The leaves of maize drought-tolerant inbred line H8186 and control inbred line B73 were taken at the three-leaf stage and sequenced. The sequencing was entrusted to Mega Biotech. The transcriptome data is not yet public. Data is stored in 10.6084/m9.figshare.12309998.

Ninety Arabidopsis WRKY genes, one hundred twenty-four Maize WRKY genes, and one hundred sixteen Millet WRKY genes were aligned using ClustalW in MEGA 7.0 [23]. A neighbor-joining (NJ) phylogenetic tree was then constructed based on conserved residues with 1000 replicates. In addition, potential WRKY domains including WRKY domain and plant zinc cluster domain were detected using Pfam (https://www.ebi.ac.uk/interpro/) (accessed on 02 April 2024). The ZmWRKY25 protein sequence was fed into the online analysis software String (https://string-db.org/) (accessed on 02 April 2024) for network analysis. In order to further investigate the transcriptional regulation mechanism of ZmWRKY25, the promoter region of ZmWRKY25 was predicted as reported by Zhao et al. [24].

Maize inbred line B73, Tobacco NC89 and nicotiana benthamiana, Escherichia coli strain E. coli DH5α, agrobacterium tumefaciens strain EHA105, Cloning vector pMD18-T, expression vector pCAMBIA3301-Bar, expression vector pCAMBIA1302-GFP, by the Jilin Agricultural University Plant Biotechnology Center Laboratory to provide and save. PCR Kit, Restriction endonucleases (BglII, BstEII, NcoI) were purchased from Thermo Fisher Scientific (Shanghai, China); Construction vector was cloned from Clone Smarter with a seamless cloning kit. The Plant Genome Extraction Kit was purchased from Kang for Century, Primers (Table S1), synthesized by Kumei Corporation.

The expression patterns of ZmWRKY25 were analyzed using qRT-PCR with a primer specifically designed for this gene. The study aimed to investigate the expression levels of ZmWRKY25 under various stress conditions, including drought, salt, and ABA. The experimental design and data analysis followed the methodology detailed by Chen et al. [25]. Maize inbred line B73 at the three-leaf stage was utilized to investigate the expression patterns of ZmWRKY25. The seedlings were subjected to various stress treatments—drought treatment using PEG6000 (10%), ABA (120 μM) treatment, and salt stress using NaCl (260 mM). The expression levels of ZmWRKY25 were assessed by collecting leaves at different time points (0, 1, 3, 6, 12 h) during the treatments. Additionally, a tissue-specific expression analysis was performed by sampling roots, stems, and leaves of maize inbred line B73 to examine the expression of ZmWRKY25 in different tissues.

In this study, following Lagace M’s method of material stress treatment, maize roots were washed and then immersed in a 25% PEG 6000 aqueous solution for 2 h [26]. Total RNA was extracted from young and vigorous maize leaves, followed by reverse transcription into cDNA. Specific primers with homologous arm sequences were designed based on the sequence of the ZmWRKY25 gene in GenBank (accession number NM_001158417) and the expression vector p3301-Bar sequence (Table S1). The cDNA of maize inbred line B73 was used as a template for amplifying the ZmWRKY25 gene coding region sequence. The PCR reaction product was ligated with the cloning vector pMD18-T, then transferred to E. coli DH5α competent cells. These cells were plated on LB-AMP solid medium and cultured overnight, and a single colony was picked. Bacterial plasmids were extracted, and the plasmid was confirmed by PCR before being sent to Changchun Kumei Co. (Changchun, China) for sequencing.

Utilizing the principle of homologous recombination with a seamless kit, the plasmid pMD-18T-ZmWRKY25 served as the template for amplifying the ZmWRKY25 gene using specific primers (Table S1). Subsequently, the expression vector p3301-Bar was subjected to digestion with the restriction endonucleases BglII and BstEII at 37°C for 2 h. The purified gene fragments and the large fragment of the digested product were then recovered. Following the instructions of the seamless cloning connection kit, the recombinant plant expression vector p3301-ZmWRKY25-Bar was constructed. The identity of the plasmid was confirmed through PCR and double digestion before being sent to Changchun Kumei Co., Ltd. (China) for sequencing. In order to determine the subcellular localization of ZmWRKY25, we constructed the pCAMBIA1302-ZmWRKY25-GFP vector. The coding region of ZmWRKY25 was inserted downstream of the 35s promoter in the pCAMBIA1302 vector. The construction process for the pCAMBIA1302-ZmWRKY25-GFP vector followed the same protocol as the pCAMBIA3301 expression vector, involving digestion with BglII and NcoI.

The target gene was successfully introduced into tobacco plants using Agrobacterium tumefaciens. For details on the selection and disinfection methods of wild-type tobacco NC89 seeds, refer to the protocol for Agrobacterium tumefaciens-mediated transformation of tobacco leaves and subsequent transformation of tobacco tissue. Additionally, positive plants were selected by culturing transformed seeds on MS medium with 10 mg/mL Bar. Three T3 generation overexpression lines (OE-ZmWRKY25-1, OE-ZmWRKY25-2, OE-ZmWRKY25-3) showing increased expression of ZmWRKY25 were identified through qRT-PCR analysis for further study. For information on tobacco transient transformation, see the work by Huo et al. [27].

Seed germination rate and root growth experiments were conducted to analyze the impact of drought stress on the phenotype of tobacco plants. Tobacco seeds, including both wild-type (WT) and transgenic varieties, were cultured on MS medium containing either 5% or 10% PEG6000 at 26°C for a duration of one week. To determine root growth, 7-day-old seedlings were moved to MS medium containing 10% PEG6000 for another week, and the tap root length was measured. After 13 days in the same conditions, the survival rates of both transgenic and WT tobacco plants were recorded. The seedling roots after drought stress were washed to remove the medium and put into a root scanner (Epson Perfection V800 Photo) to measure the root phenotype. For the drought treatment, 10-day-old transgenic and WT tobacco seedlings were transferred to soil. After three weeks of incubation at 40% humidity and 26°C (16 h light/8 h dark), watering was stopped for 13 days. The water loss rate and other physiological and biochemical indicators were measured for three periods (before the drought treatment, 13 days after the drought treatment and three days after rewatering). The root morphology of tobacco after 13 days of drought treatment was scanned with a root scanner. All drought stress experiments were repeated 3 times.

To elucidate the potential molecular mechanism of ZmWRKY25 in the ABA signaling pathway, we utilized qRT-PCR to evaluate the expression levels of NtMYB4a, NtDREB4, NtABF, and NtERF105 genes in transgenic tobacco and wild type plants under normal conditions. The specific primers used for this analysis can be located in Table S1.

The peroxidase activity (POD), superoxide dismutase activity (SOD), and malondialdehyde levels (MDA), proline and chlorophyll content (Pro and Chl), as well as relative conductivity (REC) and relative water content (RWC) in the leaves of both transgenic plants and control plants were measured following the protocols outlined by Qin et al. [28] and Zhang [29]. These parameters were assessed at 0, 3, and 13 days post rehydration.

In order to identify genes in maize that respond to drought stress, the study halted watering three-leaf seedlings for a period of 7 days and examined the changes in transcription levels through transcriptome sequencing analysis. The results indicated significant alterations in the expression of numerous genes following the drought treatment. These differentially expressed genes were categorized into functional groups using Gene Ontology (GO) and further analyzed to determine the pathways they may be associated with by referencing the KEGG database. As a result, the gene ZmWRKY25 was singled out from the pool of DEGs for additional investigation (Fig. S1). The results of qRT-PCR verification for differential gene expression in the transcriptome are presented in Fig. S2.

Phylogenetic trees show that ZmWRKY25 with AtWRKY11, 17, 15, 39, 74, 21 are phylogenetically close (Fig. 1). A comprehensive analysis of protein sequences was conducted to investigate the correlation between phylogeny and sequence structure among WRKY members. The study revealed a close phylogenetic relationship between ZmWRKY25, AtWRKY11, and AtWRKY17.

The WRKY domain sequences of WRKY IIb were analyzed by comparing multiple sequences and conducting conserved domain analysis (Fig. 2). Transcription-factor gene families generally contain one or more highly conserved domains that play a role in DNA binding. It is important to note that sequence similarity may decrease beyond these crucial domains (Fig. 2). The monophyletic group formed by AtWRKY11 and AtWRKY17 (Fig. 2) shares a common ancestor with ZmWRKY25, which is supported by the presence of highly conserved motif patterns.

Through protein interaction analysis(Fig. 3, Table S2), ZmWRKY25 protein is mainly associated with EREB61, Ethylene-responsive transcription factor ERF105 interaction. Both EREB61 and ERF105 are involved in the ABA signaling pathway.

The cis-regulatory element of the ZmWRKY25 gene was identified using the plantCARE online database (available online: http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) (accessed on 02 April 2024). (Table 1). Analysis of Table 1 reveals that the ZmWRKY25 promoter region contains multiple abiotic stress-related elements, such as the MYB binding site involved in drought inducibility (MBS), ABA-responsive elements (ABRE), and the MeJA-responsive element (CGTCA-motif), suggesting that ZmWRKY25 may play a role in drought stress response.

To confirm the successful construction of the cloning vector and expression vector, we utilized PCR and sequencing techniques for validation. The cloning outcomes of the target gene are depicted in Fig. S2. The RNA extraction, target gene cDNA, and PCR outcomes of the cloning vector exhibit clear and accurate band positions, confirming the successful construction of the cloning vector. The PCR, double restriction enzyme digestion, and sequencing results of the target genes in the pCAMBIA3301 and pCAMBIA1302 plant expression vectors indicated successful vector construction (Figs. S2–S6). Following Agrobacterium-mediated genetic transformation, a total of 6 transgenic lines were obtained in the T3 generation. The qPCR expression detection results of the positive strains are presented in the figure, revealing significantly elevated expression levels of ZmWRKY25 in strains 1, 2, and 3 compared to the control (Figs. S7 and S8).

The subcellular localization of the ZmWRKY25 fusion protein was investigated through transient transformation of ZmWRKY25-green fluorescent protein (GFP) recombinant into Nicotiana benthamiana leaves, with the pCambia1302-GFP vector as a control. Fig. 4 clearly shows that the ZmWRKY25-GFP fusion protein is specifically localized in the nucleus, while the pCambia1302-GFP control is distributed throughout the entire cell. These results indicate that the ZmWRKY25-GFP fusion protein has a distinct nuclear localization, suggesting potential interactions with other nuclear proteins or involvement in gene expression regulation within the nucleus. Understanding this subcellular localization is essential for elucidating the function and behavior of ZmWRKY25.

The tissue-specific expression of ZmWRKY25 in maize was analyzed using qPCR across roots, stems, and leaves. ZmWRKY25 was mainly expressed in leaves, with lower expression levels observed in roots and stems. Furthermore, the study investigated the effects of drought, salt, and ABA stress on ZmWRKY25 expression patterns. Following ABA treatment, ZmWRKY25 showed its highest expression level after 1 h. Drought stress induced a peak in ZmWRKY25 expression at 3 h, followed by a decrease. Salt stress caused a 2-fold increase in ZmWRKY25 expression at 1 h, reaching its peak at 3 h (Fig. 5A–D).

WT as a control for genetically modified tobacco, three T3 transgenic tobacco lines that overexpress ZmWRKY25 were selected for analysis. Under normal growth conditions, no significant difference in seed germination rates was observed between wild-type (WT) and transgenic plants. However, when exposed to 5% and 10% PEG6000 stress, the seed germination rate of transgenic plants showed a notable increase compared to WT plants (Fig. 6). There was minimal variation in total root length between the transgenic lines and WT plants. Notably, after a seven-day cultivation period under 10% PEG6000 stress, the taproot lengths of transgenic lines surpassed those of WT plants (Fig. 7). Phenotypic analysis of roots at the seedling stage using a root scanner indicated that the root surface area, total root projected area, root volume, and average root diameter of transgenic plants were significantly larger than those of WT seedlings. Moreover, the total number of root tips and branches in the transgenic lines were notably higher than in WT seedlings (Fig. 8). At the seedling stage, there were no major differences in growth between transgenic plants and WT plants. However, following drought treatment, the transgenic lines showed enhanced resilience compared to WT plants, with a lower water loss rate and a more developed root system (Fig. 9).

To investigate the molecular mechanism of ZmWRKY25 stress response, the study examined the gene expression levels of stress response in transgenic and negative controls under normal conditions (see Fig. 10). The results revealed that the expression levels of stress-responsive genes NtMYB4a, NtABF, NtDREB4, and NtERF105 were more than three times higher in transgenic Tobacco compared to the wild-type.

In order to investigate the role of the ZmWRKY25 gene during drought conditions, a NBT staining experiment was conducted on both WT plants and ZmWRKY25 transgenic plants 13 days after exposure to drought stress. The results indicated a noticeable decrease in reactive oxygen species (ROS) content within the mesophyll cells of ZmWRKY25 transgenic plants compared to WT plants. Additionally, the activities of POD and SOD were assessed under the same conditions. Following 13 days of drought treatment, the POD activity in transgenic plants significantly increased compared to WT plants, while it notably decreased before drought exposure and after three days of rewatering. The changes in SOD enzyme activity closely mirrored those of POD. To further explore the impact of ROS on plant cell damage, the levels of MDA, Pro, chl, and relative conductivity were analyzed under identical conditions. After 13 days of drought treatment and three days of rehydration, the MDA content and relative electrical conductivity in transgenic plants were significantly lower than in WT plants. Conversely, the levels of Pro and chlorophyll in transgenic plants were notably higher than those in WT plants (Fig. 11).