Populus × canescens AHL17 Negatively Regulates Tolerance to Cadmium in Arabidopsis thaliana

doi:10.13560/j.cnki.biotech.bull.1985.2025-0074

Abstract

Abstract:

Objective We delved into the molecular mechanisms by which AHL17 (AT - hook motif nuclear - localized proteins, AHLs) in Populus×canescens regulates the plant’s response to cadmium stress. The findings of this research offered a reference for the breeding of stress-resistant varieties and the application of ecological restoration. Method Wild-type (WT), vector control (VC), and Arabidopsis thaliana overexpressing PcAHL17 (PcAHL17-OE1, PcAHL17-OE, and PcAHL17-OE3) were used as experimental materials, the mechanism of PcAHL17 regulating plant response to cadmium stress was studied. Result After cadmium treatment, the expressions of PcAHL17 in the roots, stems, and leaves of Populus×canescens changed. The germination rate and root length of the overexpressed lines showed a significant decrease compared to those of WT and VC. The activity and transcription level of antioxidant enzymes in the overexpressing strains both increased, but still were lower than those in WT and VC. The degree of membrane damage in the overexpressing strains was significantly higher than that in WT and VC, and the accumulation of Cd²⁺and H₂O₂ in roots, as well as the influx of Cd²⁺, were significantly higher than those in WT and VC. Overexpressed strains had lower chlorophyll content, chlorophyll fluorescence parameters, and photosynthetic rate than WT and VC. Conclusion The overexpression of PcAHL17 gene may negatively regulate tolerance of transgenic Arabidopsis thaliana to cadmium.

Key words: Populus × canescens, cadmium stress, AT-hook motif nuclear localized proteins, PcAHL17, ion homeostasis, reactive oxygen species

FENG Bing, YAN Cai-xia, LIU Yi, DONG Kai-yue, ZHAO Nan, ZHAO Rui, CHEN Shao-liang. Populus × canescens AHL17 Negatively Regulates Tolerance to Cadmium in Arabidopsis thaliana[J]. Biotechnology Bulletin, 2025, 41(6): 269-283.

Figures/Tables 15

Table 1 Primers used in this study

引物名称 Primer name	上游引物 Forward primer （5'-3'）	下游引物 Reverse primer （5'-3'）
PcUBQ	AGACCTACACCAAGCCCAAGAAGA	CCAGCACCGCACTCAGCATTAG
AtACTIN2	GGTAACATTGTGCTCAGTGGTGG	AACGACCTTAATCTTCATGCTGC
AtSOD1	AGGAAACATCACTGTTGGAGAT	GAGTTTGGTCCAGTAAGAGGAA
AtPER1	CGTGCCCTTCATATTGTTGG	GACGCCATCAACAACGAGTC
AtCAT3	CTTGTGGTTCCTGGAATCTACT	AGGATCAAACTTTGAGGGGTAG
PcAHL17	GGGGCCCGGGGTCGACATGAAAGGTGAATATGCAGAACATC	CCATGGTACCGGATCCAAAAGGCGGTGGTGGTGG
PcAHL17-RT-qPCR	CAAACCCAAACCACCCGTTAT	TGTTGCCGATGGCTGACG

Fig. 1 Sequence analysis of PcAHL17 protein in Populus × canescensA: Multiple sequence alignment of AHL17 amino acid sequences between Populus×canescens and other species. B: Construction of evolutionary tree. Pc: Populus × canescens;Pa: Populus alba; Pt: Populus trichocarpa; Pn: Populus nigra; Ss: Salix suchowensis; Sp: Salix purpurea; Me: Manihot esculenta; Gm: Glycine max; At: Arabidopsis thaliana; Nt: Nicotiana tabacum; Gh: Gossypium hirsutum; Hb: Hevea brasiliensis; Tc: Theobroma cacao; Cs: Camelina sativa

Fig. 2 Changes of PcAHL17 gene expression in the roots, stems and leaves of Populus × canescens under cadmium stressThe data are the means of three repeated experimental results, with error bars indicating the standard deviation. Different lowercase letters denote significant differences (P<0.05), the same below

Fig. 3 DNA identification and fluorescence quantitative PCR identification of A. thaliana transformed by PcAHL17 in Populus×canescensA: DNA detection of T2 genaration. B: Expression of PcAHL17 gene in wild-type, vector control, overexpressing A. thaliana, AtACTIN2 was used as internal control gene

Fig. 4 Subcellular localization analysis of PcAHL17PcAHL17-GFP co-localized with the PYR1-mCherry. Scale bar =10 μm

Fig. 5 Germination rates of A. thaliana on the media containing different concentrations of CdCl2A: Effects of CdCl2 on seed germination. B: Statistical analysis of the seed germination rate

Fig. 6 Effects of CdCl2 on the growth of root length of A.thalianaA: Effects of CdCl2 on the growth of root length. B: Analysis of root growth. The white lines indicate the growth position of Arabidopsis roots

Fig. 7 Growth state of A. thaliana after cadmium treatment

Fig. 8 Effects of CdCl2 treatment on the relative electrical conductivity of different A. thaliana lines

Fig. 9 Fluorescence intensity of H2O2 in the root-tip cells of A. thaliana after CdCl2 treatment

Fig. 10 Analysis of Cd2+ content in A. thaliana under cadmium stressA: Cd2+ content in aboveground parts of A. thaliana.B:Cd2+ content in underground parts of A. thaliana.C:Steady Cd2+ flux kinetics in A. thaliana root tips. D: The fluorescence intensity of Cd2+ in the root-tip cells of A. thaliana under CdCl2 treatment

Fig. 11 Expressions of antioxidant enzyme gene and detection of protein activity in different A. thaliana lines after CdCl2 treatment

Fig. 12 Chlorophyll fluorescence parameters in A. thaliana lines under CdCl2 treatment

Fig. 13 Chlorophyll content in A.thaliana under CdCl2 treatment

Fig. 14 Photosynthetic parameters of A.thaliana under CdCl2 treatment

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