A Dirigent Gene, ZmDIR11, Positively Regulates Drought Tolerance in Maize

Abstract

The DIR gene family, which encodes Dirigent proteins, plays a crucial role in plant development and stress responses. However, the functions and mechanisms of this family in maize remained underexplored. This study identified ZmDIR11, a member of the maize Dirigent protein family, and explored its role in drought tolerance. ZmDIR11 shared conserved regions with homologous proteins in wheat, rice, and Arabidopsis. RT-qPCR analysis revealed that ZmDIR11 expression is upregulated in leaves under drought and PEG stress, while subcellular localization confirmed its presence in the endoplasmic reticulum. Virus-induced gene silencing (VIGS) and EMS mutagenesis demonstrated that ZmDIR11 silencing or mutation significantly reduces drought tolerance in maize seedlings, indicating its positive regulatory role. Silencing or mutation of ZmDIR11 led to decreased growth parameters (plant height, root length, fresh weight, dry weight, and chlorophyll content) under drought stress, alongside a reduced antioxidant capacity, as evidenced by increased levels of MDA and ROS and decreased activities of SOD, CAT, and POD. Furthermore, ZmDIR11 mutation impaired the synthesis of ABA and zeatin, downregulating key genes in the ABA (ZmABA1, ZmNCED3, ZmSnRK2.6) and zeatin (ZmIPT4, ZmCKO5, ZmCKO4b) pathways. Drought-responsive genes (ZmRD20, ZmRD22, ZmDREB2A) and lignan biosynthesis genes (Zm4CL, ZmC3H, ZmCAD1) are also downregulated in ZmDIR11 mutants. In conclusion, ZmDIR11 enhances drought tolerance in maize by regulating antioxidant capacity, ABA and zeatin synthesis, and lignan metabolism. This study provides new insights into the role of DIR genes in drought tolerance and offers a potential genetic resource for breeding drought-resistant maize varieties.

1. Introduction

Drought is generally regarded as a stochastic natural phenomenon resulting from intense and sustained deficits in rainfall, often leading to severe destructive effects on agricultural production [1]. It is one of the most challenging natural disasters to quantify, complicating the assessment of recent changes and future conditions [2]. However, with the escalating impacts of climate change on Earth, future drought occurrences are anticipated to become more frequent and severe [3]. Drought stress can impede crop growth and development, thereby limiting agricultural productivity and increasing production costs; it is also one of the primary abiotic stress factors affecting the supply of food and feed [4]. As one of the world’s three major food crops, maize is particularly sensitive to arid environments. Drought stress significantly impacts the growth and development of maize plants at all stages, making breeding drought-resistant maize essential. Identifying maize genes associated with drought resistance and employing genetic engineering to expedite the cultivation of drought-resistant maize represents a crucial direction in contemporary maize research. Through a comprehensive review of the extensive scientific literature, we have identified that the maize drought-resistant genes primarily consist of those encoding transcription factors, protein kinases, and proteins related to metabolic processes.

Dirigent proteins, which are key metabolic proteins in the lignan biosynthetic pathway, have been functionally characterized in various plants such as Arabidopsis, rice, wheat, sugarcane, and Brassicaceae, with several members identified as being associated with plant drought resistance. However, studies focusing on DIR genes in maize remain limited. DIR genes were first identified by American scientists in Forsythia suspensa [5]. Subsequent research has demonstrated that DIR genes are nearly ubiquitous across all terrestrial plants, playing a significant role in plant growth, development, and resistance to both biotic and abiotic stresses [6]. Liu et al. conducted a study identifying 107 GbDIRs from Gossypium barbadense and 107 GhDIRs from Gossypium hirsutum, suggesting that DIRs may play a significant role in cotton fiber development. Through transgenic experiments, they validated that GbDIR78 could promote the elongation of trichomes and hypocotyl cells, indicating its potential role in regulating cell elongation [7]. Luo et al. conducted a study in which they performed HMMER searches on barley proteins, identifying 64 HvDIRs. Their research indicates that the HvDIR family has significantly expanded in barley and may be involved in various developmental processes and stress responses [8]. Li et al. compared 57 NtDIRs and 33 StDIRs and found that three clustered genes (NtDIR2, NtDIR4, StDIR3) exhibited a strong response to pathogen infection, highlighting their important role in disease resistance [9]. Another study on sugarcane DIR genes revealed that ScDIR5, ScDIR7, ScDIR11, and ScDIR40 could enhance the drought resistance of transgenic tobacco, with ScDIR7 demonstrating the strongest drought resistance [10]. Current research on maize DIR genes is limited, and the mechanisms underlying maize drought resistance remain unclear. In this study, we identified a DIR gene, ZmDIR11, from previous transcriptome data that responds to drought stress. Experimental designs showed that both silencing and mutating ZmDIR11 reduced the drought resistance of maize. These findings enhance our understanding of maize DIR genes and provide a potential avenue for improving the drought resistance of maize plants through DIR gene manipulation.

2. Materials and Methods

2.1. Maize Materials and Drought Stress Treatment

The self-pollinated lines KN5585 and B73 were utilized as wild types (WT). The Virus-Induced Gene Silencing (VIGS) line was developed based on the KN5585 background, while the Ethyl Methanesulfonate (EMS) mutant was a homozygous mutant plant from the ZmDIR11-F4 generation with a B73 background. Uniform-sized seeds were selected, disinfected with 1% sodium hypochlorite for 3 min, rinsed five times with sterile water, and then placed in a 28 °C artificial climate chamber to promote germination. The seeds were sown in 10 cm³ square pots filled with a uniform mixture of potting soil, perlite, and vermiculite in a 1:1:1 ratio. The pots were kept under a day/night temperature regime of 26/22 °C and a light/dark cycle of 14/10 h, with daily equal watering. At the three-leaf stage, maize seedlings were divided into two groups. In the experiment utilizing KN5585 as the material background, one group was subjected to drought treatment, while the other group received normal watering (control, CK) for a duration of 12 days. In the experiment utilizing B73 as the background material, one group was subjected to a 20% PEG6000 (Polyethylene Glycol) solution to simulate drought conditions, while the other group received normal watering over a duration of 7 days. At various stages, the second vegetative leaf of the maize was harvested, rapidly frozen in liquid nitrogen, and subsequently stored at −80 °C in a freezer for further research.

2.2. Tobacco Materials and Subcellular Localization Experiment

Nicotiana benthamiana was utilized for subcellular localization experiments. Tobacco plants were grown in a light incubator at 26 °C with a 14/10 h light/dark cycle, cultivated in 10 cm³ black square pots filled with nutrient soil. After four weeks, these tobacco plants were employed for subcellular co-localization experiments. The ZmDIR11 sequence was amplified and cloned into the pCAMBIA-GFP vector, while control protein genes AtH2B and AtWAK-HDEL sequences were constructed in the pCAMBIA-RFP vector. The constructed vectors were extracted and transformed into GV3101 Agrobacterium to prepare the inoculation mixture, which was subsequently injected into the leaves of the four-week-old tobacco plants. The plants were then further cultivated in the dark for two days, after which the marked leaves were harvested to prepare slides for confocal laser scanning microscopy observation and photography [11]. The control protein AtH2B, a histone structural component of the nucleosome, is localized in the nucleus [12]. Another control protein, AtWAK, is a type of wall-associated kinase (WAK) associated with the endoplasmic reticulum (ER) [13]. HDEL serves as an ER retention signal typically found at the C-terminus of proteins, facilitating their localization or retention within the ER [14]. The AtWAK-HDEL protein is specifically localized within the ER (the experimental data were collected from December 2022 to February 2023).

2.3. Virus-Induced Silencing of ZmDIR11

According to methods reported in previous studies [15], a mixed virus system consisting of pCMV201-2bN81, pC101, and pC301 was employed at a ratio of 1:1:1 to conduct a cucumber mosaic virus (CMV)-induced gene silencing experiment in maize. The prediction of gene silencing fragments and primer design was performed using the SGNVIGS Tool website (https://vigs.solgenomics.net (accessed on 20 March 2024)), targeting approximately 250 bp fragments within the coding sequence (CDS) region of ZmDIR11, which were subsequently ligated onto pCMV201-2bN81 and transferred into Agrobacterium competent cells. The cells were resuspended in resuspension buffer at a 1:1:1 ratio with the strains of pC101 and pC301, followed by sufficient induction. GFP served as the control group and was injected into the veins of tobacco leaves at the 5–6 leaf stage. After being cultured in a light incubator with a 16 h light period at 26 °C during the day and an 8 h dark period at 24 °C for 4 days, the injected leaves were ground into powder to extract the crude virus extract, which was then used to inoculate KN5585 seeds. Following seedling emergence, preliminary identification was conducted through phenotypic observation, and samples were collected after the stress experiment to extract RNA for assessing silencing efficiency (the experimental data were collected from March to September 2024).

2.4. Establishment of ZmDIR11-EMS Mutant Lines

ZmDIR11 mutants were obtained from the maize EMS mutant library (https://elabcaas.cn/ (accessed on 15 July 2021)) as first-generation seeds [16]. In this experiment, two mutants, EMS4-3f0544 (T1) and EMS4-15dd8f (T3), were ultimately utilized (T1 and T3 only represent two ZmDIR11-EMS mutant lines with different mutation profiles). Detailed information regarding the mutations is provided in Table S1. Additionally, the alignment of the mutant and wild-type ZmDIR11 gene sequences, as well as their corresponding protein sequences, is illustrated in Figure S1. Following the acquisition of the mutant seeds, they were crossed with the B73-WT to purify the genetic background over two generations. Subsequently, the seeds were self-crossed for an additional two generations. Positive mutation sites in each generation were identified using PCR-agarose gel electrophoresis, employing the primers listed in Table S2. Selected homozygous lines of each mutant, along with the B73-WT, were used for phenotype analysis in a PEG-simulated drought experiment (the experimental data were collected from October 2021 to July 2024).

2.5. Bioinformatics Analysis Related to ZmDIR11

We queried the gene and protein information of ZmDIR11 through the MaizeGDB and NCBI databases. The protein structure of ZmDIR11 was predicted using the SWISS-MODEL online platform (https://swissmodel.expasy.org/ (accessed on 7 January 2023)). A domain schematic diagram of the ZmDIR11 protein was created using the IBS 2.0 online tool (https://www.ibs.renlab.org/#/home (accessed on 7 January 2023)). The interaction network and Gene Ontology (GO) enrichment analysis for the ZmDIR11 protein were conducted using the STRING protein database (https://cn.string-db.org/ (accessed on 16 June 2023)). The 2000 bp sequence upstream of the start codon of ZmDIR11 was retrieved, and the promoter elements within this region were analyzed using the PlantCare online tool (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 6 March 2023)), with visualization predictions performed using Tbtools (v2.0) software. The DIR amino acid sequences of rice, wheat, and Arabidopsis were obtained from the NCBI database. Multiple sequence alignment analysis was conducted using DNAMAN (v9.0) software, and a phylogenetic tree was generated using MEGA-X 5.0 based on the neighbor-joining method (the experimental data were collected from October 2022 to December 2023).

2.6. Growth Parameter Determination

Randomly select ten seedlings from each group and measure their above-ground height and root length using a tape measure. Fresh weight and dry weight are determined with an analytical balance. Chlorophyll content is measured using a kit from Yuanye Biotech (Shanghai, China) through an organic solvent extraction method. The absorbance values at 663 nm and 645 nm are recorded using a microplate reader. Chlorophyll content (mg/g) is calculated using the formula (20.2 A 645 nm + 8.02 A 663 nm) × V/(1000 × W), where A663 nm and A645 nm represent the absorbance at wavelengths 663 nm and 645 nm, respectively; V is the volume of the extraction solution in milliliters; and W is the fresh weight of the maize leaves in grams (the experimental data were collected from July 2024 to November 2024).

2.7. Determination of Physiological Indices

The contents of Proline (Pro), Malondialdehyde (MDA), H₂O₂, and superoxide anion (O²⁻) as well as the activities of superoxide dismutase (SOD), catalase (CAT), and peroxidase (POD) are determined using kits obtained from Solarbio Science & Technology (Beijing, China). In accordance with the manufacturer’s instructions, all assays are conducted using the microplate method. The final absorbance values are recorded with a microplate reader and converted to concentration using the provided formulas. Each sample group consisted of three replicates (the experimental data were collected from July 2024 to November 2024).

2.8. Extraction and Determination of Total Lignans

Total lignans are extracted using methanol as the extraction solvent. Fresh maize leaves are ground into a powder and mixed with the solvent at a ratio of 1:20 (w/v). The mixture is then subjected to ultrasonic extraction at a power of 2500 W and a temperature of 40 °C for 1 h. Following centrifugation, the supernatant is utilized for detection using the Plant Lignan ELISA KIT from Enzyme-linked Biotech (Shanghai, China). The absorbance is measured at 450 nm with a microplate reader, and the total lignan content is calculated based on the standard curve regression equation (y = 0.235x + 0.0239, R² = 0.996). Each sample group consisted of three replicates (the experimental data were collected from July 2024 to November 2024).

2.9. Determination of ABA and Zeatin Contents

The contents of ABA and zeatin were determined by Personal Biotech (Shanghai, China). The measurement of ABA was conducted using the liquid chromatography–mass spectrometry (LC–MS) method, while zeatin levels were assessed using gas chromatography–mass spectrometry (GC–MS). Each sample group consisted of six replicates (the experimental data were collected from October 2024 to November 2024).

2.10. Extraction of Total RNA and Reverse Transcription

Total RNA was extracted from maize leaves using the Tiangen TRIZol total RNA extraction kit (Tiangen, Beijing, China), and genomic DNA contamination was removed through treatment with DNase. The extracted total RNA from maize was then reverse transcribed into a cDNA template using the HiScript III All-in-One RT Supermix kit (Vazyme, Nanjing, China), following the manufacturer’s instructions.

2.11. Quantitative Real-Time PCR

RT-qPCR was conducted using the ChamQ SYBR Color qPCR Master Mix kit (Vazyme, Nanjing, China). All primers necessary for the experiment were designed with the Primer 5.0 tool (see Table S2). The primers were synthesized by Tsingke Biotech (Shanghai, China). Fluorescence quantitative PCR was performed on the Applied Biosystems Quant Studio™ 6 Flex analyzer. ZmACTIN1(Zm00001eb216070) served as the internal reference gene. The reaction conditions and systems were established following the manufacturer’s instructions. Each sample included three biological replicates and three technical replicates, with Ct values between replicates consistently below 0.5. The relative expression levels of the genes were calculated using the 2^−ΔΔCT method (the experimental data were collected from July 2024 to November 2024).

2.12. Statistical Analysis

All the experiments in this paper were repeated at least three times, and results from representative datasets are presented. GraphPad Prism (version 9.3.0) was used for the statistical analysis. The statistical evaluations used one-way analysis of variance (ANOVA) with multiple comparisons, followed by Tukey tests. The results were considered statistically significant at x p < 0.05.

3. Results

3.1. Identification and Bioinformatics Analysis of ZmDIR11

In a previous transcriptome analysis of corn subjected to drought stress, we identified a gene, Zm00001d012432, which was significantly upregulated and subsequently designated as ZmDIR11 [17]. Further investigation using MaizeGDB revealed that this gene is located on the eighth chromosome of maize. ZmDIR11 exhibits a simple structure comprising a single exon, which is characteristic of the classic structure of DIR genes [18]. We successfully amplified this gene and confirmed its full length of 639 bp through sequencing. The gene encodes a Dirigent protein consisting of 212 amino acids, which includes a single Dirigent domain located at positions 99-186 of the amino acid sequence (Figure 1A). As illustrated in Figure 1B, this represents the predicted protein structure model of ZmDIR11. Additionally, we analyzed the 2000 bp promoter region upstream of the ZmDIR11 gene and identified multiple stress-related transcription factor binding elements within this region. Specifically, we found six types of binding elements: ARE, MYB, MYC, STRE, LTR, and MBS (Figure 1C). Notably, there are nine MYB-type promoter elements, suggesting a potential association between the ZmDIR11 gene and MYB transcription factors. Previous studies have indicated that MYB transcription factors represent one of the largest families of transcription factors in plants, playing a crucial role in the response of crops to abiotic stresses [19]. By querying the NCBI database, we obtained DIR protein sequences from Arabidopsis, wheat, rice, and maize and compared them with the ZmDIR11 protein sequence, revealing highly conserved amino acid regions (Figure 1D). Furthermore, we analyzed the evolutionary relationship between ZmDIR11 and DIR genes from the aforementioned species using Tbtools software to construct a phylogenetic tree, which demonstrated that ZmDIR11 is highly homologous to the rice gene Os01g0837400 (Figure 1E). Predictions from the STRING protein database indicate that the ZmDIR11 protein may interact with three additional maize DIR proteins (Zm00001d024969, Zm00001d004753, and Zm00001d026546). This suggests that ZmDIR11 may collaborate with other DIR proteins to perform metabolic functions (Figure S2). Gene Ontology enrichment analysis suggests that the biological functions of ZmDIR11 are predominantly associated with plant defense responses and phenylpropanoid biosynthetic processes (Figure S3). This association may be linked to its role in the metabolism and synthesis of lignans.

3.2. Expression Pattern of ZmDIR11

Analysis of tissue expression patterns revealed that ZmDIR11 was expressed in various tissues, including young roots, roots, stems, young leaves, leaves, and silk, but not in pollen. Among these tissues, the expression levels were highest in roots and relatively lowest in stems (Figure 2A). Furthermore, drought conditions and treatment with 20% PEG-6000 significantly induced the expression of ZmDIR11 in maize leaves, suggesting that ZmDIR11 plays a role in response to drought stress (Figure 2B,C).

3.3. Subcellular Localization of ZmDIR11 in Tobacco

To study the subcellular localization of ZmDIR11, the target gene was cloned into the transient expression vector pCAMBFA-GFP using the GateWay technology. The AtH2B sequence and the AtWAK-HDEL gene were cloned into the transient expression vector pCAMBFA-RFP. Due to the fusion of ZmDIR11 with GFP and the fusion of AtH2B and AtWAK-HDEL with RFP, the green fluorescent characteristic of GFP and the red fluorescent characteristic of RFP were used to transform tobacco leaves and observe the subcellular localization of ZmDIR11 under a confocal microscope. Figure 2Da illustrates the co-localization results of ZmDIR11 and AtH2B proteins, where green fluorescence indicates the presence of ZmDIR11 and red fluorescence represents AtH2B. The distinct separation of the green and red fluorescence suggests that ZmDIR11 does not co-localize with AtH2B within the cell nucleus. In a similar manner, Figure 2Db depicts red fluorescence corresponding to AtWAK-HDEL, which merges with the green fluorescence of ZmDIR11, resulting in yellow fluorescence. This observation indicates that ZmDIR11 co-localizes with the AtWAK-HDEL protein in the endoplasmic reticulum. The results showed that the ZmDIR11 fusion protein was only localized in the endoplasmic reticulum (Figure 2D).

3.4. VIGS Silencing of ZmDIR11 Reduces Drought Tolerance in Maize

To preliminarily investigate the regulatory role of ZmDIR11 in drought tolerance in maize, we successfully silenced the gene using Virus-Induced Gene Silencing (VIGS) technology in the KN5585 self-pollinated line of maize. A GFP-silenced line was established as a negative control for the experiment. Drought trials were conducted on both lines. Under normal, well-watered conditions, no significant differences in growth and development were observed between the silenced and control lines, suggesting that ZmDIR11 does not influence plant growth and development under normal environmental conditions. After 12 days of water withdrawal, the growth of the ZmDIR11-VIGS line was significantly weaker than that of the mock line (Figure 3A). Subsequent expression analysis revealed that, under drought conditions, the expression of ZmDIR11 in the leaves of the mock plants was significantly higher than in the ZmDIR11-VIGS line (Figure 3B). This indicates that the target gene in the silenced lines was successfully silenced, and it was the silencing of ZmDIR11 that resulted in the observed growth differences between the silenced lines and the control under drought conditions. We continued to measure growth indicators such as plant height (Figure 3C), root length (Figure 3D), fresh weight (Figure 3E), dry weight (Figure 3F), and chlorophyll content (Figure 3G) and found no significant differences under normal conditions between the control and silenced lines. Following drought stress, the overall growth indicators of the control line were significantly higher than those of the silenced line. The silencing of ZmDIR11 diminished the plant’s drought tolerance, reduced its capacity to accumulate dry matter under drought stress, and exacerbated the loss of chlorophyll in the leaves. DIR genes are implicated in the metabolic synthesis of lignans. We quantified the total lignan content in the leaves and found that, under normal conditions, the total lignan content in the VIGS maize seedlings was slightly lower than that in the mock line, although the difference was not statistically significant. However, after drought stress, the total lignan content in the leaves of the mock line was significantly higher than that in the ZmDIR11-VIGS line (Figure 3H). This finding indicates that ZmDIR11 primarily catalyzes the synthesis of lignans under drought stress, while lignan synthesis in the ZmDIR11-VIGS line was markedly impaired. In response to drought stress, plants rapidly accumulate free proline (Pro), which can alleviate osmotic pressure and enhance drought tolerance, making it an important physiological indicator for evaluating plant drought resistance [20]. Under well-watered conditions, there was no significant difference in Pro content between the mock and ZmDIR11-VIGS lines. However, under drought treatment, the Pro content in the leaves of the mock plants was significantly higher than that in the ZmDIR11-VIGS line (Figure 3I), indicating that the mock line exhibited stronger drought resistance in terms of physiological metabolism.

In response to abiotic stress, crops accumulate significant amounts of MDA and ROS, including H₂O₂ and O²⁻. The accumulation of MDA and ROS can lead to lipid peroxidation of cell membranes, exacerbate cellular oxidative damage, and even result in cell death, thereby affecting the normal physiological and biochemical responses of the plant and reducing its ability to withstand abiotic stress [21,22]. Consequently, we continued to measure the levels of MDA (Figure 3J), H₂O₂ (Figure 3K), and O²⁻ (Figure 3L) in both the mock line and ZmDIR11-VIGS line under different treatments. Our findings revealed that under normal conditions, there was no significant difference in MDA and ROS indicators between the mock and ZmDIR11-VIGS lines. However, after drought treatment, the levels of MDA, H₂O₂, and O²⁻ in the ZmDIR11-VIGS line were significantly higher than those in the mock line. This indicates that silencing ZmDIR11 significantly reduced the overall antioxidant capacity of the plants compared with the mock line, thereby noticeably weakening their drought resistance. These results suggest that ZmDIR11 may play a crucial role in maintaining osmotic pressure and antioxidant defense capabilities in maize cells, ultimately enhancing the plant’s drought tolerance under arid conditions.

3.5. ZmDIR11 Positively Regulates Drought Tolerance in Maize

To further investigate the biological function of ZmDIR11 and maintain a consistent drought effect, we selected the reference genome inbred line B73 wild type and two homozygous ZmDIR11-EMS mutant lines, T1 and T3, which were developed from the B73 background, as our materials. We employed a 20% PEG-6000 solution to simulate drought stress in our experiments and conducted a detailed analysis of the phenotypic differences between the B73-WT and the mutant lines. As shown in Figure 4A, under normal conditions, the total lignan content in the T1 and T3 lines was slightly lower than that in the wild type; however, this difference was not significant. In contrast, under drought induction, the total lignan content in the T1 and T3 lines was significantly lower than that in the WT. This finding suggests that the mutation in ZmDIR11 influences the synthesis of total lignans in plants subjected to drought stress. We treated maize seedlings with a 20% PEG-6000 solution prepared with 1/2 Hoagland nutrient solution for 7 days. After this period, the wilting degree of the mutant plants was significantly greater than that of the WT, indicating that the WT exhibited greater drought tolerance (Figure 4B). Measurements of plant height, root length, dry weight, and fresh weight revealed that the mutant lines had significantly reduced plant height (Figure 4C), root length (Figure 4D), dry weight (Figure 4E), and fresh weight (Figure 4F) compared with the WT. This indicates that the growth and development of the mutant lines were more severely inhibited, leading to slower dry matter accumulation relative to the WT. The measurement of chlorophyll content in the lines revealed that the mutant lines exhibited significantly lower chlorophyll levels under drought stress treatment compared with the WT (Figure 4G). Additionally, further analysis of Pro metabolic levels demonstrated that WT accumulated higher levels of Pro after drought stress treatment, significantly surpassing those in the mutant lines. These findings are consistent with observations made in the silenced ZmDIR11 lines within the KN5585 background, suggesting that ZmDIR11 plays a positive role in regulating drought resistance in maize.

3.6. ZmDIR11 Enhances Drought Tolerance in Maize by Regulating Antioxidant Capacity

To further investigate whether ZmDIR11 can enhance the antioxidant capacity of maize under drought conditions in the B73 background, we continued to measure the content of MDA and ROS, as well as the expression of antioxidant-related genes and the activity of key enzymes (SOD/CAT/POD). Consistent with the results from the silencing experiments in the KN5585 background, under PEG6000 treatment, the leaves of the mutant line exhibited significantly higher levels of MDA (Figure 5A), H₂O₂ (Figure 5B), and O²⁻ (Figure 5C) compared with the WT. In contrast, under normal conditions, no significant differences were observed in these indicators between the WT and mutant lines. This suggests that the mutation in ZmDIR11 leads to an increased accumulation of MDA, H₂O₂, and O²⁻ in the plant. Typically, plants upregulate the expression of antioxidant-related genes to enhance their antioxidant capacity at the genetic level in response to oxidative stress [23]. Therefore, we further assessed the relative expression levels of ZmSOD1, ZmCAT3, and ZmPOD1 in maize leaves subjected to various treatments. Quantitative analysis revealed that, under normal conditions, there were no significant differences in the relative expression levels of ZmSOD1 (Figure 5D), ZmCAT3 (Figure 5E), and ZmPOD1 (Figure 5F) between the WT and mutant lines. However, under PEG6000 treatment, the relative expression levels of all three genes in the WT were significantly higher than those in the T1 and T3 lines. We subsequently measured the activities of the antioxidant enzymes SOD, CAT, and POD in the maize leaves, which represent the primary classes of defensive enzymes that plants employ to eliminate ROS and enhance their stress tolerance during abiotic stress (Rajput 2021). Under normal conditions, there were no significant differences in the activities of these three enzymes between the WT, the T1, and the T3 lines. However, after drought stress treatment, the activities of SOD (Figure 5G), CAT (Figure 5H), and POD (Figure 5I) in the mutant lines were significantly lower than those in the WT. These results indicate that under simulated drought conditions, ZmDIR11 can mitigate the accumulation of MDA and ROS by enhancing the expression of antioxidant enzyme-related genes and increasing the activity of antioxidant enzymes.

3.7. ZmDIR11 Enhances Drought Resistance by Promoting ABA and Zeatin Synthesis

Studies have indicated that plants typically increase the synthesis of ABA to combat biotic and abiotic stresses, activating ROS scavenging and antioxidant defense mechanisms through ABA signaling [24]. ABA is one of the most critical plant hormones involved in resistance to abiotic stress [25]. Therefore, we employed LC–MS to measure ABA content in plant leaves under various treatments. Our findings revealed that under normal conditions, there was no significant difference in ABA content between WT and the T1 and T2 lines. However, following PEG treatment, the ABA content in WT leaves was significantly higher than in the mutant lines T1 and T3 (Figure 6A). Based on previously reported research, we compiled a simplified pathway for ABA synthesis and signal transduction [26] (Figure S4). We also quantitatively analyzed key genes in this pathway: ZmABA1 (Figure 6B), ZmNCED3 (Figure 6C), and ZmSnRK2.6 (Figure 6D). We found that under normal conditions, the relative expression levels of these genes in WT and mutant lines were not significantly different. However, under PEG treatment, the relative expression levels of these three key genes in WT leaves were significantly higher than in the mutant lines T1 and T3. This suggests that in response to drought stress, WT can induce the synthesis of more ABA to enhance their defense capabilities, whereas the mutation in ZmDIR11 reduces the induced synthesis of ABA.

In addition to synthesizing ABA, plants also produce zeatin to enhance their resistance to stress [27]. Zeatin, a type of cytokinin, plays a crucial role in plant resistance to abiotic stress. It facilitates plant adaptation and resilience to drought, salt stress, and other environmental pressures by regulating metabolic accumulation, gene expression, and signal transduction pathways [28]. We measured changes in zeatin content in plant leaves under various treatments and found that, under well-watered conditions, there was no significant difference in zeatin content between WT and mutant lines. However, under PEG treatment, the zeatin content in the leaves of mutant lines T1 and T3 was significantly lower than that in WT (Figure 6E). We further analyzed the key pathway for zeatin synthesis (Figure S5) and quantitatively assessed the expression of key synthetic genes. The relative expression levels of ZmIPT4 (Figure 6F), ZmCKO5 (Figure 6G), and ZmCKO4b (Figure 6H) in the leaves of different lines under normal treatment did not show significant differences. Under drought stress, the expression of ZmIPT4, ZmCKO5, and ZmCKO4b in WT leaves was significantly upregulated, surpassing that in mutant lines T1 and T3. These results suggest that the mutation of ZmDIR11 may lead to a reduction in the synthesis of ABA and zeatin by suppressing the expression of critical genes involved in the biosynthetic pathways of these hormones, thereby diminishing the plant’s capacity to endure drought stress.

3.8. Impact of ZmDIR11 Mutation on the Expression of Drought and Water Deficit Response Genes

Plants enhance their drought tolerance through the regulation of gene expression at the transcriptional level in response to drought and water deficit stress [29]. In addition to the antioxidant defense-related genes, as well as genes related to ABA synthesis and signaling and zeatin synthesis previously verified in our experiments, we further explored whether mutations in ZmDIR11 also affect the expression of other drought-related genes. RD20, RD22, and DREB2A are key genes involved in the plant’s response to drought stress and play a significant role in dehydration responses [30,31]. Consequently, we obtained information on these genes from the reference genome in the MaizeGDB database and conducted a quantitative analysis. We found that under normal conditions, the relative expression levels of ZmRD20 (Figure 7A), ZmRD22 (Figure 7B), and ZmDREB2A (Figure 7C) did not show significant differences between the WT and mutant lines. However, under drought stress, the relative expression levels of these three dehydration response marker genes in the WT were significantly higher than those in the mutant T1 and T2 lines. This indicates that the mutation in ZmDIR11 reduces the plant’s drought tolerance, which is reflected at the transcriptional level as a decrease in the expression of dehydration response-related genes. Typically, plants upregulate the expression of certain key drought-related genes in response to drought stress; these genes serve as drought markers that enhance the plant’s drought tolerance [32]. Based on previous studies, we selected the lipid transfer protein (LTP) gene ZmLTP3 [33], the galactinol synthase gene ZmGOLS3 [34], and the small G protein gene ZmRAB18 [35] for validation, all of which are confirmed to play active roles in plant responses to drought stress. RT-qPCR analysis revealed that, under normal conditions, there was no significant difference in the relative expression levels of the three drought marker genes between WT and mutant lines. However, under PEG treatment, the relative expression levels of ZmLTP3 (Figure 7D), ZmGOLS3 (Figure 7E), and ZmRAB18 (Figure 7F) in the mutant lines T1 and T3 were significantly lower than those in WT. This finding indicates that the mutation in ZmDIR11 leads to a reduction in plant drought tolerance. DIR proteins are essential components of the lignan synthesis pathway [36], and the previously mentioned measurement of total lignan content further demonstrates that the mutation in ZmDIR11 impacts lignan synthesis under drought stress. Moreover, prior reports indicate that the expression levels of two key enzyme genes, Zm4CL and ZmC3H, in the lignan synthesis pathway are significantly upregulated under water-deficient conditions [37]. This suggests that lignans may play a role in the plant’s response to drought stress. Consequently, we retrieved key enzyme genes in the lignan synthesis pathway and conducted expression analyses of Zm4CL, ZmC3H, and ZmCAD1 genes. The simplified model of the lignan monomer synthesis pathway is shown in Figure S6. Under normal conditions, we found no significant differences in the relative expression levels of these three enzyme genes. However, under PEG treatment, the relative expression levels of Zm4CL (Figure 7G), ZmC3H (Figure 7H), and ZmCAD1 (Figure 7I) in WT were significantly higher than those in the mutant lines T1 and T3. This indicates that, in addition to Zm4CL and ZmC3H being involved in the response to drought stress, ZmCAD1 may also play a role in lignan synthesis and stress response. Furthermore, the mutation in ZmDIR11 may suppress the expression of Zm4CL, ZmC3H, and ZmCAD1 to some extent. These results suggest that the mutation in ZmDIR11 may influence the expression of genes related to water deficit and drought response in plants, thereby affecting the plant’s drought tolerance.

4. Discussion

The DIR gene family has been extensively identified and validated across various crops, playing a significant role in both plant growth and development as well as in responses to biotic and abiotic stresses [9]. In soybeans, 54 GmDIRs have been characterized, with GmDIR27 identified as crucial for pod dehiscence [38]. In mung beans, 25 CcDIRs have been identified, among which CcDIR2 and CcDIR9 specifically respond to salt stress [39]. In green grams, 37 VrDIRs have been characterized, and expression profiling indicates that VrDIR genes exhibit differential responses under various stress conditions, with some genes responding specifically to drought or salt stress [40]. In potatoes, 31 StDIRs have been identified, which display specific responses to cold stress, salt stress, ABA, and drought stress, thereby providing new candidate genes for enhancing potatoes’ resistance to environmental stresses [41]. In foxtail millet, 38 SiDIRs have been identified, and real-time quantitative PCR (RT-qPCR) analysis reveals that SiDIR genes respond to stresses such as CaCl2, CdCl, NaCl, and PEG6000, elucidating the function of SiDIR genes in responding to abiotic stresses and demonstrating their regulatory potential in root development [42]. Currently, there is no definitive information regarding the number of ZmDIRs identified in maize, and research exploring the relationship between ZmDIRs and drought tolerance remains insufficient.

This study has identified a maize DIR gene, ZmDIR11, which demonstrates responsiveness to drought stress, as evidenced by previous transcriptome data. By constructing a silenced line using the KN5585 inbred line as the material background and a mutant line based on the reference genome B73 inbred line, we have validated that the silencing and mutation in this gene reduce plant drought tolerance across different genetic backgrounds. The silencing and mutation in ZmDIR11 result in slower plant growth under drought stress conditions (Figure 3A and Figure 4B) and increased chlorophyll loss (Figure 3G and Figure 4G). Physiologically and metabolically, this is evidenced by a relative decrease in Pro accumulation (Figure 3I and Figure 4H), a dramatic increase in MDA, H₂O₂, and O²⁻(Figure 3J–L and Figure 5A–C), and a relative reduction in the activity of antioxidant enzymes. In terms of hormone metabolism, the ZmDIR11 mutation is associated with a relative decrease in the synthesis of ABA and zeatin (Figure 6A,E). Genetically, there is a relative reduction in the expression levels of genes linked to ABA, zeatin, and lignan metabolism, as well as those involved in dehydration response and various drought marker genes. Overall, the mutation of ZmDIR11 leads to diminished drought tolerance in plants, indicating that this gene plays a positive regulatory role in the drought resistance of maize. We employed PCR and sequencing to analyze the ZmDIR11 gene in the KN558 and B73 inbred lines and found no variation between them, suggesting that ZmDIR11 may be a conserved functional gene across these genetic backgrounds. Furthermore, we utilized ZmDIR11-VIGS or mutant lines in various maize backgrounds to successfully validate that this gene positively regulates maize drought resistance. Unlike the functional identification of other maize drought-resistance genes, the mutation of ZmDIR11 has been shown to weaken drought resistance in two well-known maize inbred lines. Regarding the relationship between ZmDIR11 and lignan, our experiments did not yield detailed detection results. However, they confirmed that the mutation of ZmDIR11 affects the total lignan content, with this effect being more pronounced under drought stress. This may relate to one or several lignans associated with ZmDIR11, which respond specifically to environmental stress. Furthermore, yeast two-hybrid experiments did not reveal any interacting proteins with the ZmDIR11 protein. Additionally, we are currently unable to ascertain whether the ZmDIR11 protein functions independently, aggregates with multiple copies of itself to exert its effects, or synergistically interacts with other DIR proteins. The functional analysis of ZmDIR11 at the protein level remains incomplete; currently, we can only conclude that mutations in this gene influence the regulation of certain genes, thereby affecting plant drought resistance at both physiological and metabolic levels. Notably, this study observed a significant increase in total lignan content in response to drought stress. However, it remains unclear how many types of lignan exist in maize and which specific lignan type corresponds to ZmDIR11, which will be a primary focus of our future research. Additionally, we need to clarify whether this particular lignan enhances plant drought resistance, potentially offering a new metabolic indicator for drought identification in maize.

In summary, the ZmDIR11 gene structure comprises a single exon, and the protein structure domain also contains only one Dirigent domain, which is characteristic of DIR genes and Dirigent proteins. Subcellular localization analysis indicates that the ZmDIR11 protein is situated in the endoplasmic reticulum, suggesting a potential role in the metabolism and synthesis of lignans. Our experiments have demonstrated that ZmDIR11 influences the synthesis of total lignans in plants under drought stress. By silencing and mutating ZmDIR11, we have validated the gene’s function in regulating the drought tolerance of maize plants, revealing that this gene positively impacts drought resistance. The functional analysis of ZmDIR11’s role in drought resistance not only addresses a gap in the research on DIR genes related to maize drought resilience but also provides new insights into the mechanisms of action of the DIR gene family in plant stress responses. This study highlights the potential of DIR genes in enhancing the drought resistance of maize and offers a novel genetic resource for future efforts in cultivating drought-resistant maize varieties through genetic engineering.

5. Conclusions

In conclusion, this study identifies ZmDIR11 as a pivotal drought-responsive DIR gene in maize, demonstrating its conserved role in enhancing drought tolerance across distinct genetic backgrounds (KN5585 and B73). Functional validation through silencing and mutation revealed that ZmDIR11 deficiency compromises drought resilience by impairing physiological responses, including reduced Pro accumulation, elevated oxidative stress markers (MDA, H₂O₂, O²⁻), diminished antioxidant enzyme activity, and altered hormone metabolism (ABA and zeatin). ZmDIR11, encoding a single-exon Dirigent domain protein localized to the endoplasmic reticulum, influences lignan metabolism under drought, though its specific lignan targets and protein interaction mechanisms remain unresolved. Despite these gaps, our findings underscore ZmDIR11’s critical role in drought adaptation and highlight its potential as a genetic resource for breeding drought-resistant maize. Future studies should focus on elucidating ZmDIR11-linked lignan diversity, protein functionality, and stress-specific metabolic pathways to advance its application in crop resilience strategies. This work expands the understanding of DIR gene roles in plant stress responses and provides a foundation for leveraging DIR family members in agricultural biotechnology.