Identification and characterization of soybean phytochrome-interacting factors and their potential roles in abiotic stress

Abstract

Abstract

Phytochrome-interacting factors (PIFs) belong to a subfamily of the bHLH transcription factor family and play a pivotal role in plant light signal transduction, hormone signal pathways, and the modulation of plant responses to various abiotic stresses. The soybean (Glycine max) is a significant food crop, providing essential oil and nutrients. Additionally, it is a vital industrial raw material and a lucrative cash crop. Nevertheless, research on PIFs in soybean is relatively scarce. In this study, we conducted a comprehensive analysis of the gene structure, chromosomal location, conserved motifs, phylogenetic relationships, and expression patterns of the Glycine max PIF (GmPIF) genes. A total of 20 GmPIF genes were identified in the soybean genome. These are unevenly distributed on 12 soybean chromosomes. The analysis of gene duplication events revealed the existence of five pairs of duplicated genes within the GmPIF gene set. Conserved motif analysis demonstrated the presence of several conserved motifs that were generally aligned with the classification of PIF protein. Cis-acting elements in the GmPIF promoters were found to be responsive to light, heat, drought, and phytohormone signaling. The expression levels of certain GmPIF genes were significantly induced under shade, high temperature and drought stress conditions. The heterologous expression of the GmPIF6c/GmPIL1 in an Arabidopsis mutant resulted in a reduction in the elongation of the hypocotyl in response to shade. It is proposed that GmPIF6c/GmPIL1 may exert an inhibitory effect on shade avoidance. This study elucidated the evolution, structural and functions of GmPIF family members.

Clinical trial number

Not applicable.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12870-024-05950-4.

Introduction

Soybeans (Glycine max L.) are a commercially significant crop due to their high protein and oil content [1]. Statistics show that soybeans account for 61% of oilseed production, 70% of protein meal and 28% of vegetable oil used across the world [2]. Furthermore, soybeans contain a variety of active substances that are beneficial to the human health, including lecithin, saponins, isoflavones and other physiological active substances [3]. In order to meet the significant and increasing demands of a growing global population, the overall cultivation of soybeans must an annual augmentation of approximately 2.4%, notwithstanding the constraints imposed by finite arable [4, 5]. High-density planting and intercropping are essential means to increase soybean yield on limited farmland. However, the potential yield of these two cultivation modes is limited by environmental factors, including light and temperature. Under these high density cultivation conditions, soybean are susceptible to shade stress from adjacent canopies or high-growing crops [6, 7].

When plants thrive amidst high-density conditions, adjacent vegetation absorbs red (R) light and reflects or transmits far-red (FR) light, thereby instigating a series of characteristic responses known as the shade avoidance syndrome (SAS) [8], including elongation of the hypocotyl (or stem) and petiole, reorientation of leaf or branch growth, and accelerated flowering [9, 10]. Numerous studies have demonstrated that phytochromes (phys), the primary photoreceptor of red and far-red light signal, play critical roles in plant growth and development [11, 12]. phyB is activated by red light signal, which enables it to enter the nucleus and inhibit the phytochrome-interacting factor (PIF) activity of bHLH family transcription factors. This subsequently inhibits the shade-avoidance response [12, 13]. PIFs induce SAS by initiating an expression cascade of genes involved in auxin biosynthesis and signaling, such as YUCCA8 (YUC8), YUCCA9 (YUC9), INDOLE-3-ACETIC ACID INDUCIBLE 19 (IAA19), INDOLE-3-ACETIC ACID INDUCIBLE 29 (IAA29) [14, 15]. Arabidopsis thaliana has eight PIF members (AtPIF1 to AtPIF8), which are encoded by AtPIF1 to AtPIF8 genes. AtPIFs exhibit several fundamental structural characteristics, including the basic helix-loop-helix (bHLH) domain, as well as active binding domains for phytochrome A (APA) and phytochrome B (APB) that interact with PHYA and PHYB. The bHLH domain has the capacity to binds to cis-acting elements, including the G-box (5’-CACGTG-3’) and the E-box (5’-CANNTG-3’), which are located in the promoters of target genes [16–19]. The functions of PIF have been studied in many crops, eight PIF members have been identified in tomato, designated SlPIF1a, SlPIF1b, SlPIF3, SlPIF4, SlPIF7a, SlPIF7b, SlPIF8a and SlPIF8b. The three SlPIFs (SlPIF1a, SlPIF1b, and SlPIF3) contain APA and APB motifs [20]. There are seven PIFs in rice, designated OsPIL11 to OsPIL16 and OsPIF8. All OsPIFs possess an APB motif, while only OsPIL15 has an additional APA motif [21]. The Corn plant has seven PIFs: ZmPIF3.1 to ZmPIF3.3, ZmPIF4.1, ZmPIF4.2, ZmPIF5.1, and ZmPIF5.2. All ZmPIFs have conserved APB motifs and interact with ZmphyB1 and ZmphyB2. ZmPIF3.1 and ZmPIF3.2 contain additional APA motifs [22]. Numerous studies have demonstrated that PIFs play a pivotal role in a multitude of signaling pathways in response various external stimuli, including light, temperature, hormones and biological stresses [23]. Under shade conditions, the protein complex of PIF4 and CDF2 increased the expression of auxin synthesis gene YUCCA8, thus promoting hypocotyl elongation [24]. The DELLA protein has the capacity to bind to the DNA-binding domain of the PIF protein, preventing the PIF protein from binding to its downstream DNA, thereby negatively regulating the expression of genes involved in cell elongation [25]. The DELLA-BZR1-PIF4 module regulates plant growth by modulating light signals, thereby activating growth-regulatory programs and enhancing auxin signaling, which in turn upregulates genes that play a role in vertical expansion [10]. PIF1 has been demonstrated to interact with physically interacts with Abscisic Acid Insensitive 3 (ABI3) in seeds, thereby exerting a synergistic regulatory effect on the expression of SOMUS (SOM), a negative regulator of seed germination [26]. PIFs have been extensively studied in Arabidopsis, maize, rice, grape, and other plants. However, they are less studied in soybean, an important grain and oil crop. Hina Arya conducted a preliminary identification of GmPIF family members, a total of 15 GmPIF genes were identified [27]. In their study, the PIF genes were identified through use of the keywords “PIF,” “Phytochrome Interacting factors,” and blast searches against Arabidopsis PIFs in the proteome database of the latest version of the soybean genome (Wm82.a2.v1). This result is incomplete. Currently, the soybean phytochrome-interacting factor (GmPIFs) gene family members have not been systematically identified, and their functions in regulating development and responding to stress remain under investigation. In this study, a total of 20 GmPIF genes were identified in soybean. Subsequently, a comprehensive analysis was conducted to elucidate the functional roles of GmPIFs, encompassing gene duplication, structural, syntenic and cis-element investigations. In order to further verify the functions of these genes in light response and stress responses, we conducted a detailed analysis of the expression patterns of GmPIFs under a range of treatments. Furthermore, the overexpression of GmPIF6c/GmPIL1 in an Arabidopsis pil deletion mutant resulted in the inhibition of shade hypocotyl length. This finding suggests that GmPIF6c/GmPIL1 may negatively regulate plant shade avoidance. This study can provide guidance for further research on the biological functions of GmPIFs.

Materials and methods

Identification of PIF family members in soybean

The full-length AtPIF protein sequences of Arabidopsis thaliana were obtained from TAIR (https://www.arabidopsis.org/) and used as the reference sequences. The GmPIF homologues were identified through a BLASTp search conducted in the Phytozome v13 database (https://phytozome-next.jgi.doe.gov/) [28]. Additionally, the soybean genome and genome annotation files were obtained from the Phytozome database. The amino acid composition, theoretical isoelectric point (pI) and molecular weight (Mw) of the identified GmPIF proteins were evaluated using the ExPASy website (http://expasy.org/tools/) [29]. The subcellular localizations of GmPIFs were predicted using the PSORT tool on the GenScript online platform (https://www.genscript.com/wolf-psort.html).

Phylogenetic analyses and classifications of the GmPIFs proteins

All PIFs protein sequences of A. thaliana (At) and Glycine max (Gm) were aligned using ClustalX software with default settings [30]. The Neighbor-joining (NJ) phylogenetic tree of PIFs was constructed by MEGA 6 with the bootstrap test of 1000 [31]. The accession number of PIFs used for the phylogenetic analysis are listed in supplementary Table 1.

Conserved motif and gene structures analysis

The conserved domains of PIF protein were predicted by MEME online website (http://meme-suite.org/tools/meme) using the protein sequences of PIF. The visualization of MEME motifs and the Seq Logos were generated using Adobe Illustrator 2021. Additionally, the structure of the genes was visualized using GSDS v2.0. This visualization process utilized soybean genomic DNA sequences, coding sequences (CDS) and a phylogenetic tree as templates.

GmPIFs chromosomal locations, duplication events and divergence rates analyses

To analyze the chromosomal locations and duplications of GmPIFs based on the soybean genome information available on Phytozome, we utilized MapGene2Chrom, which allowed us to map and display the chromosomal locations and duplications of these genes. The analysis was conducted using the virtual machine Bio-Linux system. Subsequently, an investigation was conducted into the duplicated genes and the nucleotide non-synonymous (Ka) to synonymous (Ks) ratios (Ka/Ks) of duplicated gene pairs. The calculations and subsequent analysis of the evolutionary relationship were conducted using TBtools. This comprehensive approach facilitated a detailed exploration of the genetic landscape and evolutionary dynamics of GmPIFs in soybean. Subsequently, the Circos diagram was generated using Tbtools. The FPKM values of GmPIFs gene expression in different tissues were obtained from the Phytozome database, and then TBtools was employed to create heat maps.

qPCR analyses of GmPIFs

The specific quantitative RT-PCR primers for the selected GmPIFs were designed by Sangon Biotech (Shanghai, China). Total RNA was isolated with EasyPure Plant RNA Kit (Transgen Biotech, Lot#O101221). 1 µg of total RNA were used for reverse transcription by MightyScript Plus cDNA Kit (Sangon, B639252), qRT-PCR was performed using SYBR qPCR Master Mix (Vazyme, Q711-02) with a QuantStudio Real-Time PCR software (Thermo Fisher Scientific). GmACT3 (Glyma09g17040) was used as a reference gene, and the expression level was calculated based on the 2^−∆∆Ct method [32–34]. The whole above-ground parts of soybean seedlings after different treatments were collected for qPCR assay.

Plant materials and growth conditions

The soybean material employed in this experiment was Guixia 7 provided by Guangxi Academy of Agricultural Sciences. The soybean cultivar Guixia 7 was selected as the experimental material. Soybean seeds with full and uniform sizes were selected and placed in a petri dish, where they were sterilized with chlorine for a period of 16 h.

All A. thaliana plants used in this study were of the Col-0 ecotype. The Arabidopsis mutants pil1-4 was used in this experiment, which were provided by Li Lin from Fudan University.

Shade stress

The soybean seedlings were grown in standard illumination condition (total light: 450–540 µmol·m^− 2·s^− 1, far-red light: 20–80 µmol·m^− 2·s^− 1, red light: 250–280 µmol·m^− 2·s^− 1, blue light:140–150 µmol·m^− 2·s^− 1). The photoperiod is 16/8 h (day and night). Until V1 stage (unifoliate leaf fully grown, the first compound leaf margin separated), then the soybean seedlings were transferred to shade condition (total light: 450–540 µmol·m^− 2·s^− 1, far-red light: 320–350 µmol·m^− 2·s^− 1, red light: 180–210 µmol·m^− 2·s^− 1, blue light:140–150 µmol·m^− 2·s^− 1) for 0 h, 1 h, 6 h and 10 h. Sample the entire above-ground portion. The Arabidopsis seeds were allowed to germinate on plates containing 1/2 MS medium. The plates were incubated in growth chambers under white light (total light: 60–70 µmol·m^− 2·s^− 1, far-red light: 5–10 µmol·m^− 2·s^− 1, red light: 35–45 µmol·m^− 2·s^− 1, blue light:15–20 µmol·m^− 2·s^− 1) for 4 days at 22℃ under a 14/10 h photoperiod. The plates were moved to shade condition (total light: 40–50 µmol·m^− 2·s^− 1, far-red light: 30–45 µmol·m^− 2·s^− 1, red light: 20–30 µmol·m^− 2·s^− 1, blue light: < 10 µmol·m^− 2·s^− 1) or white light for 5 days before measuring the hypocotyl elongation.

Heat stress

The soybean plants were cultivated under standard conditions until they reached the V1 stage of growth, with a photoperiod of 16/8 h and a temperature of 25 °C. The following treatments were applied when the soybean reached the V1 stage: a control temperature of 25 °C and a heat stress temperature of 42 °C. Samples were taken from the control and heat stress groups at 0 h, 1 h, 3 h, and 6 h after treatment [35]. The entire aerial samples was sampled.

Drought stress

The soybean plants were cultivated under normal conditions until they reached the V1 stage of growth, which was marked by a photoperiod of 16/8 h and a temperature of 25 °C. The following treatments were administered to the soybean plant when it reached the V1 stage: the control treatment involved the application of 500 ml of water, while the drought stress treatment involved the application of 500 ml of a 20% PEG-6000 solution. Samples were collected from the control and drought stress treatments at 0 h, 1 h, 3 h, and 6 h after treatment [36]. The entire aerial samples was sampled.

Subcellular localization analysis

Coding sequences of each GmPIF without the terminator were cloned into the pEarleyGate103 binary vector through the Xho1 restriction site to generate the 35 S::GmPIFs-GFP, and all binary vectors were introduced into Agrobacterium tumefaciens strain GV3101. For subcellular localization analysis of GmPIFs, the GV3101 cells harboring 35 S: GmPIFs-GFP or a nuclear protein marker construct 35 S: mRFP-AHL22 were co-injected into Nicotiana benthamiana leaf epidermal cells [37]. GFP and RFP signals in transient transformed plants were observed under an inverted fluorescence microscope after 2 to 3 day.

Expression vector construction and Arabidopsis transformation

The coding sequences of GmPIF6c/GmPIL1 were ligated into the pEarleyGate 103 (35 S::GFP) vector. The vector was transformed into Arabidopsis pil1 mutant via the Agrobacterium-mediated floral dip method using GV3101, and a homozygous T3 generation was produced as previously described.

Statistical analysis

Excel 2021 was used for data statistics, Graphpad Prism 9.5 was used for data analysis. A two-tailed Student’s t-test was used to evaluate significant differences between two groups.

Results

Identification of GmPIF proteins

A total of twenty GmPIFs were identified following the screening of GmPIF homologs using the complete amino acid of eight AtPIF proteins as a reference. The potential GmPIFs were designated as GmPIF1 to GmPIF8 according to the Arabidopsis orthologs and GmPIFs were further marked with an a or b subgenomic label. The GmPIFs were classified into six groups compared with AtPIFs. The sequence length and molecular weight of the GmPIF proteins exhibited considerable variation. The GmPIF proteins displayed a wide range of amino acid lengths, from 154 amino acid (aa) to 723 aa, and molecular masses range from 17.6 KDa to 77.2 KDa. The isoelectric points of these GmPIF proteins spanned a range from 5.68 to 9.87 (Table 1).

Table 1.

Information about the PIF members found in soybean

Gene ID	Name	Chr	Genomic position	MW (kDa)	Protein localization	PI
Glyma.02G160200	GmPIF1a	2	18095264.18096522	17.6	Nucleus	9.45
Glyma.03G170300	GmPIF1b	3	38522369.38529232	55.0	Nucleus	5.86
Glyma.13G130100	GmPIF1c	13	24305965.24311744	56.4	Nucleus	5.68
Glyma.10G042800	GmPIF1d	10	3838844.3844535	54.3	Nucleus	5.84
Glyma.03G227800	GmPIF3a	3	42980227.42984347	67.8	Nucleus	5.75
Glyma.19G224700	GmPIF3b	19	47691501.47696511	69.0	Nucleus	5.72
Glyma.10G142600	GmPIF3c	10	37701441.37706329	74.2	Nucleus	6.43
Glyma.20G091200	GmPIF3d	20	33451707.33456649	77.2	Nucleus	5.95
Glyma.02G282100	GmPIF4a	2	46437888.46443042	62.2	Nucleus	6.58
Glyma.14G032200	GmPIF4b	14	2338828.2344252	62.1	Nucleus	6.97
Glyma.08G303900	GmPIF4c	8	42217649.42223362	57.8	Nucleus	6.41
Glyma.18G115700	GmPIF4d	18	14099161.14105084	60.5	Nucleus	6.58
Glyma.19G222000	GmPIF6a	19	47396909.47399851	57.4	Nucleus	8.87
Glyma.03G225000	GmPIF6b	3	42726377.42729203	57.4	Nucleus	8.75
Glyma.10G138800	GmPIF6c	10	37245347.37247924	49.2	Nucleus	8.33
Glyma.01G133500	GmPIF8c	1	45366512.45368816	20.9	Nucleus	9.87
Glyma.03G034000	GmPIF8d	3	3971585.3975700	44.2	Nucleus	9.14
Glyma.11G166128	GmPIF8a	11	1986303.1993346	50.0	Nucleus	8.63
Glyma.01G076900	GmPIF8b	1	18297565.18303581	49.2	Nucleus	8.88
Glyma.06G199600	GmPIF7	6	18231850.18238006	44.0	Nucleus	8.49

Chr, chromosome; PI, theoretical isoelectric point; MW, molecular weight

Structural analysis of GmPIFs

The exon-intron patterns of soybean GmPIFs were obtained through the screening of corresponding genomic DNA sequences and annotation files, with the objective of investigating the diversity of GmPIF gene structures (Fig. 1A). The GmPIFs were found to contain four to eight exons. GmPIF1a and GmPIF8d possessed only four exons, GmPIF3a had eight, and other GmPIFs had six to seven exons. To further demonstrate the structure of the GmPIF proteins, a scheme was constructed based on the MEME-motif scanning result (Fig. 1B). In this study, the highly conserved bHLH domains were identified in all GmPIF proteins. Additionally, GmPIF proteins possessed the distinctive phyA and/or phyB binding motifs APA and/or APB. However, the number of these motifs present in each GmPIF was different. For example, with the exception of GmPIF1a and GmPIF8d, all the remaining GmPIFs possessed APB motif. GmPIF3s and GmPIF4s even contained APA domains, and GmPIF1c and GmPIF1d also possessed APA domains which differs from GmPIF1a and GmPIF1b, respectively. The existence of APA and APB motifs suggests that GmPIFs are extensively involved in light regulation pathways.

Fig. 1.

The phylogenetic tree, gene structure and conserved motifs analysis of GmPIFs. A. Analysis of gene structure of GmPIF gene family. B. The unrooted phylogenetic tree constructed using GmPIF protein sequences. C. Conservative motifs analysis of GmPIFs. The top 15 motifs identified by the MEME are represented by the number

Analysis of cis-acting elements in the GmPIF gene promoters

Analyzing the cis-acting in promoters helps to understand the precise regulation of gene. Consequently, our investigation delved into the cis-acting elements within the promoter regions of GmPIF genes to elucidate their prospective regulatory patterns. We analyzed the 2.0-kb upstream sequences from the translation start sites of GmPIF genes with the Plant CARE website. There are abundant light response elements, stress response and hormone response elements in promoter regions (Fig. 2). The most prevalent among these putative cis-acting elements were light-responsive elements, including G-box (TACGAT), ACE (CTAACGTATT), LAMP (CTTTATCA), as well as others. The presence of these cis-acting elements suggests that GmPIFs may be involved in a number of different responses, the full implications of which require further investigation.

Fig. 2.

Cis-acting element analysis of the GmPIF genes from upstream 2000 bp sequence to the transcription start site. The number at the bottom indicates to the translation initiation codon, ATG. Different cis-acting element were indicated by distinct colors and shapes

Chromosome distribution of GmPIF genes

A chromosomal location analysis showed that the 20 GmPIF genes were unevenly distributed on twelve chromosomes of Glycine max (Fig. 3). For example, there are four GmPIF genes (GmPIF1b, GmPIF3a, GmPIF6b, GmPIF8c) on chromosomes 3 (Chr 3). Only one GmPIF was located on the Chr 6, Chr 8, Chr 13, Chr 14, Chr 18, Chr 20 and Chr 21. Likewise, there were two GmPIFs on Chr 1, Chr 2 and Chr 19. And three GmPIFs were found on Chr 10.

Fig. 3.

Chromosomal localization of GmPIF genes. Individual chromosomes are represented by white bar, with the chromosome number marked to the up is in millions (Mb)

Phylogeny and synteny of PIF-coding genes

A phylogenetic analysis was subsequently conducted based on the protein sequences of PIF from Glycine max and Arabidopsis to investigate their evolutionary relationship. We constructed a neighbor-joining phylogenetic tree with MEGA6 software. As shown in Fig. 4. Twenty GmPIFs were clustered into six subgroups, Group I to Group VI. Group I, Group II, Group III, and Group VI each contain four GmPIF members, while Group IV contains three.

Fig. 4.

The phylogenetic tree of the PIF proteins from soybean and Arabidopsis. Unrooted phylogenetic tree of total 28 (8 Arabidopsis thaliana PIF and 20 Glycine max PIF) proteins. The AtPIF and GmPIF protein sequences were aligned by MEGA-X with neighbor-joining method. Roman numerals line up with the PIF subfamily. The tree demonstrated the six phylogenetic subfamilies marked with different colors

Gene duplication analysis of PIFs in soybean

To explore GmPIF gene duplication during evolution, MCSanX software was used to analyze PIF gene replication events and calculate Ka/Ks (Table 2) in Glycine max. Ultimately, we identified 5 paralogous gene pairs in Glycine max (Fig. 5). Furthermore, we calculated the substitution rates of nonsynonymous (Ka) versus synonymous (Ks) mutations in order to estimate the selection pressure on GmPIF sequences, with a Ka/Ks ratio less than 1 indicative of neutral selection. The results demonstrated that all Ka/Ks values were less than 1, suggesting that intense purifying selection pressure was exerted on the duplicated GmPIFs to eliminate deleterious mutations.

Table 2.

Ka/Ks values of GmPIFs duplicate genes

Gene A	Gene B	Ka	Ks	Ka/Ks
GmPIF3d	GmPIF3c	0.039642724	0.081837348	0.484408707
GmPIF4a	GmPIF4b	0.051554515	0.115726141	0.44548721
GmPIF4c	GmPIF4d	0.02581761	0.141043388	0.183047292
GmPIF6b	GmPIF6a	0.041589443	0.097858466	0.424995857
GmPIF8b	GmPIF8a	0.030803174	0.191510019	0.16084367

Fig. 5.

Synteny analysis of PIF genes in Glycine max. Five segmental duplication pairs of GmPIF genes are highlight with blue lines. Tandem duplicated genes are indicated on the outer ring of the circle

Expression profiles of the GmPIF genes

To explore the possible biological functions of GmPIFs, the expression patterns of these GmPIFs in nine soybean tissues were analyzed, including flowers, leaves, nodules, pods, roots, root hairs, seeds, apex meristem and stem. As shown in Fig. 6, GmPIF1c showed the highest expression level in flowers, whereas GmPIF4b, GmPIF8a and GmPIF6c/GmPIL1 exhibited the highest expression level in leaves, suggesting that GmPIFs may play differential roles in various stages of soybean growth and development.

Fig. 6.

Expression profiles of GmPIFs in various tissues. The heatmap showed the expression levels of GmPIF genes in nine tissues, including flowers, leaves, nodules, pods, roots, root hairs, seeds, shoot apical meristems and stems

Expression analysis of GmPIFs in response to shade stress

Previous studies in other species has demonstrated that the expression of certain PIFs are involved in light regulatory pathways, and most of them were sensitive to shade condition, therefore, we analyzed the expression mode of GmPIFs in shade stress. We examined the changes in 20 GmPIFs expression levels under shade stress by qRT-PCR. The results showed that GmPIF3b, GmPIF6a, GmPIF6b and GmPIF6c/GmPIL1 increased significantly after shade stress, with the extension of time, the expression level gradually increased, which increased by 6, 3, 24 and 9 times at 10 h, respectively (Fig. 7). In addition, after 1 h of shade treatment, the expression of GmPIF4a was significantly increased, there was an eightfold increase at the 1 h time point. Subsequently, the expression level exhibited a gradual decline over time. The expression of other GmPIF members, including GmPIF1s, GmPIF3a, GmPIF3c, GmPIF3d, GmPIF4b, GmPIF4c, GmPIF4d, GmPIF7 and GmPIF8s were not significantly induced by shade signal. Based on the expression patterns of GmPIFs, GmPIF3b and GmPIF6s may play important roles under shade conditions.

Fig. 7.

The gene expression of GmPIF genes in response to shade stress. Soybean plants in V1 stage were placed in growth chambers at white light (WL) and shade stress (SD), and aerial samples were taken at 0 h, 1 h, 6 h and 10 h respectively. Total RNA was isolated from the samples, and the transcription level of GmPIF genes was detected. The bar indicate the mean ± SD of three independent raplicates. Student’s t-test: * p < 0.033; ** p < 0.002; and *** p < 0.0002, **** p < 0.0001

Subcellular localization of GmPIF6a GmPIF6b and GmPIF6c

All members of the GmPIF6 subgroup exhibited sensitivity to shade response. Therefore, we conducted further investigation into the subcellular localization of GmPIF6a, GmPIF6b and GmPIF6c/GmPIL1. Nuclear localization is one of the key common features of transcription factors. GmPIF-green fluorescent protein (GFP) constructs were injected into N. benthamiana leaves through agroinfiltration. Following a two-day dark growth, the samples were observed under a confocal microscope. Remarkably, all the three GmPIF6 homologs were detected within the nucleus (Fig. 8).

Fig. 8.

Subcellular localization of GmPIF6a, GmPIF6c and GmPIF6c. Green Fluorescent Protein (GFP) fluorescence signals was observed in epidermal cells isolated from infiltrated tobacco leaves. The nucleus was marked by Red Fluorescent Protein (RFP) fluorescence

GmPIF6c/GmPIL1 complement the arabidopsis pil1 mutant phenotype

Research has demonstrated that PIL1 can bind to its own promoter and negatively regulate its own expression under shade, PIL1 collaborates with PIF4, forming interactions that collectively act to inhibit plant growth [38]. GmPIF6 contains three homologous proteins, GmPIF6a, GmPIF6b and GmPIF6c/GmPIL1, which was in the same branch of the evolutionary tree as Arabidopsis PIL1. AtPIL1 was the closest to GmPIF6c/GmPIL1. To study the regulatory mechanism of GmPIF6c/GmPIL1 under shade stress. We overexpressed GmPIF6c/GmPIL1 in the pil1 Arabidopsis mutant. Under white light conditions, the hypocotyl length of pil1 mutant and the GmPIF6c/GmPIL1 overexpression (GmPIF6c/GmPIL1-OE) in pil1 transgenic lines showed a similar phenotype (Fig. 9A). Under shade stress, the wild-type (WT) Arabidopsis exhibited a notable increase in hypocotyl length. Furthermore, the pil1 mutant hypocotyl exhibited a notable increase in length when compared to the wild-type. Interestingly, the GmPIF6c/GmPIL1-OE transgenic lines exhibited shorter hypocotyls than the pil1 mutant (Fig. 9B). Therefore, we suspect that GmPIF6c/GmPIL1 could negatively regulate soybean shade avoidance.

Fig. 9.

GmPIF6c/GmPIL1 rescued the phenotype of Arabidopsis pil1 mutant seedings. Hypocotyl phenotypes of wild-type (WT), pil1 mutant and GmPIF6c in pil1 under white light and shade stress. A. Represent images of 9-day-old seedings. B. Display corresponding hypocotyl lengths. The bar indicate the mean ± SD of six independent raplicates. Student’s t-test: * p < 0.033; ** p < 0.002; and *** p < 0.0002, **** p < 0.0001

Expression analysis of GmPIFs in response to heat stress

The analysis of cis-acting elements revealed the presence of temperature-responsive elements in the promoter region of GmPIFs. Consequently, we investigated the response of GmPIFs to heat stress. Under heat stress, there was a significant up-regulation in the expression levels of GmPIF3c, GmPIF4b, GmPIF4c, and GmPIF4d, which exhibited the most pronounced increase (Fig. 10). In contrast, Other GmPIF members did not show a significant induction by heat stress (Fig. 10).

Fig. 10.

The gene expression of GmPIF in response to heat stress. Soybean plants in V1 stage were placed in growth chambers at 25℃ and 45℃, and aerial samples were taken at 0 h, 1 h, 3 h and 6 h, respectively. Total RNA was isolated from the samples, and the transcription level of GmPIF genes was detected. The bar indicate the mean ± SD of three independent raplicates. Student’s t-test: * p < 0.033; ** p < 0.002; and *** p < 0.0002, **** p < 0.0001

Expression profiles of GmPIFs in response to drought stress

The promoter of GmPIFs contained cis-acting elements that respond to drought stress. Thus, the response of GmPIFs to drought stress was studied. Under drought stress, the expression levels of most GmPIFs initially increased before subsequently declining (Fig. 11). Specifically, the transcript levels of GmPIF1s, GmPIF4c, GmPIF4d, GmPIF8a, GmPIF8b, GmPIF8c, and GmPIF7 exhibited the greatest increase at 1 h. GmPIF3c, GmPIF3d, GmPIF4a and GmPIF4b reached their peak expression at 3 h. Other GmPIF members were not significantly induced by drought stress.

Fig. 11.

The expression patterns of GmPIFs under drought stress. Soybean plants in V1 stage were placed in growth enviroment at drought stress, and aerial samples were collected after 0 h, 1 h, 3 h and 6 h drought stress respectively. Total RNA was isolated from the samples, and the transcription level of GmPIF genes was detected. The bar indicate the mean ± SD of three independent raplicates. Student’s t-test: * p < 0.033; ** p < 0.002; and *** p < 0.0002, **** p < 0.0001

Discussion

Plant PIFs are bHLH family transcriptional factors that play significant roles in plant growth and development. They participate in the regulation of a number of processes, including the regulation of light signals network, circadian rhythm, flowering, seed germination, shade avoidance and abiotic stress responses [39–41]. There are eight PIF members in Arabidopsis including PIF1-PIF8. According to previous reports, PIF1 has been reported to inhibit seed germination, and PIF3 regulates photomorphogenesis by repressing photomorphogenic development in the dark and promoting light-induced responses under light [42, 43]. PIF4 and PIF5 have been demonstrated to play a role in the regulation of shade avoidance and high temperature responses [44]. PIF2/PIL1 is a relatively special member of the PIF family. In recent years, PIF2/PIL1 has been found to inhibit plant shade avoidance response and negatively regulate the whole signaling pathway [38]. To date, the PIF family has been studied in multiple species such as maize, rice and tomato [20–22]. Nevertheless, there is a paucity of research on PIFs in soybean. Based on homology search and conserved domain confirmation, twenty PIF genes were identified in Glycine max, which were classified into six subfamilies according to their structures. Including GmPIF1s, GmPIF3s, GmPIF4s, GmPIF6s, GmPIF7 and GmPIF8s.

Sequence comparison showed that GmPIFs and AtPIFs had high similarity in amino acid sequence and gene structure. All GmPIFs members had bHLH domain, which suggested GmPIFs had the ability to bind to downstream genes. In addition, some GmPIFs contained APA and APB domains, indicating that they have the ability to participate in light signaling pathway by binding to phyA and phyB.

The analysis of GmPIFs expression patterns under shade stress is valuable for elucidating the regulatory mechanism of GmPIFs in shade avoidance response. Previous studies have identified PIL1, PIF4, PIF5 and PIF7 as the most responsive PIFs to plant shade signals. Under shade conditions, PIF4 and PIF5 regulate elongation growth by directly controlling auxin biosynthesis and the expression of genes encoding auxin signaling elements [14]. In this study, we found that GmPIF3b, GmPIF4a, and GmPIF6s all exhibited sensitivity to shade stress, consistent with previous findings in Arabidopsis. PIL1 functions as a negative regulator of the shade avoidance response and exhibits robust expression upon shade induction. The elongation response in lines overexpressing PIL1 was attenuated under shade conditions, whereas deletion mutants displayed enhanced elongation [38]. Furthermore, PIL1 has the capacity to directly bind to the G-box motif, a crucial element of the PIF pathway, indicating its ability for direct DNA binding and regulation. Previous studies have demonstrated that PIL1 indirectly modulates shading responses by interacting with other members of the PIF family (such as PIF4) to regulate downstream gene expression [45]. In this study, it was evident from the phylogenetic tree that AtPIL1 and GmPIF6c/GmPIL1 coexist on the same branch, which indicated their close relationship. The ectopic expression of GmPIF6c/GmPIL1 gene was observed to inhibit hypocotyl elongation under shade stress in Arabidopsis. Therefore, it could be speculated that soybean GmPIF6c/GmPIL1 played a pivotal role in the shade avoidance response of plants. Based on these findings, we will further investigate the regulatory mechanism underlying the involvement of GmPIF6c/GmPIL1 in soybean’s shade avoidance response.

Thermomorphogenesis is similar to the shade avoidance response, PIF4 is a central regulator of this process in plants. The expression of PIF4 is stimulated by high temperatures. High temperature induced hypocotyl and petiole growth, leaf hyponasty and stomatal index are repressed in the pif4 null mutant [46–48]. In this study, we found that the expression of GmPIF4b, GmPIF4c and GmPIF4d was significantly up-regulated under heat stress. This result is similar to previous studies in Arabidopsis. Intriguingly, the expression of GmPIF3c was significantly higher than GmPIF4s. Previous investigations on PIF3 primarily focused on its role in photomorphological establishment and low-temperature response, while no relevant literature was found regarding its involvement in high temperature response. Therefore, GmPIF3c may be involved in the regulation of the plant’s high-temperature response, but the specific molecular mechanisms need to be further studied.

Studies have demonstrated that PIFs play a crucial role in facilitating plant adaptation to drought stress. AtPIF3 in Arabidopsis has the closest genetic relationship to the ZmPIF1 and ZmPIF3 genes of maize. It also reduces the rate of transpiration in response to drought stress and improves the drought tolerance of plants [48–50]. In this study, the expression levels of GmPIF1s, GmPIF3s, GmPIF4s, GmPIF8s and GmPIF7 were significantly up-regulated following drought treatment. Moreover, multiple GmPIFs were found to be involved in the response to drought stress in soybean, suggesting a more intricate drought response system compared to Arabidopsis.

Conclusion

In summary, our investigation represents a comprehensive and systematic exploration of the PIF transcription factor family in Glycine max L. We successfully identified a total of 20 GmPIF genes, which were distributed across the soybean genome. We proceeded to conduct a comprehensive analysis of the distinctive characteristics displayed by each member within the GmPIF gene family. GmPIFs were distributed on 12 chromosomes and categorized into six subgroups. These proteins contained conserved bHLH domains in their C-terminal regions, some GmPIFs contained APA/APB domains. The preliminary investigation indicated that the GmPIFs genes play a role in responses to environmental stresses, including shade, heat, and drought. Meanwhile, GmPIF6c/GmPIL1 may negatively regulate plant shade avoidance response. Our findings provide a basis for future studies aimed at elucidating the biological functions of PIF transcription factors in Glycine max.

Electronic Supplementary Material

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Author contributions

JB D designed the research, DW M and ZW S performed the experiments, DW M, HK J and HY G wrote the paper with contributions from the other authors. YH L, LQ L, YP Z, XX Z, SL L, JX Y, L Y, CY L analyzed the data. All authors read and approved the final manuscript.

Funding

The work was supported by funding from the National Natural Science Foundation of China (32171939, 32372059), Sichuan Science and Technology Program (2023YFH0011).

Data availability

Data availability statement: All relevant data are within the manuscript and its Additional files.

Declarations

Ethics approval and consent to participate

All studies for using plants were carried out following the relevant guidelines. All plant materials are permitted.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.