Screening and Functional Analysis of a Triphenyl Phosphate-tolerant Mutant in Arabidopsis thaliana

doi:10.13560/j.cnki.biotech.bull.1985.2025-1096

Abstract

Abstract:

Objective Triphenyl phosphate (TPHP) is a common environmental organic pollutant that poses threats to both human health and ecological security. Elucidation of the molecular mechanism underlying Arabidopsis thaliana tolerance to TPHP toxicity provides a reference for future phytoremediation strategies for TPHP-contaminated soil. Method Wild-type A. thaliana (Col-0) was used as the experimental material for TPHP exposure treatment and phenotypic analysis of root length, fresh weight, and chlorophyll content. A mutant with tolerance to TPHP toxicity was identified from an A. thaliana mutant library based on significantly altered phenotype. Map-based cloning and whole-genome resequencing analyses were then employed to identify the candidate mutant gene, with validation via exogenous hormone application. Finally, molecular docking, overexpression lines, and mutant analyses were conducted to preliminarily investigate the potential molecular mechanism of TPHP tolerance in A. thaliana. Result The root length, fresh weight, and chlorophyll content in wild-type Col-0 seedlings reduced significantly after treatment with 2 mg/L TPHP. A TPHP-tolerant mutant, designated ems-29d (ems-29 dwarf), was isolated based on phenotypic screening. This mutant was dwarf with dark green and wrinkled leaves. Resequencing analysis revealed the key gene DWF4 involved in brassinosteroids synthesis in mutants, containing C1179T single-nucleotide variation, resulting in a leucine-to-phenylalanine substitution at position 306 (L306F) in the DWF4 protein. Exogenous application of 2,4-epibrassinolide (eBL) partially restored the growth inhibition phenotype of ems-29d. Molecular docking simulations revealed a lower binding energy between TPHP and the mutant DWF4^L306F compared to that with the wild-type DWF4. However, the overexpression of DWF4^L306F in transgenic lines did not enhance TPHP tolerance, indicating that the resistance was not target-site mediated. Finally, following TPHP treatment, the suppression of chlorophyll content was significantly milder in the DWF4 T-DNA insertion mutant dwf4 than in the wild-type, demonstrating that impairment of BR biosynthesis markedly enhances A. thaliana tolerance to TPHP. Conclusion The mutation in DWF4 in ems-29d disrupts brassinosteroids synthesis, thereby increasing plant tolerance to TPHP toxicity.

Key words: Arabidopsis thaliana, triphenyl phosphate, map-based cloning, DWF4, brassinosteroid, molecular docking, overexpression line, phytoremediation

FAN Fan, LI Meng-jiao, YANG Xian-peng, CUI Li-li. Screening and Functional Analysis of a Triphenyl Phosphate-tolerant Mutant in Arabidopsis thaliana[J]. Biotechnology Bulletin, 2026, 42(7): 226-235.

Figures/Tables 9

Table 1 Fine mapping of the gene controlling the dwarf phenotype of ems-29d

Sample	3‒11.22 M	3‒14.95 M	3‒17.36 M	3‒17.857 M	3‒18.89 M	3‒19.131 M	3‒19.679 M	3‒20.575 M	3‒20.8 M
1	A	A	A	A	A	A	A	A	H
2	A	A	A	A	A	A	H	H	H
3	A	A	A	A	A	A	H	H	H
7	A	A	A	A	A	H	H	H	H
8	H	H	H	H	A	H	H	H	H
12	H	H	H	H	A	A	A	A	A
16	H	H	H	H	A	H	H	H	H
18	A	A	A	A	A	A	H	H	H
19	B	B	H	H	A	A	A	A	A
21	A	A	A	A	A	A	A	H	H
24	H	H	H	H	A	A	A	A	A
27	H	H	H	H	A	A	A	A	A
32	H	H	H	A	A	A	A	A	A
33	A	A	A	A	A	A	A	A	H
36	H	H	A	A	A	A	H	H	H

Fig. 1 Wild-type Col-0 and Ler-0 of A. thaliana grown on the medium containing TPHP for 7 dA: Phenotype. B: Root length. C: Fresh weight per plant. D: Chlorophyll content. The different lowercase letters indicate significant differences among samples (P<0.05). The same below

Fig. 2 Wild-type Col-0 and ems-29 mutant of A. thaliana after 7 d of TPHP treatmentA: Phenotype. B: Root length. C: Fresh weight per plant. D: Chlorophyll content. * indicates the ems-29d mutant

Fig. 3 Preliminary mapping of the DWF4 gene in the ems-29d mutant* indicates the Col-0 band, moving to the right are the ems-29 mutant band and the Ler-0 band, respectively

Fig. 4 Analysis of mutation sites of DWF4 gene in ems-29dA: Schematic representation of DWF4 gene structure. B: Mutation site on DWF4 gene in ems-29d

Fig. 5 Wild-type Col-0 and ems-29d mutant of A. thaliana after 4 d of treatment with 0.1 μmol/L eBLA: Phenotype. B: Root length. C: Fresh weight per plant

Fig. 6 Interaction analysis of TPHP with DWF4 (A) and DWF4L306F (B)

Fig. 7 Wild-type Col-0 and DWF4L306F transgenic lines of A. thaliana after 7 d of TPHP treatmentA: Molecular identification of DWF4L306F transgenic lines. B-E: Phenotypes (B), root length (C), fresh weight per plant (D) and chlorophyll content (E) of wild-type Col-0 and the DWF4L306F OE transgenic lines of A. thaliana after treatment with 2 mg/L TPHP

Fig. 8 Wild-type Col-0 and dwf4 mutants of A. thaliana grown for 7 d on media containing different concentrations TPHPA: Phenotype. B: Chlorophyll content. C: Chlorophyll content inhibition rate. *, **, and *** indicate significant differences at P<0.05, P<0.01, and P<0.001 levels, respectively

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