Heterologous Expression of Arabidopsis AtARA6 in Soybean Enhances Salt Tolerance

Abstract

Salt damage is an important abiotic stress affecting the agronomic traits of soybean. Soybeans rapidly sense and transmit adverse signals when salt-damaged, inducing a set of response mechanisms to resist salt stress. AtARA6 encodes a small GTPase, which plays an important role in Arabidopsis vesicle transport and salt tolerance. In this study, we transformed the Arabidopsis gene AtARA6 into the cultivated soybean Shen Nong 9 (SN9). To investigate the salt tolerance pathways affected by AtARA6 in soybean, we performed transcriptome sequencing using transgenic soybean and wild-type (SN9) under salt treatment and water treatment. Our results suggest that AtARA6 is involved in the regulation of soybean SNARE complexes in the vesicle transport pathway, which may directly strengthen salt tolerance. In addition, we comprehensively analyzed the RNA-seq data of transgenic soybean and SN9 under different treatments and obtained 935 DEGs. GO analysis showed that these DEGs were significantly enriched in transcription factor activity, sequence-specific DNA binding, and the inositol catabolic process. Three salt-responsive negative regulator transcription factors, namely MYC2, WRKY6, and WRKY86, were found to be significantly downregulated after salt treatment in transgenic soybeans. Moreover, four genes encoding inositol oxygenase were significantly enriched in the inositol catabolic process pathway, which could improve the salt tolerance of transgenic soybeans by reducing their reactive oxygen species content. These are unique salt tolerance effects produced by transgenic soybeans. Our results provide basic insights into the function of AtARA6 in soybeans and its role in abiotic stress processes in plants.

Keywords: AtARA6; RAB GTPase; RAB5; SNARE pathway; salt tolerance; sybean.

FIGURE 1

Genetic transformation of AtARA6. Agrobacterium tumefaciens EHA101-mediated genetic transformation of soybean, including exosome infiltration, clump shoot induction, clump shoot screening, and rooting culture.

FIGURE 2

Confirmation of AtARA6 transgenesis. (A,B) Positive transgenic soybean progeny Lines1-6: 404 bp of GmActin was used to detect plant genomic DNA, AtARA6 445 bp, Bar 443=bp (M, Trans2K Plus II DNA marker; +, AtARA6 expression vector plasmid DNA; −, WT genomic DNA). (C) LibertyLink test strips for genetically transformed soybeans. (D) RT-PCR analysis of AtARA6 gene expression at different times under 200 mM NaCl treatment. (E) qRT-PCR analysis of the relative expression of AtARA6 in different durations under salt treatment.

FIGURE 3

Salt treatment phenotypes of transgenic soybean and WT during germination. (A) Phenotypes of transgenic soybean Lines1-3 and WT treated with 200 mM NaCl, 100 mM NaCl, or water for 7 days at the germination stage. (B) Root length phenotypes of WT and transgenic soybean under salt stress during germination (p < 0.05).

FIGURE 4

Salt treatment phenotypes of transgenic soybean and WT at emergence stages. (A–C) Phenotypes of transgenic soybean Lines1-3 and WT (left) treated with 200 mM NaCl at emergence stages for 1, 2, and 3 weeks.

FIGURE 5

Determination of physiological parameters of transgenic soybean and WT treated with salt stress. (A) Superoxide dismutase (SOD) activity. (B) Peroxidase (POD) activity. (C) Catalase (CAT) activity. (D) Proline content in leaves (µg/g). (E) Malondialdehyde content in leaves (µmol/g). (F) Chlorophyll content of leaves after salt treatment (mg/g). All values were measured after 21 days of salt treatment. Statistical analysis was performed using one-way ANOVA, *p < 0.05, **p < 0.01, ***p < 0.001, p > 0.05 indicates the difference between transgenic line and WT was not significant.

FIGURE 6

Comparison of DEGs between transgenic positive lines and SN9 under salt treatment and water treatment.

FIGURE 7

qRT_PCR analysis of homologous genes of SYP121 and VAMP727. The homologous genes of SYP121, Glyma.02G069700 and Glyma.16G151200. The homologous genes of VAMP727, 01G179300 and Glyma.11G062900. They were significantly up-regulated under in transgenic soybean after salt treatment. Three biological replicates were used. Data was calculated using the 2^−ΔΔCT method, Statistical analysis was performed using ANOVA (p < 0.05), *p < 0.05, **p < 0.01, and ***p < 0.001.

FIGURE 8

RNA-seq reveals the expression levels of DEGs enriched in the Rho/Ras/GTPase regulator/GTPase activity pathways under different treatments.

FIGURE 9

GO enrichment analysis of 935 DEGs including BP, CC and MF. p-values < 0.01 for all GOs. Transcription factor activity, sequence-specific DNA binding (GO:0003700, p = 5.2e-11, MF) and inositol catabolic process (GO:0019310, p = 4.0e-10, BP) are the most significant.

FIGURE 10

Kyoto Encyclopedia of Genes and Genomes analysis of up-regulation and down-regulation of DEGs. (A,B) KEGG analysis on up-regulated (left) and down-regulated (right) DEGs under salt stress in transgenic soybean compared to WT.

FIGURE 11

Salt tolerance pathway of AtARA6 transgenic soybean.