Identification of WRKY gene family in Dioscorea opposita Thunb. reveals that DoWRKY71 enhanced the tolerance to cold and ABA stress

Identification of WRKY gene family in D. opposita

Due to the lack of genomic data of this species, the WRKY gene family sequence of yam was identified by the full-length transcriptome data of yam tuber at different developmental stages in our laboratory (Nuohezhiyuan Company, Beijing in 2020). Hidden Markov model (HMM) profiles based on the Oryza sativa WRKY (PFAMID: PF03106.15) protein from the PFAM database (http://pfam.xfam.org/) were used (Ross, Liu & Shen, 2007) (Table S1). BLASTp analysis of the D.opposita transcriptome database was performed using HMMER 3.0., setting the cut-off E-value as 0.1. The presence of WRKY motif was confirmed using the online program PFAM (http://pfam.xfam.org/search), Conserved Domains DataBase Search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) and SMART tool (http://smart.embl-heidelberg.de/). The open reading frame (ORF) of the candidate sequences with complete WRKY transcription factors was predicted from the search results using ORF Finder (https://www.ncbi.nlm.nih.gov/gorf/gorf.html) (Portions of this text were previously published as part of a preprint (https://www.researchsquare.com/article/rs-2164173/v1)) (Xing et al., 2022).

Sequence analyses and conserved motif detection of D. opposita

Biophysical properties of WRKY members, such as peptide length, molecular weight (MW), and isoelectric point (pI) were predicted using the online ExPasy program (http://www.expasy.org/tools/) (Wilkins et al., 1999). Protein subcellular localization prediction was done using an online website (https://wolfpsort.hgc.jp/). The online software MEME (https://meme-suite.org/meme/) was employed to identify conserved motifs among all DoWRKY genes and annotated with InterPro Scan. The parameters used in this study were as follows: 0 or 1 occurrence per sequence; maximum number of motifs: 10; and other optional parameters were set to default (Bailey et al., 2015).

Multiple sequence alignment and phylogenetic analysis

All 25 DoWRKYs proteins with WRKY domains and 44 selected OsWRKYs proteins were used to generate multiple protein sequence alignments using Clustal Omega with default settings. A phylogenetic tree was constructed with the maximum likelihood method with MEGAX software (Tamura et al., 2011). Branch support for the tree topology was estimated through bootstrap analysis with 1,000 replicates. The online ITOL (http://itol.embl.de/help.cgi) tool was used to visualize the phylogenetic tree.

Plant materials and treatments

Tissue culture seedlings of D. opposita (Dioscorea opposita Thunb., Dahechangyu, D. opposita) was used as the experimental material. The seedlings were cultured in an artificial climatic chamber at 25 °C with a 16/8 h photoperiod (day/night). For stress treatments, 6-week-old seedlings were exposed to different conditions: ABA (100 μmol/L), cold (4 °C), MeJA (100 μmol/L), and cultured for a total of 24 h. Leaves were collected at 0, 1, 6, 12 and 24 h after treatment. For different developmental stages, yam tubers of each cultivar were randomly sampled in the morning at six different stages: after 90, 105, 120, 135, 150 and 165 days. All samples were immediately frozen in liquid nitrogen and stored at −80 °C for subsequent total RNA extraction and gene expression analysis. Three biological replicates were used for each sample.

RNA extraction and quantitative real-time PCR

Total RNA was extracted using RNA extraction kit (9769; TaKaRa, Beijing, China) according to the manufacturer’s instructions. First-strand cDNA synthesis was performed using TransScript One-Step gDNA Removal and cDNA Synthesis SuperMix (RR047A; TaKaRa, Beijing, China). The quality of the RNA samples were tested using NanoDrop2000c (Thermo Fisher Scientific, Waltham, MA, USA) and gel electrophoresis. Quantitative real-time PCR (qRT-PCR) was carried out using TB Green Premix Ex Taq II (6210A; TaKaRa, Beijing, China) on an FTC-3000P system (Funglyn Biotech, Toronto, Canada). Design the primer used Primer Premier 5.0 with the primers listed in Table S2. The PCR conditions consisted of denaturation at 95 °C for 30 s, followed by 40 cycles of 5 s at 95 °C, and 30 s at 60 °C, and a final step at 4 °C. All samples were tested with three technical replicates and three independent biological replicates. The relative expression level was calculated using the 2^−ΔΔCT method with yam ribosomal RNA (18S) as the internal control (internal reference genes screened by our laboratory) (Shao et al., 2021).

Subcellular localization analysis

After screening the transcriptome sequencing results of yam and subsequent studies, the DoWRKY71 gene was found to be responsive to tuber expansion, cold, ABA, and MeJA stresses, and was selected for further research. Subsequently, its full length was cloned, and it was named DoWRKY71 (Genbank number: OP380252) after comparison with NCBI. The ORF of DoWRKY71 was amplified using the specific primers, DoWRKY71-SF/SR, which contained restriction enzyme cutting sites (Table S2). Subsequently, the amplified ORF was inserted into the CaMV35S::GFP vector. Following sequence confirmation, the CaMV35S::GFP- DoWRKY71 recombinant plasmid and CaMV35S::GFP (control) plasmids were transferred into Agrobacterium tumefaciens LBA4404 (Weidi Biotechnology, Shanghai, China). The empty CaMV35S::GFP vector was used as the control. Two types of Agrobacterium tumefaciens were then used to infect Nicotiana benthamiana leaves. After 2–3 days, infected parts of leaves were cut and used for fluorescent signal detection using a laser scanning confocal microscope (C2-ER; Nikon, Tokyo, Japan) (Aung et al., 2017).

Generation and identification of transgenic plants

The encoding sequence of DoWRKY71, along with the restriction sites of KpnI and BamHI (specific primers, DoWRKY71-ZF/ZR), was cloned into pPZP221 to construct the expression vector pPZP221-DoWRKY71. The Agrobacterium tumefaciens strain EHA101 (Weidi Biotechnology, Shanghai, China) was used to transform the wildtype (WT) tobacco via the freeze-thaw method. The plants were selected on MS medium supplemented with cefotaxim (500 mg/L) and gentamicin (50 mg/L). DNA and RNA from the leaves of transgenic and WT tobacco were detected using Plant Kit (DNA: DP305; TIANGEN, Beijing, China; RNA: 9769; TaKaRa, Beijing, China). Three positive transgenic lines (T2, T3 and T4) were selected for further gene-functional verification.

Treatment and functional analysis of overexpressed DoWRKY71 in tobacco

The 4-week-old transgenic and WT tobacco plants were transplanted into plug trays and allowed to grow for 2 weeks. Transgenic and WT tobacco plants with similar growth activity were selected and treated with ABA (100 μmol/L) and 4 °C, and their leaves were selected at 0, 1, 6, 12 and 24 h after treatment. Collection and storage methods are the same as plant materials and treatments. WT tobacco was used as control to determine various physiological indicators, including chlorophyll content, soluble protein and malondialdehyde (MDA) content, peroxidase (POD), superoxide dismutase (SOD) (Li, 2000). The content of gibberellin (GA) and ABA were measured using the kit (Shanghai Best Biotechnology Co., Ltd., Shanghai, China). The production rate of superoxide anion radical (O₂·-) and the content of hydrogen peroxide (H₂O₂) were detected by staining of nitroblue tetrazolium (NBT) and diaminobenzine (DAB) (Yang et al., 2018). The experiments were repeated at least three times, with three biological replicates for each sample. Stomatal openings of transgenic and WT tobacco leaves under ABA treatment were observed and photographed with an electron microscope (C2-ER; Nikon, Tokyo, Japan). The index of pore size was calculated using Digimizer software. The relative expression levels of DoWRKY71 gene in transgenic tobacco were determined at different time points during 4 °C and ABA treatments.

Statistical analysis

All data were based on three replicates. Excel 2019 was used for data processing and mapping. SPSS 25 (IBM, Armonk, NY, USA) was used for statistical analysis. The gene expression and physiological indicator data were tested with p < 0.05 or p < 0.01 significance level.

Identification and sequence analysis of D. opposita

After manually removing redundant entries through screening (manually deleting the genes without ORF/WRKY motif) and validation of the search results, a number of 25 WRKY family genes were characterized from D. opposita. 25 proteins were named according to NCBI database comparison and their basic characteristics are shown Table 1. The coding sequence lengths of DoWRKY proteins varied from 191 to 718 amino acids, from 21.56 to 78.02 kDa in molecular weight, and from 4.88 to 9.93 in their isoelectric points. Different amino acids have different isoelectric points due to their different structures. The isoelectric point of acidic amino acid solution must be less than seven, and the isoelectric point of basic amino acid must be greater than seven. Of the 25 proteins we isolated, 12 were basic and 13 were acidic. Protein molecular weight is mainly used to determine the size of proteins, and can be used to measure the complexity of proteins. Members of the same gene family have differences in protein size and complexity. The subcellular localization of the DoWRKY proteins was predicted by using WOLF PSORT, which indicated that most of the DoWRKY proteins were likely located in the nucleus, with only a few in the plasmolemma (Chang et al., 2022; Li et al., 2024).

Phylogenetic analysis and multiple sequence alignment of DoWRKY proteins

To investigate the evolutionary relationships and classification of the DoWRKY and Oryza WRKY (OsWRKY) proteins, a phylogenetic tree was constructed with the 25 DoWRKY proteins and OsWRKY proteins (Fig. 1). The results indicated that the 25 DoWRKY proteins were divided into three categories (I–III) according to the groups in Oryza. Group I contained the highest WRKY members with 7 DoWRKYs, containing one C₂H₂ zinc-finger motif and two WRKY domains. A total of 15 DoWRKY proteins included one WRKY domain and one C₂H₂ zinc-binding motif, which were considered to be Group II. Among them, Group lla contains 5 DoWRKY proteins, Group IIb contains none, Group IIc contains three DoWRKY proteins, Group IId contains five DoWRKY proteins, and Group IIe contains two DoWRKY proteins. There are three DoWRKY proteins classified as Group III, containing a single WRKY domain and a C₂HC zinc-binding motif Table 1.

Based on previous studies, we randomly selected the representative sequence of Oryza for comparison with yam. In addition, conserved amino acids in the WRKY domain analysis via multiple sequence alignments of the DoWRKY proteins were performed. Among the 25 DoWRKY proteins in the yam, 24 proteins have the “WRKYGQK”, the core dominant terminal. While only one protein (DoWRKY50) has single amino acid substitutions in the WRKYGQK domain and “WRKYGKK” domain. The 22 DoWRKY proteins had C₂H₂ zinc-finger, and 3 DoWRKY proteins (DoWRKY70, DoWRKY38 and DoWRKY46) had C₂HC zinc-finger at the N-terminal (Figs. 2 and 3).

The classification of DoWRKYs confirmed the diversity of their protein structures, suggesting that different subfamily members may have different regulatory functions, and that orthologous genes are likely to have similar functions. These results revealed that WRKY proteins existed in various plant species.

Conserved motifs and sequence alignment of DoWRKY proteins

To further analyze the characteristics of DoWRKYs, the MEME online tool was used to predict the potential conserved motifs. A total of 10 motifs were identified, with lengths ranging between 16 and 50 amino acids (Fig. 4). Motif 1 represents the WRKY domain, and Motif 2 represents the zinc finger motif. These two domains were the two highly conserved motifs contained present in every WRKY member. DoWRKY40a, DoWRKY40b and DoWRKY40c contained the highest number of conservative motifs, including six. DoWRKY38, DoWRKY69, DoWRKY46 and DoWRKY50 contained the fewest number of conservative motifs, only two. The clustered DoWRKY pairs, such as DoWRKY2a/2b, DoWRKY51a/51b, and DoWRKY40a/40c, displayed a highly similar motif distribution. The conserved motifs of the same subgroup of DoWRKY proteins are similar, but discrepant between groups. This conserved domain analysis reflected the similarity of amino acid sequences within groups and the difference of amino acid sequences between groups. This also suggested that the phylogenetic classification was reliable.

Expression analysis of DoWRKYs in different developmental stages of yam tuber

By comprehensively considering the protein family classification and subcellular localization, we selected eight genes from six groups to analyze the expression level of yam under different growth and development stages. The expression of DoWRKY40b was down-regulated at 105 days and up-regulated at other periods, reaching the highest value at 165 days. DoWRKY69 was down-regulated in the whole process. DoWRKY33 reached its peak expression at 105 d, which is 8.11 times higher than at 90 days. Three genes DoWRKY20, DoWRKY21 and DoWRKY71, showed the same trend: up-regulation expressed in the earlier stage and down-regulation expression in the mature stage (Fig. 5).

DoWRKYs gene expression following abiotic stress by qRT-PCR

To further investigate the role of the 8 DoWRKYs in abiotic stress responses, we subjected them to various treatments including cold, ABA and MeJA.

Following cold treatment (Fig. 6A), the expression levels of two genes (DoWRKY40b and DoWRKY71) increased by more than twofold, among which the expression of DoWRKY40b was 6.38 times and DoWRKY71 was 11.53 times higher than that of the control. The expression of two genes (DoWRKY33 and DoWRKY69) increased significantly at 1 h, decreased at 6 h, and then their expression significantly increased and peaked at 24 h. The expression levels of DoWRKY22 and DoWRKY40b genes showed an overall upward trend, and became stable at 12 h. The expression levels of DoWRKY21 and DoWRKY22 genes showed an overall upward trend, reaching the highest level at 12 h and becoming stable.

After ABA treatment, the expressions of two genes (DoWRKY20 and DoWRKY71) first increased and then decreased (Fig. 6B). The expression levels of DoWRKY21 increased significantly over the control. The expression levels of DoWRKY33 and DoWRKY70 were about eight times higher than that of the control 24 h after spraying treatment. The expression levels of DoWRKY69 decreased significantly compared to the control.

For the case of MeJA stress (Fig. 6C), the expression of DoWRKY21 and DoWRKY40b showed a downward trend. DoWRKY33 was up-regulated during the whole process, and the expression at hour 24 was 8.21 times that of 0 h. The expression level of the three genes DoWRKY20, DoWRKY33 and DoWRKY69 reached the highest value at 24 h.

Subcellular localization of DoWRKY71

To furnish additional evidence for the plausible involvement of DoWRKY71 in transcriptional regulation, we constructed a DoWRKY71-GFP fusion vector alongside a GFP fusion expression empty vector for use as a control. These vectors were driven by the CaMV35S promoter and were subsequently introduced into the epidermis of tobacco leaves through the Agrobacterium-mediated transient expression method. Subsequently, they were then visualized under a confocal microscope. The GFP fluorescence emanating from the CaMV35S-GFP-DoWRKY71 exhibited a robust signal solely within the nucleus, whereas the CaMV35S-GFP vector displayed a more even distribution throughout the tobacco cells (Fig. 7).

Identification and regeneration of transgenic tobacco with DoWRKY71 gene

To examine the cold stress tolerance of DoWRKY71 in transgenic tobacco, we employed the recombinant vector pPZP221-DoWRKY71. Transgenic lines were meticulously selected for PCR and RT-PCR detection, which indicated the successful integration of the DoWRKY71 gene into the tobacco genomic DNA and its expression at the transcriptional level. Subsequently, three independent transgenic lines overexpressing DoWRKY71(T2-T4) were acquired through resistance selection to facilitate subsequent experiments (Fig. 8).

Physiological characteristics of overexpressed DoWRKY71 tobacco under cold stress

During our investigation of the cold stress tolerance of DoWRKY71 in transgenic tobacco, we observed that the growth of transgenic tobacco under control conditions surpassed that of the wild-type tobacco, and the cold tolerance of transgenic tobacco exhibited notable enhancement (Fig. 9A). Furthermore, the chlorophyll content of the transgenic plants was significantly higher than that of the wild-type plants after 6 h of treatment (Fig. 9B). Notably, the soluble protein content initially declined, reaching its peak 24 h after treatment began, and the transgenic plants displayed significantly higher levels in comparison to the wild-type plants (Fig. 9C). Moreover, the activities of SOD and POD demonstrated an upward trend, with the transgenic plants exhibiting significantly higher levels than the wild-type plants (Figs. 9D and 9E).

Low temperature typically induces membranous peroxidation and overproduction of reactive oxygen species, eventually leading to oxidative stress. NBT and DAB staining were performed on tobacco subjected to cold treatment (Fig. 9F), and while staining was evident in both control wild-type and transgenic plants, the O₂·- and H₂O₂ levels were lower than those observed in the wild-type plants. Additionally, MDA content in both transgenic and wild-type plants increased with time during low-temperature stress, but experienced a slight decrease after 24 h. Notably, the MDA content accumulated by transgenic lines in control and experimental groups was significantly lower than that of the wild-type plants (Fig. 9G). These findings suggest that overexpression of DoWRKY71 regulates reactive oxygen species homeostasis and reduces oxidative damage in tobacco.

Physiological characteristics of overexpressed DoWRKY71 tobacco under ABA treatment

As the spraying time extended, the soluble protein content initially declined and then rose. In both the control and treatment groups, the transgenic plants exhibited significantly higher soluble protein content than that observed in the wild-type tobacco (Fig. 10A). Furthermore, the trends in SOD and POD activity were essentially similar to those observed under cold treatment, with POD activity significantly higher than that observed in the wild-type tobacco (Figs. 10B and 10C). As for GA content, it displayed an overall upward trend, peaking at 12 h, whereas ABA content initially increased and then decreased. The GA content in the transgenic lines, both in the control and experimental groups, was significantly lower than that observed in the wild-type plants (Fig. 10D), while ABA content was significantly higher than that in the wild-type plants (Fig. 10E).

Microscopic observation (100×) of the stomata in DoWRKY71 transgenic plants and wild-type plants under ABA treatment revealed that, under normal conditions, the stomatal pore size index of T3 and T4 was significantly larger than that observed in the wild-type plants (Fig. 11A). However, upon treatment with 100 μmol/L ABA spray, the stomatal index of DoWRKY71 transgenic lines was significantly smaller than that observed in the wild-type plants (Fig. 11B). These results provide compelling evidence that the overexpression of DoWRKY71 promotes ABA-mediated stomatal closure in tobacco.

Expression analysis of DoWRKY71 gene in transgenic tobacco at cold and ABA treatment

Under cold and ABA treatment, the expression level of this gene initially increased and then decreased, demonstrating a trend of fluctuation. The expression level of treated plants was higher than that of untreated plants (0 h), except for those treated at 4 °C for 1 h; this difference was significant (Fig. 12).