Metabolite Changes and Tolerance Mechanisms in Cynodon dactylon Roots Under Pb-stressed Soils Based on LC-MS Non-targeted Metabolomics
YANG Xiao-Rong, WEN Shao-Fu, HAN Zeng, ZHANG Cai-Long, ZHANG Mei, HOU Xiu-Li*
College of Agronomy and Life Sciences/Collaborative Innovation Center for Plateau Lake Ecology and Environmental Health (at Universities in Yunnan Province), Kunming University, Kunming 650214, China
Abstract：Plant root exudates are the key chemical signals and regulatory factors for plant response to environmental stress, and their composition changes directly affect the bioavailability of heavy metals in the rhizosphere and plant adaptability. In this study, using liquid chromatography-mass spectrometry (LC-MS) untargeted metabolomics, the metabolite changes in root exudates of Cynodon dactylon were systematically analyzed under soil Pb stress at concentrations of 500 and 1 000 mg/kg, and the tolerance mechanism of C. dactylon to soil Pb was revealed. The results showed that C. dactylon in the 500 mg/kg Pb treatment group exhibited higher root length, plant height, and leaf biomass compared with the 1 000 mg/kg Pb treatment group. Additionally, soil Pb stress reduced the contents of nitrogen, phosphorus and chlorophyll in leaves and roots of C. dactylon, indicating that soil Pb stress inhibited the absorption of nitrogen and phosphorus in soil and photosynthesis of C. dactylon. Different concentrations of soil Pb stress changed the metabolite components of C. dactylon root exudates, and a total of 1 028 differential metabolites were identified (VIP>1, P<0.05 ). The relative contents of DL-malic acid and γ-aminobutyric acid (GABA) in root exudates increased at 500 mg/kg soil Pb treatment group, but decreased at 1 000 mg/kg soil Pb treatment group. The metabolic response of C. dactylon to soil Pb stress revealed 2 key metabolic pathways associated with GABA: The GABAergic synapse pathway and butanoate metabolism pathway. These significantly enriched pathways under metal stress contributed to alleviating Pb-induced oxidative damage and ameliorating the imbalance in energy metabolism, and DL-malic acid had the ability to chelate heavy metals to reduce the bioavailability of Pb. Therefore, the organic acids (DL-malic acid, fumaric acid ) and GABA in the root exudates of C. dactylon jointly mediated the formation of a synergistic detoxification network of 'chelation-transport-antioxidation' to alleviate the stress of soil heavy metal Pb on plants. This study provides a theoretical basis for the regulation of plant rhizosphere metabolism on soil heavy metal Pb remediation technology.
[1] 江润海, 姜冉冉, 朱城强, 等. 2023. 微生物强化植物修复铅污染土壤的机制研究进展[J]. 生物技术通报, 39(08):114-125. (Jiang R H, Jiang R R, Zhu C Q, et al.2023. Research progress in mechanisms of microbial-enhanced phytoremediation for lead-contaminated soil[J]. Biotechnology Bulletin, 39(08):114-125.) [2] 姜冉冉, 江润海, 朱城强, 等. 2022. EDTA对铅胁迫下狗牙根根际土壤质量及微生物的影响[J]. 农业环境科学学报, 41(12):2722-2732. (Jiang R R, Jiang R H, Zhu C Q,et al.2022. Effects of EDTA on soil quality and microorganisms in rhizosphere of Cynodon dactylon under lead stress[J]. Journal of Agro-Environment Science, 41(12):2722-2732.) [3] 寇乐勇, 赵宽, 操璟璟, 等. 2019. 低分子量有机酸提取土壤中部分重金属的拟合模型研究[J]. 环境科学学报, 39(07):2260-2268. (Kou L Y, Zhao K, Cao J J, et al.2019. The fitting model of low molecular weight organic acids extracts heavy metals in soil[J]. Acta Scientiae Circumstantiae, 39(07):2260-2268.) [4] 李亚藏. 2012. 铅胁迫对茶条槭与五角槭的叶片叶绿素含量和膜脂过氧化及保护酶活性的影响[J]. 湖南农业大学学报(自然科学版), 38(04):404-407. (Li Y Z.2012. Effect of lead stress on chlorophyll content,membrane lipid peroxidation and protective enzyme activity in leaves of Acer ginnala Maxim and Acer mono Maxim[J]. Journal of Hunan Agricultural University(Natural Sciences), 38(04):404-407.) [5] 刘长风, 段士鑫, 张晓宇, 等. 2021. 植物根系分泌物在重金属胁迫下的响应研究进展[J]. 福建农业学报, 36(12):1506-1514. (Liu C F, Duan S X, Zhang X Y, et al.2021. Research advances on plant root exudates in response to heavy metal stress[J]. Fujian Journal of Agricultural Sciences, 36(12):1506-1514.) [6] 孟静, 叶星, 林鹏程, 等. 2025. 茉莉酸甲酯诱导马尿泡根系分泌物的差异含量及分析[J]. 广西植物, 45(01):172-184. (Meng J, Ye X, Ling P C,et al.2025. Differential content and analysis of methyl jasmonate induced root exudates in Przewalskia tangutica[J]. Guihaia, 45(01):172-184.) [7] 莫思琪, 曹旖旎, 谭倩. 2022. 根系分泌物在重金属污染土壤生态修复中的作用机制研究进展[J]. 生态学杂志, 41(02):382-392. (Mo S Q, Cao Y N, Tan Q.2022. Research progress on root exudates and their effects on ecological remediation of heavy metal contaminated soil[J]. Chinese Journal of Ecology, 41(02):382-392.) [8] 宋艺君, 孙婧, 郭涛, 等. 2024. 基于植物代谢组学方法的陕产黄精不同炮制品化学差异研究[J]. 中国药学杂志, 59(21):2011-2021. (Song Y J, Sun J, Guo T, et al.2024. Analysis of chemical variability on different processed products of Polygonati rhizoma produced in Shaanxi province based on plant metabolomics[J]. Chinese Pharmaceutical Journal, 59(21):2011-2021.) [9] 王莎, 杜致辉, 陈之林. 2021. 基于GC-MS代谢组学技术2种香型美花石斛代谢物的对比分析[J]. 分子植物育种, 19(09):3081-3089. (Wang S, Du Z H, Chen Z L.2021. Comparison of metabolites of two aromatic Dendrobium loddigesii species based on GC-MS metabonomics[J]. Molecular Plant Breeding, 19(09):3081-3089.) [10] 王亚, 冯发运, 葛静, 等. 2022. 植物根系分泌物对土壤污染修复的作用及影响机理[J]. 生态学报, 42(03):829-842. (Wang Y, Feng F Y, Ge J, et al.2022. Effects and mechanisms of plant root exudates on soil remediation[J]. Acta Ecologica Sinica, 42(03):829-842.) [11] 吴清莹, 林宇龙, 孙一航, 等. 2021. 根系分泌物对植物生长和土壤养分吸收的影响研究进展[J]. 中国草地学报, 43(11):97-104. (Wu Q Y, Lin Y L, Sun Y H,et al.2021. Research progress on effects of root exudates on plant growth and soil nutrient uptake[J]. Chinese Journal of Grassland, 43(11):97-104.) [12] 杨红霞, 陈俊良, 刘崴. 2019. 镉对植物的毒害及植物解毒机制研究进展[J]. 江苏农业科学, 47(02):1-8. (Yang H X, Chen J L, Liu W.2019. Research progress on cadmium toxicity and detoxification mechanism in plants[J]. Jiangsu Agricultural Sciences, 47(02):1-8.) [13] 杨秀敏, 胡振琪, 胡桂娟,等. 2008. 重金属污染土壤的植物修复作用机理及研究进展[J]. 金属矿山, (07):120-123. (Yang X M, Hu Z Q, Hu G J,et al. 2008. Progress in research on and mechanism of phytoremediation for heavy metal-polluted soil[J]. Metal Mine, (07):120-123.) [14] 于飞, 马海天才, 钟沃秀,等. 2018. 铅胁迫下金发草和狗牙根耐受性的对比[J]. 草业科学, 35(11):2602-2613. (Yu F, MaHai T C, Zhong W X,et al.2018. Effect of lead stress on the growth and physiological characteristics of Pogonatherum paniceum and Cynodon dactylon[J]. Pratacultural Science, 35(11):2602-2613.) [15] 周可欣, 王小瑞, 米世灿, 等. 2025. 土壤污染根际修复研究进展[J]. 中国生态农业学报(中英文), 33(04):769-782. (Zhou K X, Wang X R, Mi S C,et al.2025. Research progress on rhizosphere remediation of soil pollution[J]. Chinese Journal of Eco-Agriculture, 33(04):769-782.) [16] Ali H,Khan E.2019. Trophic transfer, bioaccumulation, and biomagnification of non-essential hazardous heavy metals and metalloids in food chains/webs-concepts and implications for wildlife and human health[J]. Human and Ecological Risk Assessment:An International Journal, 25(6):1353-1376. [17] Ashraf S, Ali Q, Zahir Z A,et al.2019. Phytoremediation:Environmentally sustainable way for reclamation of heavy metal polluted soils[J]. Ecotoxicology and Environmental Safety, 174:714-727. [18] Balyan G,Pandey A K.2024. Root exudates, the warrior of plant life:Revolution below the ground[J]. South African Journal of Botany, 164:280-287. [19] Benidickson K H, Raytek L M, Hoover G J,et al.2023. Glutamate decarboxylase-1 is essential for efficient acclimation of Arabidopsis thaliana to nutritional phosphorus deprivation[J]. New Phytologist, 240(6):2372-2385. [20] Chai Y N, Schachtman D P.2022. Root exudates impact plant performance under abiotic stress[J]. Trends in Plant Science, 27(1):80-91. [21] Chen L, Luo S, Li X, et al.2014. Interaction of Cd-hyperaccumulator Solanum nigrum L. and functional endophyte Pseudomonas sp. Lk9 on soil heavy metals uptake[J]. Soil Biology and Biochemistry, 68:300-308. [22] Ding K, Ji J, Xie G,et al.2021. Lipid biosynthesis mediated by the cyclic adenosine monophosphate (cAMP) signaling pathway in Chlorella pyrenoidosa under salt-induced osmotic stress[J]. Renewable Energy, 180:222-231. [23] Dunn W B, Broadhurst D, Begley P, et al.2011. Procedures for large-scale metabolic profiling of serum and plasma using gas chromatography and liquid chromatography coupled to mass spectrometry[J]. Nature Protocols, 6(7):1060-1083. [24] Edelstein M, Ben-Hur M.2018. Heavy metals and metalloids:Sources, risks and strategies to reduce their accumulation in horticultural crops[J]. Scientia Horticulturae, 234:431-444. [25] Feng J, Zhou T, Gu Y,et al.2024. γ-Aminobutyric acid alleviates salinity-induced impairments in rice plants by improving photosynthesis and upregulating osmoprotectants and antioxidants[J]. Agronomy, 14(11):2524. [26] Gupta M, Dwivedi V, Kumar S, et al.2024. Lead toxicity in plants:Mechanistic insights into toxicity, physiological responses of plants and mitigation strategies[J]. Plant Signaling & Behavior, 19(1):2365576. [27] Haroun M, Xie S, Awadelkareem W,et al.2023. Influence of biofertilizer on heavy metal bioremediation and enzyme activities in the soil to revealing the potential for sustainable soil restoration[J]. Scientific Reports, 13(1):20684. [28] Hazrati H, Fomsgaard I S, Ding L, et al.2021. Mass spectrometry-based metabolomics unravel the transfer of bioactive compounds between rye and neighbouring plants[J]. Plant, Cell & Environment, 44(12):3722-3731. [29] Herrero S, González E, Gillikin J W, et al.2011. Identification and characterization of a pyridoxal reductase involved in the vitamin B6 salvage pathway in Arabidopsis[J]. Plant Molecular Biology, 76(1):157-169. [30] Javed M T, Akram M S, Tanwir K,et al.2017. Cadmium spiked soil modulates root organic acids exudation and ionic contents of two differentially Cd tolerant maize (Zea mays L.) cultivars[J]. Ecotoxicology and Environmental Safety, 141:216-225. [31] Li W, Finnegan P M, Dai Q,et al.2021. Metabolic acclimation supports higher aluminium-induced secretion of citrate and malate in an aluminium-tolerant hybrid clone of Eucalyptus[J]. BMC Plant Biology, 21(1):14. [32] Liu D, Li T, Yang X,et al.2007. Enhancement of lead uptake by hyperaccumulator plant species Sedum alfredii Hance using EDTA and IAA[J]. Bulletin of Environmental Contamination and Toxicology. 78(3):280-283. [33] Liu L, Li J, Yue F,et al.2018. Effects of arbuscular mycorrhizal inoculation and biochar amendment on maize growth, cadmium uptake and soil cadmium speciation in Cd-contaminated soil[J]. Chemosphere, 194:495-503. [34] Lü G, Liang Y, Wu X,et al.2019. Molecular cloning and functional characterization of mitochondrial malate dehydrogenase (mMDH) is involved in exogenous GABA increasing root hypoxia tolerance in muskmelon plants[J]. Scientia Horticulturae, 258:108741. [35] Michaeli S, Fromm H.2015. Closing the loop on the GABA shunt in plants:Are GABA metabolism and signaling entwined?[J]. Frontiers in Plant Science, 6:419. [36] Moon D H, Koutsospyros A.2022. Stabilization of lead-contaminated mine soil using natural waste materials[J]. Agriculture, 12(3):367. [37] Nagajyoti P C, Lee K D,Sreekanth T V M.2010. Heavy metals, occurrence and toxicity for plants:A review[J]. Environmental Chemistry Letters, 8(3):199-216. [38] Park W, Kim H S, Park T W,et al.2017. Functional characterization of plasma membrane-localized organic acid transporter (CsALMT1) involved in aluminum tolerance in Camelina sativa L[J]. Plant Biotechnology Reports, 11(3):181-192. [39] Peng A, Yu K, Yu S,et al.2023. Aluminum and fluoride stresses altered organic acid and secondary metabolism in tea (Camellia sinensis) plants:Influences on plant tolerance, tea quality and safety[J]. International Journal of Molecular Sciences, 24(5):4640. [40] Ramesh S A, Tyerman S D, Gilliham M, et al.2017. γ-Aminobutyric acid (GABA) signalling in plants[J]. Cellular and Molecular Life Sciences, 74(9):1577-1603. [41] Ramesh S A, Tyerman S D, Xu B, et al.2015. GABA signalling modulates plant growth by directly regulating the activity of plant-specific anion transporters[J]. Nature Communications, 6(1):7879. [42] Seifikalhor M, Aliniaeifard S, Bernard F,et al.2020. γ-Aminobutyric acid confers cadmium tolerance in maize plants by concerted regulation of polyamine metabolism and antioxidant defense systems[J]. Scientific Reports, 10(1):3356. [43] Shelp B J, Aghdam M S, Flaherty E J.2021. γ-aminobutyrate (GABA) regulated plant defense:Mechanisms and opportunities[J]. Plants, 10(9):1939. [44] Shi X, Wang S, He W, et al.2023. Lead accumulation and biochemical responses in Rhus chinensis Mill to the addition of organic acids in lead contaminated soils[J]. RSC Advances, 13(7):4211-4221. [45] Shi Y, Wang S, Guo J, et al.2022. Effects of arbuscular mycorrhizal inoculation on the phytoremediation of PAH-contaminated soil:A meta-analysis[J]. Chemosphere, 307:136033. [46] Shi Y, Zhang X, Zhao M, et al.2024. The Status of research on the root exudates of submerged plants and their effects on aquatic organisms[J]. Water, 16(13):1920. [47] Solomon W, Janda T, Molnár Z.2024. Unveiling the significance of rhizosphere:Implications for plant growth, stress response, and sustainable agriculture[J]. Plant Physiology and Biochemistry, 206:108290. [48] Sun L, Cao X, Tan C, et al.2020. Analysis of the effect of cadmium stress on root exudates of Sedum plumbizincicola based on metabolomics[J]. Ecotoxicology and Environmental Safety, 205:111152. [49] Tan Z, Wu C, Xuan Z,et al.2022. Lead exposure dose-dependently affects oxidative stress, AsA-GSH, photosynthesis, and mineral content in pakchoi (Brassica chinensis L.)[J]. Frontiers in Plant Science, 13:1007276. [50] Trivedi P, Leach J E, Tringe S G,et al.2020. Plant-microbiome interactions:From community assembly to plant health[J]. Nature Reviews Microbiology, 18(11):607-621. [51] Ullah A, Ali I, Noor J,et al.2023. Exogenous γ-aminobutyric acid (GABA) mitigated salinity-induced impairments in mungbean plants by regulating their nitrogen metabolism and antioxidant potential[J]. Frontiers in Plant Science, 13:1081188. [52] Usman K, Abu-Dieyeh M H, Zouari N, et al.2020. Lead (Pb) bioaccumulation and antioxidative responses in Tetraena qataranse[J]. Scientific Reports, 10(1):17070. [53] Want E J, Masson P, Michopoulos F, et al.2013. Global metabolic profiling of animal and human tissues via UPLC-MS[J]. Nature Protocols, 8(1):17-32. [54] Wen S, Jiang R, Yang X,et al.2025. Bacillus velezensis combined with phosphogypsum promotes the growth of Cynodon dactylon L. by influencing rhizosphere soil nutrients, Pb bioavailability and microbial community composition in lead-contaminated soil[J]. Environmental Technology & Innovation, 37:103956. [55] Wu B, Li X, Lin S, et al.2024. Miscanthus sp. root exudate alters rhizosphere microbial community to drive soil aggregation for heavy metal immobilization[J]. Science of The Total Environment, 949:175009. [56] Wu X, Jia Q, Ji S, et al.2020. Gamma-aminobutyric acid (GABA) alleviates salt damage in tomato by modulating Na+ uptake, the GAD gene, amino acid synthesis and reactive oxygen species metabolism[J]. BMC Plant Biology, 20(1):465. [57] Yao Y, Yuan H, Liu D,et al.2024. Response of soybean root exudates and related metabolic pathways to low phosphorus stress[J]. PLOS ONE, 19(12):e0314256. [58] Zelena E, Dunn W B, Broadhurst D, et al.2009. Development of a robust and repeatable UPLC-MS method for the long-term metabolomic study of human serum[J]. Analytical Chemistry, 81(4):1357-1364. [59] Zeng L S, Liao M, Chen C L,et al.2007. Effects of lead contamination on soil enzymatic activities, microbial biomass, and rice physiological indices in soil-lead-rice (Oryza sativa L.) system[J]. Ecotoxicology and Environmental Safety, 67(1):67-74.
