporting plant survival in challenging environments.

Mechanisms of cystamine-modified biochar in alleviating cadmium stress

Previous studies have established that raw biochar can alleviate Cd stress in plants by improving nutrient uptake, adjusting physiological parameters, and limiting Cd absorption and translocation (Yu et al. 2024; Wang et al. 2024). Our findings advance the understanding of the specific mechanisms through which Cys-BC mitigates Cd stress. While earlier research has generally demonstrated positive effects of biochar on plant growth under Cd exposure, our study uniquely highlights significant enhancements in root morphology, including increased root length, surface area, and volume that have not been extensively documented. Notably, Cys-BC not only promoted root growth but also significantly elevated chlorophyll-a content and net photosynthetic rates, suggesting a substantial improvement in photosynthetic efficiency and overall plant vitality.

A key distinction in our findings relates to the alleviation of oxidative stress. Cd-induced oxidative damage is well-documented, with the accumulation of reactive oxygen species (ROS) leading to cellular injury (Mansoor et al. 2023; Rudenko et al. 2023). While prior studies have emphasized the general role of antioxidant enzymes in mitigating this stress, our research demonstrates a comprehensive enhancement of the plant’s antioxidant defense (Kesawat et al. 2023). Cys-BC results in a substantial reduction in malondialdehyde (MDA) and hydrogen peroxide (H₂O₂) levels, indicating decrease lipid peroxidation and oxidative stress. Furthermore, the simultaneous upregulation of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) activities, along with enhanced components of the ascorbate-glutathione cycle components-including elevated concentrations of reduced glutathione (GSH), cysteine (Cys), and glutathione reductase (GR) activity-provides a detailed understanding of how Cys-BC accelerates ROS scavenging through sulfur-containing ligands. These findings extend beyond previous observations by identifying specific biochemical pathways that are enhanced, clarifying how Cys-BC strengthens the plant's defense against Cd-induced oxidative damage.

Mechanisms for reducing soil-to-plant cadmium transfer with cystamine-modified biochar

The role of biochar in modifying soil properties to reduce heavy metal bioavailability is well-established (Zhou et al. 2024). Our study offers new insights into the efficacy of Cys-BC in altering soil chemistry to limit Cd uptake by plants. Previous research has shown that biochar can increase soil pH and CEC, thereby reducing Cd availability (Huang et al. 2024; Zhang et al. 2024). In contrast, our results demonstrate a more pronounced effect of Cys-BC on these soil properties, with a pH increase of 0.10–0.32 units and a CEC rise of 3.73–6.37 mol kg^− 1, leading to a 15.81–59.44% reduction in available Cd. These alterations in soil parameters are significantly greater than those reported for unmodified or other modified biochars, indicating that cystamine modification enhances the capacity of biochar to immobilize Cd effectively.

A notable distinction in our findings is the role of functional groups introduced by cystamine modification. While previous studies have broadly linked biochar’s functional groups to Cd immobilization, our linear regression analysis establishes a quantitative relationship between increased CEC and reduced Cd bioavailability, with highly significant correlations (P = 0.0014–0.0273, R²=0.9865–0.9972). This suggests that the functional groups of Cys-BC exert a more targeted and efficient influence on enhancing CEC compared to unmodified biochar. Furthermore, the combined effects of reduced nutrient loss and lowered Cd bioavailability in our study emphasize the enhanced resilience of lettuce to Cd toxicity, demonstrating the superior performance of Cys-BC in comparison to results from previous biochar research. These findings contribute novel insights into how specific biochar modifications can more effectively mitigate Cd uptake in plants.

Effects of cystamine-modified biochar on soil microbial diversity

Soil microorganisms are sensitive to Cd stress, which significantly impacts their biomass, community structure, diversity, and functionality (Goncharuk et al. 2023). Biochar can influence microbial activity and biomass in soil, alter the ratio of soil bacteria to fungi, enhance soil enzyme activity, and reshape microbial community structure (Alotaibi et al. 2023).

Our new finding demonstrates the abundance of different bacterial species in Cys-BC compared to raw BC highlights a specific class, Deltaproteobacteria. In raw BC treatment, the mean abundance of Deltaproteobacteria is lower than that of Gammaproteobacteria, whereas Deltaproteobacteria becomes more dominant in the Cys-BC treatment. This shift can be attributed to their differing metabolic pathways: Gammaproteobacteria are versatile in degrading organic pollutants, including heavy metals, while Deltaproteobacteria specialize in sulfur metabolism and can help stabilize heavy metals like Cd in less harmful forms (Sun et al. 2022). This specialization may explain the enhanced effectiveness of Cys-BC in improving soil conditions and increasing plant resilience during Cd stress.

Additionally, the differences in bacterial and fungal composition between raw BC and Cys-BC treatments may arise from: (1) Ecological Niche Differences: Certain microbial groups, such as Planctomycetacia and Bacilli, as well as Mucoromycete and Basidiomycota, may thrive under favorable conditions such as organic matter enrichment or optimal pH levels. Conversely, groups like Nitrospira, Chloroflexia, and Tremellomycetes may be better adapted to Cd-stressed environments (Palansooriya et al. 2019). (2) Microbial Interactions: The relationships among different bacterial and fungal species-whether competitive, synergistic, or inhibitory-can also contribute to the observed differences in community composition (Song et al. 2021).

Cys-BC enhances plant fresh weight and root parameters while increasing chlorophyll content, thereby promoting photosynthetic activity under Cd stress. It effectively reduces oxidative stress and increases antioxidant enzyme activities, highlighting the biochemical pathways activated by Cys-BC and providing a robust mechanism for mitigating Cd-induced oxidative damage. Furthermore, Cys-BC raises soil pH and CEC, significantly decreasing Cd bioavailability. The functional groups resulting from cystamine modification play a crucial role in this enhanced Cd immobilization, outperforming raw biochar. Additionally, Cys-BC positively influences soil microbial diversity, increasing populations of beneficial bacteria and fungi that are essential for nutrient cycling and plant health. These shifts in microbial community structure illustrate the complex interactions between soil amendments and microbial dynamics in Cd-contaminated environments. Overall, this research underscores the potential of cystamine-modified biochar as an effective strategy for reducing Cd toxicity in agriculture, ultimately contributing to improved food safety and environmental sustainability. Future studies should explore the long-term effects of Cys-BC on soil health and plant productivity, as well as its applications across diverse cropping systems.

Supplementary Materials The following supporting information can be downloaded at https://, Table S1: Effect of biochar addition on lettuce biomass; Table S2: Soil treatment design; Table S3: Effects of different treatments on lettuce growth indexes in different cadmium-contaminated soils. Fig. S1 Effects of different biochar treatments on morphology of lettuce roots, Cd addition level of 5 mg kg^-1and growth period from 14 to 28 days (from top to bottom); Fig. S2 Variation trend curves of net photosynthetic rate of lettuce leaves under different Cd level: 1 mg kg^-1 (a), 3 mg kg^-1 (b), and 5 mg kg^-1 (c); Fig. S3 Effects of different biochar treatments on Intercellular CO2 concentration (a), Stomatal Conductance (b), and transpiration rate (c).

Author Contributions Conceptualization, R.C. and T.Z.; methodology, X.D. and R.X.; formal analysis, R.C. and T.Z.; funding acquisition, R.C.; investigation, R.X.; data curation, R.C. and T.Z.; validation, R.C. and X.D.; writing-original draft, T.Z.; writing-review and editing, R.C. and T.Z.

Funding The present work was supported by the Shandong Agriculture and Engineering University Start-Up Fund for Talented Scholars (No. BSQJ202325); the Basic Research Pilot Project on Integration of Science, Education, and Industry of Qilu University of Technology (Shandong Academy of Science) (No. 2023PX098); the Shandong Modern Agricultural Technology and Industry system (SDAIT-17-10).

Data availability The datasets analyzed during the study are available from the corresponding author upon reasonable request.

Acknowledgments We sincerely thank the Ji'nan Academy of Agricultural Sciences for providing the research site and express our gratitude to Pan Ma for the valuable support in managing the planting activities.

Competing interests The authors declare no conflict of interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Open Access This article is licensed under a Creative Com mons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution, and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third-party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/

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Enhancing lettuce resilience to cadmium stress: Insights from raw vs. cystamine-modified biochar

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