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Transcriptome analysis reveals differing response and tolerance mechanism of EPSPS and GAT genes among transgenic soybeans

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Abstract

Background

Glyphosate is a broad-spectrum, non-selective systemic herbicide. Introduction of glyphosate tolerance genes such as EPSPS or detoxification genes such as GAT can confer glyphosate tolerance on plants. Our previous study revealed that co-expression of EPSPS and GAT genes conferred higher glyphosate tolerance without “yellow flashing”. However, the plant response to glyphosate at the transcriptional level was not investigated.

Methods and results

To investigate the glyphosate tolerance mechanism, RNA-seq was conducted using four soybean genotypes, including two non-transgenic (NT) soybeans, ZH10 and MD12, and two GM soybeans, HJ698 and ZH10-6. Differentially expressed genes (DEGs) were identified in these soybeans before and after glyphosate treatment. Similar response to glyphosate in the two NT soybeans and the different effects of glyphosate on the two GM soybeans were identified. As treatment time was prolonged, the expression level of some DEGs involved in shikimate biosynthetic pathway and herbicide targeted cross-pathways was increased or declined continuously in NT soybeans, and altered slightly in HJ698. However, the expression level of some DEGs was altered in ZH10-6 at 12 hpt, while similar expression level of some DEGs involved in shikimate biosynthetic pathway and herbicide targeted cross-pathways was observed in ZH10-6 at 0 hpt and 72 hpt. These observations likely explain the higher glyphosate tolerance in ZH10-6 than in HJ698 and NT soybeans.

Conclusions

These results suggested that GAT and EPSPS genes together play a crucial role in response to glyphosate, the GAT gene may work at the early stage of glyphosate exposure, whereas the EPSPS gene may be activated after the uptake of glyphosate by plants. These findings will provide valuable insight for the molecular basis underlying glyphosate tolerance or glyphosate detoxication.

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Data availability

Data pertaining to the study have been included in this manuscript and supplementary materials.

Abbreviations

COG:

Clusters of Orthologous Groups

DEGs:

Differentially expressed genes

EPSPS:

5-Enolypyruvylshikimate-3-phosphate synthase

FDR:

False discovery rate

FPKM:

Fragments per kilobase of transcript per million fragments mapped

GAT:

Glyphosate N-acetyltransferase

GM:

Genetically modified

GO:

Gene ontology

GOX:

Glyphosate oxidoreductase

KEGG:

Kyoto encyclopedia of genes and genomes databases

NT:

No-transgenic

RNA-Seq:

Transcriptome sequencing

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Acknowledgements

This work was supported by the National Key R&D Program of China, Project of Sino-Uruguayan Joint Laboratory (2018YFE0116900); National Transgenic Major Program of China (2016ZX08004001); Young Science Fund Project of Jiangxi (20192ACBL21025) and the Agricultural Science and Technology Innovation Program (ASTIP) of Chinese Academy of Agricultural Sciences.

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Author notes

  1. Bing-Fu Guo and Hui-Long Hong have contributed equally to this work.

Authors and Affiliations

  1. The National Key Facility for Crop Gene Resources and Genetic Improvement (NFCRI) and MOA Key Lab of Soybean Biology (Beijing), Institute of Crop Science, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Haidian District, Beijing, 100081, People’s Republic of China

    Bing-Fu Guo, Hui-Long Hong, Yong Guo & Li-Juan Qiu

  2. Jiangxi Province Key Laboratory of Oilcrops Biology, Crops Research Institute of Jiangxi Academy of Agricultural Sciences, Nanchang, China

    Bing-Fu Guo & Li-Ping Sun

  3. College of Agriculture, Northeast Agricultural University, Harbin, People’s Republic of China

    Hui-Long Hong

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  1. Bing-Fu Guo
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  4. Yong Guo
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Contributions

BG, YG, HH, and LJQ conceived and designed the experiments. BG, HH, and YG performed the experiments. BG and YG analyzed the data. BG, YG and LJQ wrote the manuscript. LS revised the manuscript, and added some experimental data.

Corresponding authors

Correspondence to Yong Guo or Li-Juan Qiu.

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Conflict of interest

The authors declare that they have no conflict of interest.

Ethical approval

These soybeans used for this study were permitted for scientific research. The experimental research on soybean complied with Chinese legislation and field studies were in accordance with guidelines of Institute of Crop Science, Chinese Academy of Agricultural Sciences. No experiments involved endangered or protected species.

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Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 Supplementary Table S1. Primers information for qRT-PCR (XLSX 11 kb)

11033_2021_6742_MOESM2_ESM.xlsx

Supplementary file2 Supplementary Table S2. Sequencing information and clean reads mapped to soybean genome (XLSX 11 kb)

Supplementary file3 Supplementary Table S3. All DEGs identified in four soybeans (XLSX 3876 kb)

Supplementary file4 Supplementary Table S4. Validation of 15 differentially expressed genes by qRT-PCR (XLSX 12 kb)

11033_2021_6742_MOESM5_ESM.tif

Supplementary file5 Supplementary Fig S1. Overview of RNA-seq quality. Schematic representation of genome-wide distribution of mapped reads coverage in different chromosomes (a), and proportions of mapped reads in the regions of exon, intron and intergenic regions (b) (TIF 361 kb)

11033_2021_6742_MOESM6_ESM.tif

Supplementary file6 Supplementary Fig S2. Correlation analysis of replicated samples of ZH10-6_12hpt (a) and HJ698_12hpt (b) (TIF 195 kb)

11033_2021_6742_MOESM7_ESM.tif

Supplementary file7 Supplementary Fig S3. GO enrichment analysis of DEGs in ZH10_12hpt (a), ZH10_72hpt (b), MD12_12hpt (c) and MD12_72hpt (d) response to glyphosate (TIF 1264 kb)

11033_2021_6742_MOESM8_ESM.tif

Supplementary file8 Supplementary Fig S4. GO enrichment analysis of DEGs in HJ698_12hpt (a), HJ698_72hpt (b), ZH10-6_12hpt (c) and ZH10-6_72 hpt (d) response to glyphosate (TIF 1611 kb)

11033_2021_6742_MOESM9_ESM.tif

Supplementary file9 Supplementary Fig S5. KEGG enrichment analysis of DEGs in ZH10_12hpt (a), ZH10_72hpt (b), MD12_12hpt (c) and MD12_72hpt (d) response to glyphosate. The Y-axis represents the pathway name, and the X-axis represents the enrichment factor. The sizes of dots indicate numbers of DEGs in the pathway, and the colors of the dots correspond to q-value (corrected P-value) range (TIF 1116 kb)

11033_2021_6742_MOESM10_ESM.tif

Supplementary file10 Supplementary Fig S6. KEGG enrichment analysis of DEGs in HJ698_12hpt (a), HJ698_72hpt (b), ZH10-6_12hpt (c) and ZH10-6_72 hpt (d) response to glyphosate. The Y-axis represents the pathway name and the X-axis represents the enrichment factor. The sizes of dots indicate numbers of DEGs in the pathway, and the colors of the dots correspond to q-value (corrected P-value) range (TIF 1023 kb)

11033_2021_6742_MOESM11_ESM.tif

Supplementary file11 Supplementary Fig S7. Heatmap of FPKM values of DEGs involved in auxin biosynthesis pathways. Genes involved in auxin biosynthesis pathways were selected by homolog identification. Heatmap was conducted by R Project. Blue color indicates low expression level and red color indicates high expression level (TIF 261 kb)

11033_2021_6742_MOESM12_ESM.tif

Supplementary file12 Supplementary Fig S8. Heatmap of FPKM values of DEGs involved in cellulose and microtubule system. Genes involved in cellulose and microtubule system was selected by homolog identification. Heatmap analysis was conducted by R Project. Blue color indicated low expression level while red color indicated high expression level (TIF 324 kb)

11033_2021_6742_MOESM13_ESM.tif

Supplementary file13 Supplementary Fig S9. Expression analysis of genes involved in shikimate pathway differentially expressed in ZH10-6 and HJ698 (TIF 69 kb)

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Guo, BF., Hong, HL., Sun, LP. et al. Transcriptome analysis reveals differing response and tolerance mechanism of EPSPS and GAT genes among transgenic soybeans. Mol Biol Rep 48, 7351–7360 (2021). https://doi.org/10.1007/s11033-021-06742-x

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