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. 2023 Mar 18;192(2):1099–1114. doi: 10.1093/plphys/kiad170

Transcription factors GmERF1 and GmWRKY6 synergistically regulate low phosphorus tolerance in soybean

Ruiyang Wang 1, Xiaoqian Liu 2, Hongqing Zhu 3, Yuming Yang 4, Ruifan Cui 5, Yukun Fan 6, Xuhao Zhai 7, Yifei Yang 8, Shanshan Zhang 9, Jinyu Zhang 10, Dandan Hu 11,✉,b,c, Dan Zhang 12,✉
PMCID: PMC10231356  PMID: 36932694

Abstract

Soybean (Glycine max) is a major grain and oil crop worldwide, but low phosphorus (LP) in soil severely limits the development of soybean production. Dissecting the regulatory mechanism of the phosphorus (P) response is crucial for improving the P use efficiency of soybean. Here, we identified a transcription factor, GmERF1 (ethylene response factor 1), that is mainly expressed in soybean root and localized in the nucleus. Its expression is induced by LP stress and differs substantially in extreme genotypes. The genomic sequences of 559 soybean accessions suggested that the allelic variation of GmERF1 has undergone artificial selection, and its haplotype is significantly related to LP tolerance. GmERF1 knockout or RNA interference resulted in significant increases in root and P uptake efficiency traits, while the overexpression of GmERF1 produced an LP-sensitive phenotype and affected the expression of 6 LP stress-related genes. In addition, GmERF1 directly interacted with GmWRKY6 to inhibit transcription of GmPT5 (phosphate transporter 5), GmPT7, and GmPT8, which affects plant P uptake and use efficiency under LP stress. Taken together, our results show that GmERF1 can affect root development by regulating hormone levels, thus promoting P absorption in soybean, and provide a better understanding of the role of GmERF1 in soybean P signal transduction. The favorable haplotypes from wild soybean will be conducive to the molecular breeding of high P use efficiency in soybean.

A phosphate starvation response mechanism mediated by GmERF1 and GmWRKY6 regulates low phosphorus tolerance in soybean.

Introduction

Phosphorus (P) is one of the essential macroelements in plants. As a key structural component of organic compounds such as ATP, nucleic acids, phospholipids, and proteins, P is involved in multiple metabolic and biosynthetic processes required for the functioning of plant cells (Yang et al. 2021). Although P is abundant in biological systems, P in the soil is usually in the least soluble form of organic P, which cannot be acquired and utilized by plants unless mineralized to inorganic P (Schachtman et al. 1998). The lack of available P is an important factor restricting plant growth and development. Low phosphorus (LP) stress can reduce stomatal conductance and mesophyll conductance and inhibit photosynthesis (Wu et al. 2004), resulting in growth retardation, limited plant height, and reduced environmental adaptability (Abelson 1999). Understanding the mechanisms of P acquisition by plants will help us to develop P-efficient and environmentally friendly crops through the optimization of P utilization efficiency (Tian et al. 2012).

The effective uptake and transport of P by plants is necessary for the normal operation of cells. Plants have evolved a series of complex mechanisms to enhance P uptake and utilization efficiency to cope with LP stress (Liang et al. 2014), including adaptive changes in root morphology, increasing organic acid secretion, and enhancing high-affinity phosphate transporter expression, and the former is the most important way to improve P uptake efficiency. Studies in several crops, including Arabidopsis (Arabidopsis thaliana) and wheat (Triticum aestivum), have shown that plants can increase the number and length of lateral roots, root shoot ratio, root hair length, and density to enhance the uptake of soil P under LP stress (Chandrika et al. 2013; Song et al. 2016). Transcription factors (TFs) play essential roles in adapting to LP stress by modulating the expression of LP stress-responsive genes (Ding et al. 2016). These TFs include P starvation-induced TF 1 (OsPTF1) (Yi et al. 2005), MYB5 phosphate-responsive gene (OsMYB5P), and phosphate starvation response 2 (OsPHR2) (Wu and Wang 2008; Yang et al. 2018) in rice (Oryza sativa L.); MYB62 (Devaiah et al. 2009), WRKY75 (Devaiah et al. 2007a), zinc finger of Arabidopsis 6 (ZAT6) (Devaiah et al. 2007b), and helix-loop-helix type TF 32 (BHLH32) (Chen et al. 2007) in Arabidopsis; and TaZAT8 in wheat (Ding et al. 2016). Interestingly, some of the identified P response regulators belong to the ethylene response factor (ERF) family of TFs with AP2/ERF-type DNA-binding domains. Several ERF proteins have been shown to bind to GCC-box (core sequence GCCGCC) cis-regulatory elements in the promoter regions of transcriptionally regulated downstream genes (Hao et al. 2002).

As an important plant hormone, ethylene is involved in the regulation of many plant growth and development processes, such as seed germination, root growth, the flowering and fruiting of plant reproductive organs, and the response mechanism of plants in the face of adversity (Benavente and Alonso 2006). Previous studies in Arabidopsis and rice have shown that P transport from senescent tissues during plant aging is inseparable from ethylene content (Jia et al. 2011; Nagarajan et al. 2011). ERF is induced by ethylene and has the ability to bind to biotic stress resistance gene promoters (Ohme-Takagi and Shinshi 1995). ERF is a subfamily of the AP2/ERF family of macroplant-specific TFs, and its most prominent feature is the highly conserved AP2 domain at the N-terminus (Sakuma et al. 2002), which typically contains 60 to 70 amino acid residues and forms a typical 3D structure with 1 α-helix and 3 β-folds (Abiri et al. 2017). ERF with conserved DNA-binding domains plays important roles in tolerance to a variety of abiotic stresses, such as salt, drought, heat, cold, and root development (Licausi et al. 2013; Debbarma et al. 2019). For example, ERF109 regulates auxin content by directly binding to the promoter of YUC2 to regulate the structure of the root system in A. thaliana (Cai et al. 2014). MITOGENACTIVATED PROTEIN KINASE 14 (MPK14) interacts with ERF13 to regulate auxin signaling, thereby regulating the growth of lateral roots (Lv et al. 2021). The overexpression of OsAP37 (OsERF3) promotes drought tolerance in rice during the nutrient and production stages (Oh et al. 2009). Several ERF TFs have been found to be associated with LP stress responses, including AtERF070, which affects P accumulation and the development of primary roots and lateral roots in A. thaliana (Ramaiah et al. 2014), and 22 ERF genes with increased expression in response to LP stress were identified in physic nut (Jatropha curcas L.) (Tang et al. 2016). These findings indicate that ERF TFs are associated with the adaptation of plants to LP stress.

WRKYs are important TFs in plant signaling networks, which are involved in a variety of physiological processes and are associated with plant tolerance to biotic and abiotic stresses (Ulker and Somssich 2004; Rushton et al. 2010; Viana et al. 2018). They interact with DNA-binding and non–DNA-binding proteins and act as activators and repressors of gene expression, depending on their interacting partners and target genes. A number of WRKY TFs have been shown to play important roles in the regulation of LP stress responses (Gu et al. 2016). In Arabidopsis, AtWRKY45 positively regulates the expression of AtPHT1;1 by directly binding to the W-box (core bases, TTGAC[CT]) in the promoter of AtPHT1;1 in response to LP availability (Wang et al. 2014), and AtWRKY75 positively regulates AtPHT1;1 and AtPHT1;4 as transcriptional activators, mainly under P-deficient conditions (Devaiah et al. 2007a). AtWRKY42 promotes AtPHT1;1 expression and interacts with AtWRKY6 to negatively regulate PHOSPHATE 1 (AtPHO1) expression by binding to the AtPHO1 promoter under P-replete conditions (Su et al. 2015). In rice, OsWRKY21 and OsWRKY108 directly and redundantly upregulate the expression of OsPHT1;1 (Zhang et al. 2021). This evidence suggests that WRKY TF family members may be closely related to plant adaptation to LP stress, including P sensing and signal transduction, P absorption, and intracellular P homeostasis.

Soybean (Glycine max) is one of the most important grain and oil crops worldwide (Yuan et al. 2017). Its growth, development, and productivity are seriously affected by P availability in many areas worldwide. To excavate the genes related to LP stress, we selected LP-resistant and LP-sensitive soybean cultivars for LP stress analysis and transcriptome sequencing analysis and obtained multiple nodes of the coexpression network of P efficiency-related genes, including the AP2/ERF TF GmERF1. The ERF gene regulates plant root development. For example, AtERF1 inhibits root growth by affecting auxin and ethylene biosynthesis in Arabidopsis (Mao et al. 2016), so we selected the homologous gene GmERF1 as a candidate gene for further analysis. In this study, we found that GmERF1 was expressed mainly in roots and was a P-sensitive gene. Interference with GmERF1 in soybean caused greater LP tolerance by activating LP stress-induced genes and modulating root architecture. Furthermore, yeast 2-hybrid (Y2H) analysis indicated that GmERF1 interacts with GmWRKY6 in a joint role. These results demonstrate that GmERF1 is an important negative regulator of the LP stress response and that GmERF1 and GmWRKY6 synergistically regulate LP tolerance in soybean and provide an elite haplotype for the molecular breeding of P-efficient soybean.

Results

Expression pattern of GmERF1 under different phosphorus supplies

Our previous transcriptome sequencing studies found that the expression of a TF with 1 AP2/ERF domain (named GmERF1) was substantially varied in varieties with different levels of LP stress resistance after LP treatment (Zhang et al. 2017). The expression level of GmERF1 in the roots of the sensitive soybean varieties B18, GDW022, and Bogao was much higher than that of the corresponding 3 resistant varieties B20, GDW006, and Nannong94-156 (Fig. 1A), indicating that GmERF1 might play a negative regulatory role in the response to LP stress.

Figure 1.

Figure 1.

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Localization and expression of GmERF1. A) The expression (in FPKM) of GmERF1 in different soybean varieties with different P efficiencies after LP treatment. Differences in GmERF1 expression, as determined by RNA-Seq, in 2 groups of soybean varieties with contrasting P efficiency, including the tolerant soybean genotypes GDW006, B20, and Nannong94-156 and the corresponding sensitive soybean genotypes GDW022, B18, and Bogao. B) Gene expression of GmERF1 for Williams 82 in roots, flowers, pods, leaves, and shoots. Error bars indicate Sd values from 3 independent biological replicates. C) RT-qPCR analyses of the expression of GmERF1 during LP stress treatment in Nannong94-156 (LP-tolerant genotype) and Bogao (LP-sensitive genotype). The root tissues were collected for analysis at 0 h, 3 h, 1 d, 7 d, and 14 d under NP and LP treatments. Data are presented as the mean ± Sd of 3 replicated experiments. D)GmERF1 was localized to the nucleus. GFP, green fluorescent protein; BF, bright field. Scale bars, 20 µm. E) GUS staining of the T3 generation of GmERF1 pro:GmERF1::GUS transgenic Arabidopsis plants after 7 d of NP and LP treatment. NP, normal P (500 µ M P); LP, low P (5 µ M P). F) GUS staining of GmERF pro:GmERF::GUS in NP and LP treatments in transgenic soybean hairy roots for 14 d. NP, 500 µ M P; LP, 5 µ M P. Scale bars = 500 µm.

To characterize the spatial patterns of GmERF1 expression in response to LP stress, roots, stems, leaves, flowers, and pods of Williams 82 were collected for GmERF1 transcription. The reverse transcription quantitative PCR (RT-qPCR) analysis showed that GmERF1 was expressed in all examined organs and was mainly expressed in roots at least 20 times that in the other 4 organs (Fig. 1B). Subsequently, the temporal expression patterns of GmERF1 under NP and LP conditions were evaluated by RT-qPCR using RNA samples extracted from the roots of the LP-sensitive genotype Bogao and LP-tolerant genotype Nannong94-156. As shown in Fig. 1C, LP stress-induced expression of GmERF1 in roots was basically the same in the 2 materials within 7 d. However, the expression of GmERF1 was rapidly induced at 7 d post in the LP-sensitive genotype Bogao, with the peak occurring at 14 d. In contrast, GmERF1 expression showed a continuous downward trend in the LP-tolerant genotype Nannong94-156 from 7 to 14 d (Fig. 1C). The results suggested that GmERF1 might be an LP-sensitive gene and serve as a negative regulator to adapt to LP stress.

We constructed the GmERF1 fusion green fluorescent protein (GFP) expression vector 35S:GmERF1:GFP under the control of the cauliflower mosaic virus 35S (CaMV 35S) promoter to explore the subcellular localization of the protein encoded by GmERF1. The control vector 35S:GFP was constitutively expressed, while the GmERF1 fusion protein only detected a GFP signal in the nucleus, indicating that GmERF1 was located in the nucleus (Fig. 1D). The prediction of subcellular localization also indicated that GmERF1 is mainly distributed in the nucleus, which is consistent with its role as a TF to regulate gene expression and with the majority of experimental results showing that ERF TFs mainly function in the nucleus (Gao et al. 2015; Jin et al. 2018).

To further verify the expression of GmERF1, we evaluated GmERF1 promoter activity under different P treatments in Arabidopsis and soybean. We found that the GmERF1-GUS signal was observed in the whole transgenic A. thaliana plant and was stronger in plants treated with LP than in those treated with NP (Fig. 1E). In addition, the promoter of GmERF1 was more strongly expressed in soybean hairy roots under LP stress than under NP treatment (Fig. 1F). These results demonstrated that GmERF1 appears to participate primarily in adaptive changes in roots responding to LP stress, which was consistent with their expression patterns analyzed by RT-qPCR.

GmERF1 underwent selection during soybean domestication

GmERF1, as an ethylene pathway-related gene, has substantially different expression levels in the soybean LP-tolerant genotype Nannong94-156 and the LP-sensitive genotype Bogao under different P supply conditions (Zhang et al. 2020). To further confirm that GmERF1 is a candidate gene associated with P efficiency, we analyzed the 7-kb genomic region of GmERF1, including the promoter region and gene region, which contained 298 single-nucleotide polymorphisms (SNPs) among 360 soybean accessions, and 5 highly linked SNPs were identified in the promoter region of the gene (Fig. 2A). We compared the nucleotide diversity (FST and π) of the wild accessions, landraces, and cultivars in the 559 soybean germplasm samples and found that this gene was obviously under selection during domestication (Fig. 2, B and C). In addition, the 5 linked SNPs (Fig. 2D) were divided into 3 haplotype groups, which were significantly correlated with relative acid phosphatase activity (RAPA), relative phosphorus concentration (RPC), and 100 seed weight (HSW) traits, indicating that the natural sequence variation of this gene affected its RPC and RAPA in soybean roots. Hap1 was the best haplotype, with significantly higher RPC, RAPA, and HSW than Hap2 and Hap3 (Fig. 2E), and the Hap2 genotype (154 accessions, including Nannong94-156) was the second best and had significantly higher performance than Hap3 (n = 181 accessions, including Bogao). However, the frequency of the best Hap1 was decreased from wild soybean to landraces, and it disappeared in cultivated soybean (Fig. 2F), while the frequency of the second best Hap2 increased from wild to cultivated soybean. These assays revealed that the haplotypes were more abundant in wild soybean. The second best Hap2 was selected and retained during the breeding process, but the best Hap1 was lost during domestication. In addition, the geographical locations of the 559 soybeans showed that 3 haplotypes distributed in almost all regions, indicating that 3 haplotypes had no geographical region preference (Supplemental Table S1). In conclusion, GmERF1 may be a strong candidate gene related to P efficiency and has pleiotropic effects on plant development and yield, and wild soybean harbors elite alleles that were neglected during domestication and breeding.

Figure 2.

Figure 2.

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Sequence polymorphisms of GmERF1 and distributions of GmERF1 haplotypes. A) Haplotypes of GmERF1 among 360 soybean genotypes. The panel illustrates the gene structure of GmERF1, including the promoter, 5′ UTR, exon, and 3′ UTR regions. B and C) The fixation index (FST) and nucleotide diversity (π) of values in wild soybean, landraces, and cultivars across the 7-kb genomic regions surrounding GmERF1. D) Linkage disequilibrium (LD) in the GmERF1 genomic region based on pairwise r2 values. The r2 values are indicated using the color intensity index. E) Comparison of 3 traits across the 3 haplotypes. RPC represents the relative phosphorus concentration, RAPA represents the relative acid phosphatase activity, and HSW represents the 100 seed weight. Data are means ± Sds (n > 3). Means with different letters are significantly different (1-way ANOVA, Duncan, P ≤ 0.05). F) Proportions of 3 haplotypes within each of the 3 germplasm groups. Data are from the panel of 121 wild soybeans, 207 landraces, and 231 cultivars.

GmERF1 negatively regulates root development and P absorption

To determine whether GmERF1 is involved in LP stress response, we cloned GmERF1 and used an Agrobacterium tumefaciens-mediated transient transformation system to overexpress and interfere with the gene in the LP-sensitive soybean cultivar ‘Jack’ to study its function. We conducted a positive test for soybean hairy roots (Supplemental Fig. S1) and generated 18 positive composite plants per construct (overexpression and RNA interference), and 9 plants of each construct were used for NP or LP treatment. The RT-qPCR analysis showed that the expression level of GmERF1 in OE-GmERF1 (GmERF1 overexpressed) soybean transgenic hairy roots was 2 times and 25 times higher than that of the control (empty vector as the control) group under NP and LP conditions, while the expression level in Ri-GmERF1 (GmERF1 RNA interference) soybean transgenic hairy roots was 3 times and 26 times lower than that of the CK group under NP and LP, respectively (Fig. 3B). These results indicated that the gene fragments transferred into the transgenic hairy roots played roles in overexpression and silencing.

Figure 3.

Figure 3.

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Phenotypic characterization of GmERF1 transgenic hairy roots under NP and LP conditions for 14 d. A) Phenotypic comparison of transgenic hairy root plants grown in NP (500 µ M P) and LP (5 µ M P). Scale bars = 4 cm. B) RT-qPCR analysis of GmERF1 in transgenic hairy root plants. Statistical comparison of the total root length (C), root surface area (D), number of root tips (E), shoot dry weight (F), root dry weight (G), and P absorption efficiency (PAE) in roots (H) and shoots (I). OE-GmERF1 represents overexpression of GmERF1, Ri-GmERF1 represents RNA interference of GmERF1, CK represents control transgenic soybean hairy roots harboring empty vector, NP represents normal P supply (500 µ M P), and LP represents low P supply (5 µ M P). Data are means ± Sds (n = 3). Means with different letters are significantly different (1-way ANOVA, Duncan, P ≤ 0.05). Statistical methods of images were always set the same within experiments.

The Ri-GmERF1 transgenic composite plants exhibited better growth and physiological traits than CK plants regardless of P supply (Fig. 3A), especially under LP conditions, as evidenced by the greater total root length, root surface area, root tip number, dry weight, and P absorption efficiency (PAE) (Fig. 3, C to I). In contrast, the growth and physiological traits of the overexpressing transgenic plants were similar to or worse than those of the CK plants without significant differences in all 7 traits (Fig. 3, C to I). These results suggested that the overexpression of GmERF1 can reduce tolerance to LP stress, while interference with GmERF1 can enhance tolerance to LP stress by changing root architecture and increasing shoot and root biomass and PAE. Taken together, these results suggest that the expression level of GmERF1 in soybean plants is negatively associated with tolerance to LP stress.

GmERF1 affects the expression of P-related genes

To further elucidate the molecular mechanism by which GmERF1 regulates the phosphorus starvation response (PSR), the expression levels of 7 PSR genes involved in ethylene biosynthesis (GmETO1 and GmEIN3), P transport (GmPT5 [phosphate transporter 5], GmPT7, and GmPT8), and P utilization (GmACP1 and GmACP2) (Misson et al. 2004; Song et al. 2014; Zhang et al. 2014, 2020) were monitored in soybean hairy root plants under NP and LP conditions by RT-qPCR. Regardless of the P supply, 7 genes showed significant downregulation in OE-GmERF1 roots and upregulation in Ri-GmERF1 roots compared with CK plants, except for the GmACP1 gene in OE-GmERF1 roots under LP treatment, GmPT7 in OE-GmERF1 roots, and GmPT8 in the Ri-GmERF1 root under NP treatment (Fig. 4 and Supplemental Fig. S2), and the expression level under LP stress was greater than that under NP conditions, except for GmEIN3 (Fig. 4). This result suggests that GmERF1 may modulate LP stress by altering the expression of downstream PSR genes, particularly under LP conditions.

Figure 4.

Figure 4.

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Expression levels of 6 PSR genes in hairy roots of GmERF1 transgenic soybean hairy root plants under NP and LP treatments for 14 d. Expression was normalized to that of TUBULIN. The 6 genes are GmACP1 (Glyma.08G195100) (A), GmACP2 (Glyma.08G195000) (B), GmEIN3 (Glyma.02G274600) (C), GmETO1 (Glyma.14G197100) (D), GmPT5 (Glyma.10g186500) (E), and GmPT7 (Glyma.10G036800) (F). Data are means ± Sds (n = 3). Means with different letters are significantly different (1-way ANOVA, Duncan, P ≤ 0.05).

GmERF1 negatively regulates LP tolerance in soybean

To further confirm the functions of the GmERF1 gene in soybean, stable transgenic soybean plants with GmERF1 gene knockout were generated with the CRISPR-Cas9 system (Supplemental Fig. S3). Root system architecture is an important root trait that is sensitive to P status in growth medium (Lopez-Bucio et al. 2003; Jain et al. 2007). To test whether the growth of transgenic soybean plants under LP medium is related to changes in the root system architecture, wild-type (WT) and knockout transgenic lines of GmERF1 were grown in hydroponic solution containing NP and LP for 14 d. The plants showed greater growth promotion in knockout transgenic lines than in WT plants regardless of P supply (Fig. 5A). For instance, the root architecture, including total root length, root volume, root surface area, and root tip number, of knockout transgenic lines was higher than that of WT under LP conditions (Fig. 5, B to E). Moreover, root dry weight (RDW) and PAE in shoots and roots were also markedly increased in the knockout transgenic lines compared with the WT, which was consistent with the above results of hairy roots (Fig. 5, F to H). These results indicated that GmERF1 knockout could enhance the tolerance of soybean to LP stress.

Figure 5.

Figure 5.

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Phenotypic characterization of GmERF1 transgenic lines under NP and LP conditions for 8 d. A) Phenotype of GmERF1 mutants and wild-type plants grown under NP (500 µ M P) and LP (5 µ M P). Scale bars = 4 cm. Statistical comparison of total root length (B), the root volume (C), root surface area (D), number of root tips (E), P absorption efficiency (PAE) in shoots (F), PAE in roots (G), and root dry weight (H). I) 1-Aminocyclopropane-1-carboxylic acid (ACC) concentration in the leaves and roots of GmERF1 mutant and wild-type plants. KO-GmERF1, knockout of GmERF1; WT, wild-type soybean; NP, normal P supply (500 µ M P); LP, low P supply (5 µ M P). Data are means ± Sds (n = 3). Data significantly different from the corresponding controls (WT) are indicated (*P < 0.05; **P < 0.01; Student's t-test). Statistical methods of images were always set the same within experiments.

Knockout of GmERF1 increased ACC content

Ethylene signaling is a widely described signaling pathway that regulates a variety of cellular and physiological processes. It is commonly accepted that the components of the ethylene signaling pathway in plants have the potential to play key roles in abiotic stress tolerance. We speculated that GmERF1 negatively regulates root growth in an ethylene-dependent pathway. To verify this hypothesis, we detected the content of 1-aminocyclopropane-1-carboxylic acid (ACC), a precursor for ethylene synthesis. As shown in Fig. 5I, the ACC contents in both leaves and roots were increased in the knockout plants, especially in the root system, where it was twice that of WT plants. Together, these results indicate that GmERF1 promotes root growth by raising the content of ACC to adapt to LP stress.

GmERF1 physically interacts with GmWRKY6

To further study the molecular mechanism by which GmERF1 negatively regulates LP tolerance, we conducted Y2H screening to identify its potential interacting proteins using our constructed cDNA library of soybean. Before library-scale screening, truncations of GmERF1 were generated and tested for their autoactivation activity, and the results showed that the 17 amino acids at the N-terminus were required for the autoactivation activity of GmERF1 (Fig. 6A). Therefore, the truncated version of GmERF1 without 17 amino acids at the N-terminus, GmERF1-N-M17, was used for the screening. Among the identified interacting proteins (Supplemental Table S2), we found that the WRKY protein GmWRKY6 (Glyma.15G110300.1, the homologous protein of AtWRKY6) could indeed interact with GmERF1 (Fig. 6B). The interaction between GmERF1 and GmWRKY6 was further verified by luciferase complementation imaging (LCI) and bimolecular fluorescence complementation (BiFC) assays. For the LCI assay, the full-length CDS of GmERF1 and GmWRKY6 was individually fused to the C-terminal domain (cLUC-GmERF1) and N-terminal domain (GmWRKY6-nLUC) of the luciferase (LUC) gene. A strong florescence signal resulting from the reconstitution of a functional luciferase was observed when these 2 constructs were coexpressed in the leaves of Nicotiana benthamiana (Fig. 6C). In contrast, no florescence was detected when GmWRKY6-nLUC or GmERF1-cLUC was expressed with the empty vector cLUC or nLUC. The BiFC assays further confirmed that the interaction between GmERF1 and GmWRKY6 was generated in the nucleus (Fig. 6D). Together, these results demonstrated that GmERF1 could form heterodimers with GmWRKY6 to cope with LP stress.

Figure 6.

Figure 6.

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GmERF1 interacts with GmWRKY6. A) Transactivation activity of the GmERF1 proteins in yeast. Deletion of 17 amino acids (1–17 aa) in the N-terminus of the GmERF1 protein abolished the transactivation activity of GmERF1. Schematic diagrams of the full-length and truncated GmERF1 constructs are shown. The gray and blue rectangles represent the amino acids and the AP2/ERF motif, respectively. The yeast strain Y2HGold was used in the transactivation activity analysis. pGADT7 + pGBKT7-53 and pGADT7 + pGBKT7-Lam vectors were used as positive and negative controls, respectively. B) GmERF1 interacts with GmWRKY6 in yeast cells. The yeast cells were selected on SD medium lacking Leu and Trp (SD/-T-L), and the interaction was assessed based on their ability to grow on selective SD medium lacking Leu, Trp, His, and Ade (SD/-T-L-H-A) or SD medium lacking Leu, Trp, His, and Ade (SD/-T-L-H-A) but containing X-α-Gal for 3 d at 30 °C. X-α-Gal represents 5-bromo-4-chloro-3-indolyl-α-D-galactoside. C) Interaction between GmERF1 and GmWRKY6 in luciferase complementation imaging (LCI) assays. The LUCc-GmERF1 and GmWRKY6-LUCn constructs were transiently coexpressed in leaves of N. benthamiana, using LUCn and LUCc as the controls. Fluorescence signal intensities represent their binding activities. Low, weak intensity; high, strong intensity. D) Interaction between GmERF1 and GmWRKY6 was generated in the nucleus as shown in the bimolecular fluorescence complementation (BiFC) assay. GmERF1 and GmWRKY6 were both fused with the N-terminal half of yellow fluorescent protein (nYFP) and the C-terminal half of YFP (cYFP). The constructs were transiently coexpressed in leaves of N. benthamiana, using nYFP + GmERF1-cYFP and cYFP + GmERF1-nYFP as the controls. YFP, yellow fluorescent protein; BF, bright field. Scale bars = 20 µm.

GmERF1 directly interacts with GmWRKY6 to enhance GmWRKY6-mediated suppression of GmPHT1

To determine whether the transcription of GmWRKY6 is induced by LP stress, we extracted total RNA from the LP-sensitive genotype Bogao and the LP-tolerant genotype Nannong94-156 grown in hydroponic culture solution with NP and LP for RT-qPCR analysis. The expression of GmWRKY6 in soybean roots showed different trends after 12 h of LP stress. For the LP-sensitive genotype Bogao, the expression of GmWRKY6 gradually increased to a maximum on the 7th day and then returned to normal levels, while for the LP-tolerant genotype Nannong94-156, the expression of GmWRKY6 gradually decreased (Supplemental Fig. S4). The results showed that the expression of GmWRKY6 was induced by LP stress. Then, we examined the expression of GmWRKY6 in OE-GmERF1 and Ri-GmERF1 transgenic hairy roots. Under LP conditions, the expression level of GmWRKY6 in OE-GmERF1 hairy roots was higher than that in the WT, but the expression level of GmWRKY6 in Ri-GmERF1 transgenic hairy roots was the opposite (Fig. 7A), which was similar to the expression of GmERF1. Therefore, these results suggested that GmERF1 and GmWRKY6 are positively associated at the transcriptional level and participate jointly in the response to LP stress.

Figure 7.

Figure 7.

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GmERF1 interferes with the binding affinity of GmWRKY6 to the GmPT5, GmPT7, and GmPT8 promoters. A) The relative expression of GmWRKY6 in GmERF1 composite transgenic soybean seedlings under NP and LP treatments for 14 d. B) Structure of the GmPT5, GmPT7, and GmPT8 promoters. C)GmWRKY6 binds to the GmPT5, GmPT7, and GmPT8 promoter regions in the Y1H assay. Yeast cells were transformed with a bait vector containing a promoter fragment fused to the AUR1-C reporter gene and a prey vector containing GmWRKY6 fused to a GAL4 activation domain. Yeast cells were grown in liquid medium to an OD600 of 1.0 and diluted in a 10 × dilution series (10−1 to 10−3). From each dilution, 5 µL was spotted onto SD/-Leu medium to select for plasmids, and SD/-Leu was supplemented with 200- or 600-ng/mL aureobasidin A (AbA) to select for interaction. D to F) The interactions of GmWRKY6 or GmERF1 with the GmPT5, GmPT7, and GmPT8 promoters that were detected in leaves of N. benthamiana through a dual luciferase reporter system. Data in A) are means ± Sds (n = 3). Means with different letters are significantly different (1-way ANOVA, Duncan, P ≤ 0.05).

A previous study (Shang et al. 2010; Wang et al. 2014) revealed that WRKY family TFs bind to W-box cis-elements to regulate the expression of target genes. We found that some of these genes contained W-box cis-elements on their promoters by analyzing the promoters of PSR genes regulated by GmERF1. For example, in the promoter region of GmPT5, GmPT7, and GmPT8, 1 or 2 W-box elements were located upstream of the start codon (Fig. 7B). A yeast single-hybrid test showed that GmWRKY6 interact with GmPT5, GmPT7, and GmPT8 (Fig. 7C). To test whether these 3 genes were potential targets of GmERF1 and GmWRKY6, we used a dual luciferase (dual-LUC) assay system to examine whether protein–protein interactions affect the transcriptional activation of target genes. We found that the luciferase activity of GmPT5, GmPT7, and GmPT8 was slightly decreased when GmWRKY6 was expressed but was substantially impaired when GmERF1 was coexpressed with GmWRKY6 (Fig. 7, D to F). In conclusion, our study suggests that GmERF1 and GmWRKY6 together inhibited the expression of downstream genes via their interaction. These results were consistent with the expression patterns of GmPT5, GmPT7, and GmPT8 in soybean transgenic hairy roots.

Discussion

GmERF1 is a potential target to facilitate P efficiency in soybean

Soybean is an important crop worldwide and is sensitive to LP. Although P is abundant in soil, the limited available P seriously threatens soybean production. Ethylene is a stress hormone that plays an important role in various stress responses of plants and is closely related to plant nutritional stress, including LP stress (Yang et al. 2013; Romera et al. 2016). Abiotic stress can inhibit root growth by affecting ethylene biosynthesis and thus affecting plant growth and development (Swarup et al. 2007; Song et al. 2016). ERF subfamily TFs are key downstream regulators of the ethylene-mediated stress response signaling pathway (Licausi et al. 2013; Kazan 2015). Studies have reported that ERF family members regulate root development in response to LP stress in Arabidopsis (Ramaiah et al. 2014). However, their biological function in LP stress responses in soybean remained unclear. In a previous study, soybean ethylene response factor 1 (GmERF1) was obtained by preliminary transcriptome analysis (Zhang et al. 2017). Combined with the candidate interval association analysis and haplotype analysis (Fig. 2), this gene was confirmed to be a candidate gene related to phosphorus efficiency. It is worth noting that our results show that the optimal Hap1 exists mainly in WT materials and is gradually lost during domestication (Fig. 2F). One possible explanation is that this gene was not under selection pressure, owing to the ample application of phosphate fertilizer during the planting process. Another reason may be because the Hap1 in wild soybean has linkage with some unfavorable genes, which lead the Hap1 been incidentally eliminated with the linkage unfavorabe genes during the domestication. Therefore, it is promising to utilize wild relatives of crops for future breeding.

GmERF1 regulates genes related to root development, ethylene signaling, and P transport

The ERF family is a large family of ERFs, and AP2/ERF proteins in the plant ethylene signaling pathway play an important role in regulating plant growth and development and response to stress. GmERF1 may have a role in LP stress; it is mainly expressed in soybean roots and is highly induced by LP stress in the roots of low-P-sensitive soybean cultivars (Fig. 1, B and C), and its promoter was strongly induced by LP treatment in transgenic A. thaliana plant and soybean hairy roots (Fig. 1, E and F). In addition, ERF genes regulate plant root development. For example, AtERF1 in A. thaliana inhibits root growth by affecting auxin and ethylene biosynthesis (Song et al. 2016), and AtERF070 responds to LP stress by regulating root development (Ramaiah et al. 2014). We found that GmERF1 knockout in soybean can increase the ethylene precursor (ACC) level of roots (Fig. 5I) and consequently regulate root morphology. Compared with WT plants, the roots of GmERF1-interfering plants and the knockout transgenic lines were more tolerant to LP stress by increasing biomass and changing root configuration (Figs. 3 and 5). In addition, consistent with the phenotype, the overexpression of GmERF1 reduced the P uptake efficiency, while the inhibition of GmERF1 expression increased the P uptake efficiency. In response to LP stress, plants facilitate the uptake and utilization of P by secreting acid phosphatase (GmACP1/2) (Duff et al. 1994; Wang et al. 2009; Zhang et al. 2014), regulating the expression of P transporter genes (Vance et al. 2003; Lambers et al. 2006), and inducing root ethylene production (Song et al. 2016). Remarkably, GmERF1 displays 2 functions affecting P-related genes: it represses the transcription of the acid phosphatase genes GmACP1 and GmACP2, the phosphate transporters GmPT5 and GmPT7, and the ethylene-related genes GmETO1 and GmEIN3 but activates the expression of GmWRKY6 (Figs. 4 and 7A). This suggests that GmERF1 may combine with other transcription regulatory proteins to modulate the transcription of all of these genes.

GmERF1 interacts with GmWRKY6 and affects its ability to bind to downstream gene promoters

GmERF1 is a member of the ERF subfamily, and here, we demonstrate that the overexpression or knockout of this gene affects PSR gene expression levels. A recent study showed that TEOSINTE BRANCHED 1/CYCLOIDEA/PCF 15 (TCP15) and ERF4 interact to control cell cycle progression (Ding et al. 2022). However, the partners of GmERF1 and the relevant epigenetic regulation mechanism in the LP stress response remain unknown. In the present study, GmWRKY6, an AtWRKY6 homolog, was identified as the GmERF1-interacting protein and functioned as a negative regulator of LP tolerance (Supplemental Fig. S4). WRKY proteins usually bind to W-box motifs of target gene promoters (Eulgem et al. 2000). Chen et al. (2009) showed that AtWRKY6 was involved in the response to LP stress by regulating the expression of PHOSPHATE1 (PHO1). The expression level of GmWRKY6 was increased in GmERF1-overexpressing hairy roots (Fig. 7A) but decreased in roots with interference. We analyzed the promoters of stress-responsive genes and found that the GmPT5, GmPT7, and GmPT8 promoters contain W-box cis-elements, and the binding ability of GmWRKY6 to the W-box of the 3 genes was weakened in the presence of GmERF1 (Fig. 7, D to F). Based on these results, we propose a working model of how GmERF1 participates in ethylene signaling in soybean and of GmWRKY6's ability to bind target genes in response to LP stress (Fig. 8): LP induced the transcription of GmERF1, and GmERF1 regulated ACC levels to promote root development. GmWRKY6 functions as an upstream regulator that interacts with GmERF1 to repress GmPT5, GmPT7, and GmPT8 expression by directly suppressing its promoter activity and affecting P absorption and utilization efficiency, enabling the plant to cope with LP stress.

Figure 8.

Figure 8.

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Model of the roles of GmERF1, GmWRKY6, and GmPHT1s expression in the response to soybean LP stress. GmERF1 and GmWRKY6 were induced by LP stress. GmERF1 negatively regulates root development by mediating changes in 1-aminocyclopropane-1-carboxylic acid (ACC) concentration. Moreover, it also interacts with GmWRKY6 to enhance the inhibition of GmWRKY6 for downstream transcription of GmPT5, GmPT7, and GmPT8. NP represents normal P supply (500 µ M P), and LP represents low P supply (5 µ M P).

Conclusions

In brief, we identified an ERF gene, GmERF1, that negatively regulates LP tolerance in soybean by mediating changes in ACC concentration. Moreover, it has been shown to interact with GmWRKY6 to inhibit downstream gene expression of GmPT5, GmPT7, and GmPT8. This finding may provide a model for the response to LP stress in soybean and reveals Hap1 of GmERF1, which is mainly from wild soybean, as a promising candidate for the development of LP-tolerant crops.

Materials and methods

Plant materials and growth conditions

Transcriptome analysis data of LP sensitive soybean (G. max) genotypes B18, GDW022, Bogao, and LP-tolerant soybean genotypes B20, GDW006, and Nannong94-156 with contrasting P efficiency after LP treatment were used to select differentially expressed genes throughout this study. B18 and B20 were transgressive recombinant inbred lines (RILs) from the segregating soybean population consisting of 152 RILs, which derived from a cross between varieties Nannong94-156 and Bogao (Zhang et al. 2017). GDW006 and GDW022 were wild soybeans in 559 soybean accessions; their transcriptome data were supplied by Dr. Haina Song. The transcriptome data of B18, B20, Bogao, and Nannong94-156 were collected from the published article (Zhang et al. 2017). The soybean genotypes Williams 82, Bogao, and Nannong94-156 were used for physiological experiments. The soybean cultivar ‘Jack’ was used for functional analysis of soybean hairy root transformation, GUS activity assay experiments, and gene editing.

Germination and growth of seedlings were performed in an artificial climate chamber (10-h light/14-h dark and 28 °C/20 °C). The plants with different P treatment were grown in modified one-half Hoagland's nutrient solution, the LP treatment is a low supply of P (5 µ M P), and the NP treatment is a normal supply of P (500 µ M P). Soybean plants were grown in a completely randomized block design, and the solution was renewed every 3 d.

Phenotyping

Phenotyping of WT, OE-GmERF1, and Ri-GmERF1 hairy root transgenic lines was carried out as follows. After hairy root transformation, the main roots were removed, and hydroponic experiments were carried out. Half of the seedlings were transferred to LP nutrient solution, while the remaining half were kept under NP nutrient solution as controls. Plants harboring positive hairy roots with overexpressed GmERF1 were validated using RT-qPCR (Supplemental Fig. S1). Seven traits reflecting physiological responses to NP or LP were measured after 14-d NP and LP treatment. These traits included shoot dry weight (SDW), RDW, total root length, the number of root tips, root surface area, and PAE in roots and shoots. PAE was defined as total P in the plant (mg plant−1).

Phenotyping of WT and KO-GmERF1 transgenic lines was carried out as follows. For the seedlings of WT (‘Jack’) and the T3 generation knockout transgenic lines, half of them were transferred to LP nutrient solution, while the remaining half were kept under NP nutrient solution as controls. Seven traits reflecting physiological responses to NP or LP were measured after 8-d NP and LP treatment. These traits included RDW, total root length, the number of root tips, root surface area, the root volume, and PAE in roots and shoots.

Three hundred and sixty of the 559 soybean accessions were tested with HSW, RAPA, and RPC traits. Measurements of RAPA and RPC were performed as previously described (Zhang et al. 2017). HSW was measured in the field with 3 replications per genotype in 2018.

RT-qPCR

The roots, stems, leaves, flowers, and pods were collected for the tissue-specific expression analysis of target genes by investigating soybean genotype Williams 82 with NP or LP solution as described above in hydroponic experiments after 30 d of treatment. Baogao and Nannong94-156 were used for induction expression analysis of GmERF1 gene; their roots were collected at 0 h, 3 h, 12 h, 1 d, 7 d, and 14 d after NP and LP treatments and frozen in liquid nitrogen. All samples were stored at −80 °C.

Total RNA was isolated from soybean plants using the RNA Simple Total RNA Kit (TIANGEN, Beijing, China). The first chain of cDNA was synthesized using a Prime Script First-Strand cDNA Synthesis Kit (Yeasen, Shanghai, China). RT-qPCR was performed using Hieff qPCR SYBR Green Master Mix (Yeasen, Shanghai, China). The RT-qPCR procedure was as follows: 94 °C for 3 min followed by 40 cycles at 94 °C for 15 s, 60 °C for 15 s, and 72 °C for 30 s. The soybean TUBULIN gene (GenBank accession AY907703) was used as an internal control as previously described (Zhang et al. 2014, 2017). GmERF1 primers (Supplemental Table S3) were used for RT-qPCR. Three biological and technical repeats were used. The analysis of relative gene expression data using the 2(−ΔΔC[T]) method (Le et al. 2011) was performed.

Vector construction and plant transformation

To construct the overexpression vector, the full-length cDNA of GmERF1 was amplified by PCR using 2 specific primers (Supplemental Table S3) and then cloned into a pMDC83 vector that had been digested with BamHI and SalI. For the RNA interference construct, a 453-bp exon fragment encoding GmERF1 with 2 gene-specific primers (Supplemental Table S3) was inserted into pB7GWIWG2 (II) using Gateway technology. These constructs were introduced into Agrobacterium (Agrobacterium rhizogenes K599) and used for hairy root transformation in soybean cv. ‘Jack.’ For the CRISPR vector, target sequence adapters of GmERF1 were designed by CRISPR-P (http://cbi.hzau.edu.cn/crispr/). The guide DNAs (sgDNA) were inserted into gene-editing vector to construct CRISPR/Cas9 vector containing GmERF1 target sequence. For the analysis of the GmERF1 promoter, a 2,017-bp fragment upstream of the GmERF1 transcription initiation codon was amplified from Williams 82 soybean root DNA by PCR and cloned into the pCAMBIA1381z binary vector between the BamHI and SalI restriction enzyme sites in frame with the GUS reporter gene to make the pGmERF1-GUS construct. The amplified primers are listed in Supplemental Table S3. The reconstructed plasmids were transferred into A. tumefaciens strain GV3101 using a freeze–thaw procedure, and transgenic plants were produced via Agrobacterium-mediated floral dip (Clough and Bent 1998). All transgenic lines used in this study were T3 homozygous plants with a single-copy insertion.

GUS histochemical analysis of transgenic Arabidopsis and soybean

T3 GmERF1promoter-GUS (pGmERF1-GUS) transgenic Arabidopsis (A. thaliana; Columbia ecotype) were grown in a bowl with a culture medium of 1:1 nutrient soil and vermiculite and were irrigated with NP or LP solution as described above. Whole seedlings were harvested for GUS staining after 7 d. The resulting construct, pGmERF1-GUS, was transformed into A. rhizogenes strain K599 for soybean hairy roots transformation. The positive transformed roots were then transferred into modified one-half Hoagland's nutrient solution with NP or LP treatment for 14 d for GUS expression analysis. The samples were completely immersed in a reaction buffer containing 50 mmol/L phosphate buffer (NaH2PO4 and Na2HPO4), pH = 7.0, 10 mmol/L Na2EDTA, pH = 8.0, 1 mmol/L K4Fe(CN)6·3H2O, 1 mmol/L K3[Fe(CN)6], 2 mmol/L X-Gluc, and 0.1% (v/v) Triton X-100 and incubated at 37 °C overnight. After staining, the tissues were washed with ethanol (70% v/v) and then photographed.

Subcellular localization

The coding region of GmERF1 without a stop codon was amplified by PCR with specific primers (Supplemental Table S3) from soybean cDNA (Williams 82). The obtained GmERF1 fragment and GFP reporter gene were inserted into the modified pFGC5941 vector and sequenced into A. tumefaciens strain GV3101, and the control vector 35S:GFP was permeated into N. benthamiana leaves. GFP fluorescence was observed under a confocal fluorescence microscope (Zeis, Germany). The GFP was excited at 488-nm excitation and 110-nm collection bandwidth, and gain was 1.

Genetic diversity analysis and molecular evolution

The candidate–gene association analyses were performed using a mixed-model approach with TASSEL 5.0 as previously described (Yu et al. 2006; Bradbury et al. 2007; Zhang et al. 2014). A significant association threshold (P ≤ 0.05) was used to identify significant marker–trait associations. For the candidate gene-based association analysis, 298 polymorphism markers of the target gene were selected from the resequencing of 360 soybean accessions (Lu et al. 2020). SNPs with missing data >10% or MAF < 5% were filtered for the genetic diversity analysis. A linkage disequilibrium (LD) heatmap was plotted using the LD heatmap R package (Shin et al. 2006). The fixation index (FST) and nucleotide diversity (π) of 121 wild, 207 landrace, and 231 cultivated accessions were analyzed using the VCF tools package (Danecek et al. 2011). The details of 559 accessions including 121 wild, 207 landrace, and 231 cultivated accessions were shown in a previous report (Lu et al. 2020).

Detection of ACC

The WT and T3 generation knockout transgenic lines of GmERF1 were selected, and after 14 d of NP or LP treatment, the leaf and roots were taken for the test of ACC content. Each sample with 1 treatment had 3 replicates, and each replicate was taken from 3 mixed plants. The cryopreserved biological samples were ground to powder form, and 0.5-mL methanol:formic acid:water (15:4:1, V/V/V) was added for extraction. The supernatants were collected after centrifugation. Internal standards were added to plant samples. After lyophilizing for 8 h, 100-µL methxamine salt pyridine (0.015 g/mL) and 100-µL BSTFA (containing 1% TMCS) were added to the resulting solution and reacted at 37 °C for 30 min to get the derivative solution. The reaction solution was diluted to 200 µL with n-hexane, filtered through 0.22-µm filter, and tested on Agilent 7890B-7000D GC–MS/MS within 24 h.

Transcriptional activation activity and Y2H assay

The full open reading frame (ORF) of GmERF1 was amplified using gene-specific clone primers with specific restriction sites (Supplemental Table S3), which were fused to the GAL4 DNA-binding domain of the pGBKT7 vector (bait vector), and the recombinant vector was transformed into the yeast (Saccharomyces cerevisiae) strain Y2HGold. The pGBKT7-53 and pGBKT7-Lam bait plasmids were cotransformed with the pGADT7-T prey plasmid as positive and negative controls, respectively. Yeast cells and control cells carrying recombinant plasmids were grown on tryptophan-deficient and leucine (SD/-TRP/-LEU) and tryptophan-deficient histidine-adenine and leucine (SD/-TRP/-HIS/-ADE/-LEU), respectively, and vectors were used alone or in different combinations to exclude self-activation and false-positive activation. The Y2H assay was biologically repeated 3 times.

LCI assay

ORFs of GmERF1 and GmWRKY6 were cloned into JW771 and JW772 vectors, respectively. The ORF of GmERF1 was fused to the C-terminal half of luciferase (cLUC-GmERF1), and the ORF of GmWRKY6 was fused to the N-terminal half of luciferase (GmWRKY6-nLUC). cLUC and nLUC empty vectors were used as controls. The constructs were transformed into A. tumefaciens (strain GV3101) carrying a helper plasmid. The Agrobacterium strains were grown to a cell density of OD600 = 0.5 and were harvested and resuspended in infiltration buffer (10 mM MES, 0.2 mM acetosyringone, and 10 mM MgCl2) to the same concentration. Agrobacterium cells containing cLUC-GmERF1 or GmWRKY6-nLUC were suspended in infiltration buffer and then mixed at a volume ratio of 1:1. Agrobacterium mixtures were transferred to the fully expanded leaves of N. benthamiana plants and cultured as described above. The leaves were harvested and sprayed uniformly with D-luciferin (LUC) (BioVision, Yeasen, Shanghai, China). After a 5-min exposure in the dark, luminescence images were captured with an Andor iXon CCD camera (Tanon 5200, Shanghai, China). A low-light cooled CCD imaging apparatus was used to capture the LUC image as described earlier (He et al. 2004). The primers are listed in Supplemental Table S3.

BiFC assays

Construct generation for the BiFC assays was performed as follows: the full-length GmERF1 coding sequence was cloned into the pSPYNE173 vector and the GmWRKY6 cDNA sequences into the pSPYCE vector to generate GmERF-YFPN, GmWRKY6-YFPC, GmERF-YFPC, and GmWRKY6-YFPN. The leaves of 4-wk-old N. benthamiana plants were coinfiltrated with various combinations of nYFP and cYFP constructs. The fluorescence signal of YFP staining was imaged using a laser confocal microscope (LSM710, Zeiss, Germany) with a 488-nm excitation and 110-nm collection bandwidth, and gain was 1.

Dual-LUC assay

To assess transcriptional activity, a transient dual-LUC assay was performed in N. benthamiana by the method reported previously (Chen et al. 2008). Genes encoding GmERF1 and GmWRKY6 were cloned into pFGC5941-GFP, and target GmPHT1 family gene GmPT5 (phosphate transporter 5), GmPT7, and GmPT8 promoters were cloned into pGreenII-0800. Recombinant plasmids were constructed and transformed into A. tumefaciens strain GV3101. Bacteria containing plasmids were infiltrated into N. benthamiana leaves (Yu et al. 2021). After infiltration, plants were grown at 22 °C with a 16-h light/8-h dark photoperiod for 2 days. LUC (BioVision, Yeasen, Shanghai, China) was then applied to the backs of N. benthamiana leaves before imaging. LUC activity was measured using a low-light cooled CCD imaging apparatus (Tanon 5200, Shanghai, China). The primers used are listed in Supplemental Table S3.

Y1H assay

Promoter fragments of GmPT5, GmPT7, and GmPT8 were integrated into the pAbAi vector to generate the recombinant plasmid pAbAi-GmPT5, pAbAi-GmPT7, and pAbAi-GmPT8. The linearized constructs were transferred into the yeast 1-hybrid (Y1H) Gold component yeast strain, and then, the yeast transformants were tested on SD/-Ura medium with different concentrations of AbA (Aureobasidin A). The coding sequence of GmWRKY6 was cloned into the pGADT7 vector and transferred into the Y1HGold yeast strain containing pAbAi-GmPT5, pAbAi-GmPT7, and pAbAi-GmPT8, respectively. The interactions of GmWRKY6 with the promoter fragment of GmPT5, GmPT7, and GmPT8 were tested on SD/-Leu medium with the tested AbA concentration. The primers used are listed in Supplemental Table S3.

Accession numbers

Sequence data from this article can be found in the GenBank/EMBL data libraries under accession numbers: GmERF1 (Glyma.13G123100), GmWRKY6 (Glyma.15G110300.1), GmACP1 (Glyma.08G195100), GmACP2 (Glyma.08G195000), GmETO1 (Glyma.14G197100), GmEIN3 (Glyma.02G274600), GmPT5 (Glyma.10G036800), GmPT7 (Glyma.10G186500), and GmPT8 (Glyma.13G040200).

Supplementary Material

kiad170_Supplementary_Data
Click here for additional data file. (597.6KB, pdf)

Acknowledgments

We acknowledge Dr. Haina Song from Pingdingshan University for providing the transcriptome data of GDW006 and GDW022. We acknowledge the National Supercomputing Center in Zhengzhou for providing a platform for data analysis.

Contributor Information

Ruiyang Wang, Collaborative Innovation Center of Henan Grain Crops, College of Agronomy, Henan Agricultural University, Zhengzhou 450002, China.

Xiaoqian Liu, Ministry of Agriculture and Rural Affairs Key Laboratory of Soybean Biology, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing 100081, China.

Hongqing Zhu, Collaborative Innovation Center of Henan Grain Crops, College of Agronomy, Henan Agricultural University, Zhengzhou 450002, China.

Yuming Yang, Collaborative Innovation Center of Henan Grain Crops, College of Agronomy, Henan Agricultural University, Zhengzhou 450002, China.

Ruifan Cui, Collaborative Innovation Center of Henan Grain Crops, College of Agronomy, Henan Agricultural University, Zhengzhou 450002, China.

Yukun Fan, Collaborative Innovation Center of Henan Grain Crops, College of Agronomy, Henan Agricultural University, Zhengzhou 450002, China.

Xuhao Zhai, Collaborative Innovation Center of Henan Grain Crops, College of Agronomy, Henan Agricultural University, Zhengzhou 450002, China.

Yifei Yang, Collaborative Innovation Center of Henan Grain Crops, College of Agronomy, Henan Agricultural University, Zhengzhou 450002, China.

Shanshan Zhang, Collaborative Innovation Center of Henan Grain Crops, College of Agronomy, Henan Agricultural University, Zhengzhou 450002, China.

Jinyu Zhang, Collaborative Innovation Center of Henan Grain Crops, College of Agronomy, Henan Agricultural University, Zhengzhou 450002, China.

Dandan Hu, Collaborative Innovation Center of Henan Grain Crops, College of Agronomy, Henan Agricultural University, Zhengzhou 450002, China.

Dan Zhang, Collaborative Innovation Center of Henan Grain Crops, College of Agronomy, Henan Agricultural University, Zhengzhou 450002, China.

Author contributions

R.W. and D.Z. designed the experiments. R.W., X.L., H.Z., R.C., Y.F., X.Z., and Yifei Y. carried out the experiments. R.W., Yuming Y., S.Z., and J.Z. analyzed the data. R.W. wrote the manuscript. D.Z. and D.H. revised the manuscript. All authors read and approved the final manuscript.

Supplemental data

The following materials are available in the online version of this article.

Supplemental Figure S1 . PCR validation of positive hairy roots using different primer pairs.

Supplemental Figure S2 . Expression levels of GmPT8 gene in hairy roots of GmERF1 transgenic soybean hairy root plants under NP and LP treatments for 14 d.

Supplemental Figure S3 . Relative expression levels identification of knockout GmERF1 transgenic under NP condition and Cas9-mediated editing.

Supplemental Figure S4 . RT-qPCR analyses of GmWRKY6 relative expression in response to LP stress.

Supplemental Table S1 . Geographical distribution of soybean accessions with 3 haplotypes in China.

Supplemental Table S2 . Candidates from yeast 2-hybrid screening.

Supplemental Table S3 . Primers used in this study.

Funding

The work was supported by the National Natural Science Foundation of China (32072088, 32272171, 32201867, 31901895, and 32272176), Major Science and Technology Project of Henan Province (221100110300) and Henan Fine Variety Joint Tackling Key Problems Project (20220100304), and the Central Plains Talents Program Top Young Talents.

Data availability

The transcriptome data of 'Bogao', 'Nannong94-156', 'B18', and 'B20' can be found in the published paper Zhang et al. (2017). The transcriptome data of GDW006 and GDW022 were provided by Dr. Haina Song and was not published, if anyone need the raw data, please contact the corresponding author.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

kiad170_Supplementary_Data
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Data Availability Statement

The transcriptome data of 'Bogao', 'Nannong94-156', 'B18', and 'B20' can be found in the published paper Zhang et al. (2017). The transcriptome data of GDW006 and GDW022 were provided by Dr. Haina Song and was not published, if anyone need the raw data, please contact the corresponding author.

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