Construction and Validation of a Visualization System for Plants Heavy Metal Response

doi:10.13560/j.cnki.biotech.bull.1985.2025-0804

Abstract

Abstract:

Objective To develop a visual monitoring system for heavy metal ions inside plants and achieve rapid detection of heavy metal ions in rice (Oryza sativa). Method Based on the principle that metal-responsive transcription factors (MTF-1) specifically bind to metal-responsive elements (MREs) and activate transcription, a metal-responsive plant expression vector with β-glucuronidase (GUS) or enhanced green fluorescent protein (EGFP) as reporter genes was constructed, and it was introduced into rice and Arabidopsis thaliana using the Agrobacterium-mediated method. By treating the transgenic plants with different concentrations of heavy metal ions, the intensity of GUS staining or fluorescence signal was observed. RNA-seq was used to analyze the differential gene expression of transgenic rice and wild-type control before and after heavy metal treatment. The antioxidant enzyme activities and phenotypic verification were used to analyze the metal tolerance of transgenic rice and wild-type control. Result After being treated with different concentrations of As³⁺, Cd²⁺, and Cu²⁺solutions, obvious GUS staining or green fluorescence signals were observed in transgenic A. thaliana or rice. In addition, the expressions of heavy metal detoxification-related genes in transgenic rice were significantly higher than those in the control, and the activities of superoxide dismutase (SOD) and peroxidase (POD) were enhanced. Conclusion A visualization system responding to heavy metal ions was initially constructed in rice and A. thaliana, providing technical support for the rapid detection of heavy metals in rice.

Key words: heavy metals, rice, MTF-1, MREs, visualization

PENG Yan, AN Chen, SHAO Ye, MAO Bi-gang, ZHANG Xue-wen, ZHAO Bing-ran. Construction and Validation of a Visualization System for Plants Heavy Metal Response[J]. Biotechnology Bulletin, 2026, 42(7): 105-115.

Figures/Tables 7

Fig. 1 Construction of heavy-metal-responsive plant expression vectorsA-D refers to schematic diagrams of the 35S: MTF1-MRE8-mini35S: GUS, 35S: MTF1-MRE8-mini35S: EGFP, Ubi: MTF1-MRE8-mini35S: GUS,and Ubi: MTF1-MRE8-mini35S: EGFP vectors, respectively. Hyg (R) indicates the hygromycin resistance gene, MTF-1 core indicates the core sequence of the metal-responsive transcription factor MTF-1, and MRE (8×) indicates eight copies of the metal response element MRE. MRE (8×) is linked to the mini 35S promoter to form a metal-responsive synthetic promoter

Fig. 2 GUS expression analysis in transgenic A. thalianaA-C: GUS staining in transgenic A. thaliana at 7 d (A), 10 d (B), and 12 d (C)) after treated with As3+, Cd2+and Cu2+. Scale bars: 500 μm (leaf), and 1 mm (plant). D: The color intensity of the GUS staining solution at 12 d after treated with As3+, Cd2+and Cu2+. E: The relative expressions of the GUS gene in transgenic A. thaliana at 7 d (I-III), 10 d (IV-VI) and 12 d (VII-IX) after treated with As3+, Cd2+and Cu2+, respectively. The bar graph indicates the mean ± standard deviation (n = 3), the different letters on the bars indicate a statistically significant difference. F: GUS activity in transgenic A. thaliana at 7 (I-III), 10 (IV-VI), and 12 d (VII-IX) after treated with As3+, Cd2+and Cu2+, respectively. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.000 1. The same below

Fig. 3 EGFP expression analysis in transgenic A. thalianaA: Green fluorescence intensity in the root tips of transgenic A. thaliana at 7 d after treated with As³⁺, Cd²⁺, and Cu²⁺, respectively. Scale bar = 50 μm. B: Relative expression of the EGFP gene in transgenic A. thaliana at 7 d (I-III), 10 d (IV-VI), and 12 d (VII-IX) after treated with As³⁺, Cd²⁺, and Cu²⁺, respectively

Fig. 4 Verification of the expressions of heavy metal-responsive plant expression vectors inriceA: GUS activity of transgenic rice exposed to heavy metals for 7 d. B: Relative expression of the EGFP gene in transgenic rice exposed to heavy metals for 3 d (I-III), 5 d (IV-VI), and 7 d (VII-IX). C: GUS staining of transgenic rice exposed to heavy metals for 7 d, scale bars: 1 mm. D-F: Green fluorescence intensity emitted by the root tips of transgenic rice exposed to heavy metals for 3 d (D), 5 d (E), and 7 d (F), scale bars: 100 μm

Fig. 5 Differential expression and GO clustering analysisA: Number of differentially expressed genes (DEGs) among different groups. MTF(As), MTF(Cd), and MTF(Cu) refer to transgenic rice treated with As³⁺, Cd²⁺, and Cu²⁺ for 3 d, respectively. MTF(-) refers to untreated transgenic rice. WT(As), WT(Cd), and WT(Cu) refer to wild-type rice treated with As³⁺, Cd²⁺, and Cu²⁺ for 3 d, respectively. “All” refers to the total set of DEGs. “Up” and “Down” indicate the number of significantly up-regulated and down-regulated genes, respectively. B-D: GO clustering of DEGs between wild-type and transgenic rice under the same conditions. The X-axis “GeneRatio” is the ratio of the number of DEGs to the total number of DEGs. The bubble size indicates the number of genes enriched in the corresponding pathway, and the bubble color indicates the P-value adjusted for multiple hypothesis testing, with Padj < 0.05 considered significantly enriched

Fig. 6 Verification of gene expression related to heavy metal detoxificationHZ: Wild-type rice. MTF: Transgenic rice. Data are expressed as mean ± standard deviation, n = 3; * and ** indicate significant differences between wild-type rice and transgenic rice at 0.05 and 0.01 levels, respectively (t-test)

Fig. 7 Physiological characteristics and phenotypic traits of wild-type and transgenic rice exposed to different heavy metalsA: Plant height, root length, fresh weight, and antioxidant enzyme (SOD, POD, and CAT) activities of wild-type (HZ) and transgenic rice (MTF) under As, Cd, or Cu stress. Concentration gradients are 0, 50, 100, 200, 300, and 500 µmol/L. Data are presented as mean ± standard deviation, n = 3. * indicates a significant difference between wild-type (HZ) and transgenic rice (MTF) at 0.05 level (t-test). B: Phenotypic traits of wild-type (left of the white dashed line) and transgenic rice (right of the white dashed line). Scale bar = 4 cm

References 30

[1]	环境保护部, 国土资源部. 全国土壤污染状况调查公报[EB/OL]. (2014-04-17). .
	Ministry of Environmental Protection, Ministry of Land and Resources. Report on the National General Survey of Soil Contamination[EB/OL]. (2014-04-17). .
[2]	Qin GW, Niu ZD, Yu JD, et al. Soil heavy metal pollution and food safety in China: Effects, sources and removing technology [J]. Chemosphere, 2021, 267: 129205.
[3]	Yuan XH, Xue ND, Han ZG. A meta-analysis of heavy metals pollution in farmland and urban soils in China over the past 20 years [J]. J Environ Sci, 2021, 101: 217-226.
[4]	Zhao FJ, Wang P. Arsenic and cadmium accumulation in rice and mitigation strategies [J]. Plant Soil, 2020, 446(1/2): 4374.
[5]	Wen MY, Ma ZQ, Gingerich DB, et al. Heavy metals in agricultural soil in China: a systematic review and meta-analysis [J]. Eco Environ Health, 2022, 1(4): 219-228.
[6]	Yang MH, Yang YH, Liu B, et al. Amperometric glucose biosensor based on chitosan with improved selectivity and stability [J]. Sens Actuat B Chem, 2004, 101(3): 269-276.
[7]	Wang XY, Watanabe H, Uchiyama S. Amperometric l-ascorbic acid biosensors equipped with enzyme micelle membrane [J]. Talanta, 2008, 74(5): 1681-1685.
[8]	Chen JH, Chen MJ, Tong H, et al. Fluorescence biosensor for ultrasensitive detection of the available lead based on target biorecognition-induced DNA cyclic assembly [J]. Sci Total Environ, 2023, 905: 167253.
[9]	郑亚楠, 王丹. 金属调控蛋白的结构、性质及应用 [J]. 化学进展, 2019, 31(10): 1372-1383.
	Zheng YN, Wang D. Structures, properties, and applications of metalloregulatory proteins [J]. Prog Chem, 2019, 31(10): 1372-1383.
[10]	Ravikumar S, Ganesh I, Yoo IK, et al. Construction of a bacterial biosensor for zinc and copper and its application to the development of multifunctional heavy metal adsorption bacteria [J]. Process Biochem, 2012, 47(5): 758-765.
[11]	Liu XC, Hu QY, Yang JM, et al. Selective cadmium regulation mediated by a cooperative binding mechanism in CadR [J]. Proc Natl Acad Sci U S A, 2019, 116(41): 20398-20403.
[12]	Wan XY, Volpetti F, Petrova E, et al. Cascaded amplifying circuits enable ultrasensitive cellular sensors for toxic metals [J]. Nat Chem Biol, 2019, 15(5): 540-548.
[13]	Khan SS, Shen Y, Fatmi MQ, et al. Design and prototyping of genetically encoded arsenic biosensors based on transcriptional regulator AfArsR [J]. Biomolecules, 2021, 11(9): 1276.
[14]	Chen XJ, Yao H, Song D, et al. A novel antimony-selective ArsR transcriptional repressor and its specific detection of antimony trioxide in environmental samples via bacterial biosensor [J]. Biosens Bioelectron, 2023, 220: 114838.
[15]	Giedroc DP, Chen XH, Apuy JL. Metal response element (MRE)-binding transcription factor-1 (MTF-1): structure, function, and regulation [J]. Antioxid Redox Signal, 2001, 3(4): 577-596.
[16]	Laity JH, Andrews GK. Understanding the mechanisms of zinc-sensing by metal-response element binding transcription factor-1 (MTF-1) [J]. Arch Biochem Biophys, 2007, 463(2): 201-210.
[17]	Günther V, Lindert U, Schaffner W. The taste of heavy metals: Gene regulation by MTF-1 [J]. Biochim Biophys Acta, 2012, 1823(9): 1416-1425.
[18]	Koen E, Besson-Bard A, Duc C, et al. Arabidopsis thaliana nicotianamine synthase 4 is required for proper response to iron deficiency and to cadmium exposure [J]. Plant Sci, 2013, 209: 1-11.
[19]	Chen SN, Zhang M, Feng Y, et al. Nicotianamine Synthase Gene 1 from the hyperaccumulator Sedum alfredii Hance is associated with Cd/Zn tolerance and accumulation in plants [J]. Plant Soil, 2019, 443(1/2): 413-427.
[20]	Tiwari M, Kidwai M, Dutta P, et al. A tau class glutathione-S-transferase (OsGSTU5) confers tolerance against arsenic toxicity in rice by accumulating more arsenic in root [J]. J Hazard Mater, 2022, 426: 128100.
[21]	Yang Y, Li J, Li H, et al. OsGSTU5 and OsGSTU37 encoding glutathione reductases are required for cadmium tolerance in rice [J]. Int J Environ Sci Technol, 2023, 20(9): 10253-10260.
[22]	Yang M, Li YT, Liu ZH, et al. A high activity zinc transporter OsZIP9 mediates zinc uptake in rice [J]. Plant J, 2020, 103(5): 1695-1709.
[23]	Tan LT, Qu MM, Zhu YX, et al. ZINC TRANSPORTER5 and ZINC TRANSPORTER9 function synergistically in zinc/cadmium uptake [J]. Plant Physiol, 2020, 183(3): 1235-1249.
[24]	Tan LT, Zhu YX, Fan T, et al. OsZIP7 functions in xylem loading in roots and inter-vascular transfer in nodes to deliver Zn/Cd to grain in rice [J]. Biochem Biophys Res Commun, 2019, 512(1): 112-118.
[25]	张妮娜, 上官周平, 陈娟. 植物应答缺铁胁迫的分子生理机制及其调控 [J]. 植物营养与肥料学报, 2018, 24(5): 1365-1377.
	Zhang NN, Shangguan ZP, Chen J. Molecular physiological mechanism and regulation of plant responses to iron deficiency stress [J]. Plant Nutr Fert Sci, 2018, 24(5): 1365-1377.
[26]	Kobayashi T, Nagano AJ, Nishizawa NK. Iron deficiency-inducible peptide-coding genes OsIMA1 and OsIMA2 positively regulate a major pathway of iron uptake and translocation in rice [J]. J Exp Bot, 2021, 72(6): 2196-2211.
[27]	Nakanishi H, Ogawa I, Ishimaru Y, et al. Iron deficiency enhances cadmium uptake and translocation mediated by the Fe²⁺ transporters OsIRT1 and OsIRT2 in rice [J]. Soil Sci Plant Nutr, 2006, 52(4): 464-469.
[28]	彭凤, 路承凯, 梁岗. OsIMA1增强水稻对镉逆境的适应性 [J]. 广西植物, 2023, 43(6): 1097-1104.
	Peng F, Lu CK, Liang G. OsIMA1 enhances tolerance to cadmium stress in rice [J]. Guihaia, 2023, 43(6): 1097-1104.
[29]	Li ZM, Liang Y, Hu HW, et al. Speciation, transportation, and pathways of cadmium in soil-rice systems: a review on the environmental implications and remediation approaches for food safety [J]. Environ Int, 2021, 156: 106749.
[30]	Sims HI, Chirn GW, Marr MT II. Single nucleotide in the MTF-1 binding site can determine metal-specific transcription activation [J]. Proc Natl Acad Sci U S A, 2012, 109(41): 16516-16521.