The AP2/ERF GmERF113 Positively Regulates the Drought Response by Activating GmPR10-1 in Soybean

Abstract

Ethylene response factors (ERFs) are involved in biotic and abiotic stress; however, the drought resistance mechanisms of many ERFs in soybeans have not been resolved. Previously, we proved that GmERF113 enhances resistance to the pathogen Phytophthora sojae in soybean. Here, we determined that GmERF113 is induced by 20% PEG-6000. Compared to the wild-type plants, soybean plants overexpressing GmERF113 (GmERF113-OE) displayed increased drought tolerance which was characterized by milder leaf wilting, less water loss from detached leaves, smaller stomatal aperture, lower Malondialdehyde (MDA) content, increased proline accumulation, and higher Superoxide dismutase (SOD) and Peroxidase (POD) activities under drought stress, whereas plants with GmERF113 silenced through RNA interference were the opposite. Chromatin immunoprecipitation and dual effector-reporter assays showed that GmERF113 binds to the GCC-box in the GmPR10-1 promoter, activating GmPR10-1 expression directly. Overexpressing GmPR10-1 improved drought resistance in the composite soybean plants with transgenic hairy roots. RNA-seq analysis revealed that GmERF113 downregulates abscisic acid 8'-hydroxylase 3 (GmABA8'-OH 3) and upregulates various drought-related genes. Overexpressing GmERF113 and GmPR10-1 increased the abscisic acid (ABA) content and reduced the expression of GmABA8'-OH3 in transgenic soybean plants and hairy roots, respectively. These results reveal that the GmERF113-GmPR10-1 pathway improves drought resistance and affects the ABA content in soybean, providing a theoretical basis for the molecular breeding of drought-tolerant soybean.

Keywords: ABA; GmERF113; GmPR10-1; drought tolerance; soybean.

Figure 1

GmERF113 is induced by drought. Expression patterns of GmERF113 of soybean seedlings at the V2 stage were examined with 20% PEG-6000 treatment. The first trifoliate leaf samples were collected at 0, 1, 3, 6, 9, 12, and 24 h. The reference soybean gene GmActin4 (GenBank accession no. AF049106) and GmTubulin4 (GenBank accession no. XM_003554060) were used as internal controls to normalize the data. The experiment was performed on three biological replicates, each with three technical replicates, and was statistically analyzed using Student’s t-test (** P < 0.01). The bars indicate the standard deviation of the mean.

Figure 2

GmERF113 is a positive regulator of plant response to drought. (A) The relative water loss of the detached leaves in wild-type (WT) plants, GmERF113-OE, and GmERF113-RNAi transgenic soybean plants. The second trifoliate leaves of soybean seedlings at the V3 stage were cut which were weighed every hour. The experiment was carried out in three biological replicates, each with three technical replicates. The bars indicate the standard deviation of the mean. (B) Phenotypes of WT, GmERF113-OE, and GmERF113-RNAi soybean plants that were exposed to drought stress for 0, 5, and 7 days and re-watered for 2 days. (C,D) Stomatal apertures of the second trifoliate leaves of WT, GmERF113-OE, and GmERF113-RNAi soybean plants that were treated with drought for 0, 5, or 7 d and re-watered for 2 days (C), and statistical analysis of the stomatal apertures of each line (bars = 20 μm) (D). The experiment was performed on three biological replicates, each with three technical replicates, and was statistically analyzed using Student’s t-test (** P < 0.01). The bars indicate the standard deviation of the mean.

Figure 3

Overexpression or silencing of GmERF113 alters drought-related parameters that are reflective in soybean plants. (A–D) Malondialdehyde content (MDA; A), proline content (B), and superoxide dismutase (SOD; C) and peroxidase (POD; D) activities of WT, GmERF113-OE, and GmERF113-RNAi soybean plants that were exposed to drought stress for 0, 5, and 7 days and re-watered for 2 days. The experiment was performed on three biological replicates, each with three technical replicates, and was statistically analyzed using Student’s t-test (* P < 0.05, ** P < 0.01). The bars indicate the standard deviation of the mean.

Figure 4

GmPR10-1 was identified as a target of GmERF113. (A,B) ChIP analysis of GmERF113 binding to the promoter region of GmPR10-1. Chromatin from transgenic soybean plants expressing GmERF113-myc or the WT were immunoprecipitated with or without the anti-myc antibody. The precipitated chromatin fragments were analyzed with qPCR using a pair of specific ChIP-qPCR primers, which amplifies the GCC-box (highlighted in yellow) upstream of GmPR10-1, as indicated. One-tenth of the input (without antibody precipitation) of chromatin was analyzed and used as a control. A total of three biological replicates, each with three technical replicates, were averaged and statistically analyzed using Student’s t-test (** P < 0.01). The bars indicate the standard deviation of the mean. (C) Schematic representation of the reporter and effector constructs that were used in the dual luciferase assays. (D) The dual luciferase assays in tobacco leaves showing that GmERF113 activates the expression of GmPR10-1 by combining the GmPR10-1 promoter. Representative pictures were taken. (E) LUC/REN activity detection to verify GmERF113 activates the expression of GmPR10-1. The combination of the reporter construct (pGmPR10-1: LUC) and the blank effector construct [p35S] were used as the control. These experiments were performed on three biological replicates, each with three technical replicates, and were statistically analyzed using Student’s t-test (** P < 0.01). The bars indicate the standard deviation of the mean. “•” were scattered dots which can reflect the distribution of the data.

Figure 5

GmPR10-1 improves the drought tolerance in composite soybean plants with transgenic hairy roots. (A) The relative water loss of detached leaves (the second trifoliate leaves from the top) from EV, GmPR10-1-OE, and GmPR10-1-RNAi composite soybean plants with transgenic hairy roots. Transgenic soybean hairy roots of the same length (~10 cm) were selected, transferred to new pots (soil moisture contents in the pots were kept constant), and incubated for 3 days before the start of the drought treatment. (B) Phenotypes of EV, GmPR10-1-OE, and GmPR10-1-RNAi composite soybean plants with transgenic hairy roots that were exposed to drought stress for 0, 5, and 7 days and re-watered for 2 days. (C,D) Stomatal aperture of the second trifoliate leaves from the top of EV, GmPR10-1-OE, and GmPR10-1-RNAi composite soybean plants with transgenic hairy roots that were treated with drought for 0, 5, and 7 days and re-watered for 2 days (bars = 20 μm). (C), and statistical analysis of the stomatal apertures of each line. (D). The experiments were performed on three biological replicates, each with three technical replicates, and were statistically analyzed using Student’s t-test (* P < 0.05, ** P < 0.01). The bars indicate the standard deviation of the mean.

Figure 6

Overexpression or silencing of GmPR10-1 alters the drought-related parameters in composite soybean plants. (A–D) Malondialdehyde content (MDA; A), proline content (B), and superoxide dismutase (SOD; C) and peroxidase (POD; D) activities of EV, GmPR10-1-OE, and GmPR10-1-RNAi composite soybean plants with transgenic hairy roots that were exposed to drought stress for 0, 5, and 7 days and re-watered for 2 days. The second trifoliate from the top was chosen to measure physiological indicators. These experiments were performed on three biological replicates, each with three technical replicates, and were statistically analyzed using Student’s t-test (* P < 0.05, ** P < 0.01). The bars indicate the standard deviation of the mean.

Figure 7

Transcriptomic analysis of gene expression profiles in response to GmERF113 overexpression. (A) Volcano plots of significantly differentially expressed genes in GmERF113-OE vs. wild-type soybean plants after the RNA-seq analysis. (B) Heat map of significantly differentially expressed genes between the WT and GmERF113-OE transgenic soybean plants, as determined using an RNA-seq analysis. Using a false discovery rate < 0.05 and a fold change ≥1 as the screening criteria, a total of 360 differentially expressed genes (DEGs) were identified. The scale bar indicates the fold changes (log₂ values). (C) Gene Ontology functional classification of the differentially expressed genes. The differentially expressed genes were placed into the three main GO categories: biological process, cellular component, and molecular function.

Figure 8

GmERF113 and GmPR10-1 function in ABA responses. (A) The ABA contents of GmERF113 transgenic soybean plants under normal conditions and drought treatment. (B) The relative expression of three ABA-related genes that were identified by RNA-seq analysis in GmERF113 transgenic soybean plants. (C) The ABA contents of GmPR10-1 transgenic soybean hairy roots under normal conditions and drought treatment. (D) The relative expression of three ABA-related genes that were identified by RNA-seq analysis in GmPR10-1 transgenic soybean hairy roots. The reference soybean gene GmActin4 and GmTubulin4 were used as an internal control to normalize the data. These experiments were performed on three biological replicates, each with three technical replicates, and were statistically analyzed using Student’s t-test (* P < 0.05, ** P < 0.01). The bars indicate the standard deviation of the mean.

Figure 9

A molecular model of the GmERF113 in the soybean response to drought stress. When soybean plants are subjected to drought stress, GmERF113 is rapidly activated and transcribed, and then GmERF113 activates the expression of PR10-1 by binding to the GCC-box in the PR10-1 promoter, thereby enhancing the drought resistance of soybean plants. In addition, GmERF113 promotes the expression of two genes which are in the ABA signaling pathway, GmPP2C37 and GmRGLG1, also both GmERF113 and GmPR10 decrease the expression of an ABA catabolic gene, GmABA8′-OH 3, thereby the ABA content in the plants can be increased. The increased ABA level further promotes the closure of stomata and thus improves the drought tolerance of soybean plants.