The transcription factor ZmMYB3R targets CYCB1;2 to modulate maize drought tolerance

SUMMARY

The R1R2R3‐MYB (3R‐MYB) transcription factor subfamily is associated with stress tolerance; however, the underlying mechanisms in crops remain poorly understood. This study investigates the function of maize MYB3R in regulating seedling drought tolerance. We characterised MYB3R overexpression lines and CRISPR‐Cas9 loss‐of‐function mutants in maize and rice using physiological assays and transcriptome profiling. DNA affinity purification sequencing (DAP‐seq) and molecular interaction assays were employed to identify direct downstream targets. MYB3R overexpression enhanced drought tolerance by promoting root development, stomatal closure and antioxidant defence, whereas mutants displayed hypersensitivity. MYB3R binds the mitosis‐specific activator (MSA) motif to directly transactivate the B‐type cyclin gene CYCB1;2, and cycb1;2 mutants phenocopied the myb3r drought defects. These findings establish that the MYB3R–CYCB1;2 module positively regulates maize drought tolerance by coordinating developmental and physiological adaptations. This pathway provides a valuable molecular target for breeding drought‐resilient crops.

Significance Statement

Our findings uncover a novel molecular mechanism underlying the MYB3R–CYCB1;2 module confers drought tolerance in maize seedlings via synergistically regulating root development, stomatal traits, antioxidant defence capacity and the transcription of diverse drought‐responsive genes. This work provides valuable molecular candidates for molecular breeding of drought‐resilient crops.

INTRODUCTION

Drought stress is a major environmental constraint threatening global crop production and food security (He et al., 2024). To survive periods of water deficit, plants have evolved a suite of adaptive strategies, including modification of root system architecture to enhance water uptake, regulation of stomatal density and aperture to limit transpirational water loss, maintenance of cellular redox homeostasis and metabolic reprogramming. These responses are tightly coordinated through changes in growth, development and molecular and cellular processes (Gupta et al., 2020; Liu et al., 2024; Yang & Qin, 2023). Upon perception of drought signals, plants activate complex signalling cascades and induce drought‐responsive genes to initiate adaptive responses (He et al., 2024; Wang et al., 2025; Yang & Qin, 2023). Plant hormones, particularly abscisic acid (ABA) and auxin, play central roles in regulating stomatal behaviour, developmental plasticity and gene expression, thereby shaping physiological and molecular responses to drought stress (Hsu et al., 2021; Jiang & Zhang, 2002; Liu et al., 2024; Yang & Qin, 2023; Zhang et al., 2025). Prolonged drought stress also leads to excessive accumulation of reactive oxygen species (ROS), which disrupts cellular metabolism and promotes stomatal closure (Chai et al., 2021; Ma et al., 2025; Yang et al., 2024). To counteract oxidative damage, several antioxidant enzymes function cooperatively to scavenge excess ROS and preserve redox balance (Noctor et al., 2014; Xiang et al., 2025).

As key regulatory nodes and promising molecular targets for improving drought tolerance, numerous transcription factors have been shown to modulate drought stress signalling networks (He et al., 2024; Yang & Qin, 2023). Among these, MYB transcription factors, one of the largest transcription factor families in plants, play pivotal roles in responses to abiotic stress (Liu et al., 2024; Zhang et al., 2025). Based on the number of conserved MYB repeats, MYB proteins are classified into four subfamilies: R1‐MYB, R2R3‐MYB (2R), R1R2R3‐MYB (3R) and R4‐MYB (Dubos et al., 2010). Extensive evidence indicates that members of the 1R‐MYB and 2R‐MYB subfamilies participate in drought stress regulation (Cominelli et al., 2005; Zhang et al., 2025). In contrast, R4‐MYB proteins are present as single‐copy genes in only a limited number of plant genomes (Dubos et al., 2010). In plants, the R1R2R3‐MYB (3R‐MYB) subfamily comprises relatively few members, most of which are associated with the regulation of mitosis and DNA damage responses, particularly through the control of G2/M‐phase‐specific genes such as cyclins and cyclin‐dependent kinases (Bourbousse et al., 2018; Inzé & De Veylder, 2006). In Arabidopsis thaliana, five genes encode MYB3R transcription factors. With the exception of AtMYB3R2, which is linked to circadian regulation, all AtMYB3R proteins play important roles in controlling the plant cell cycle (Chen et al., 2017; Kobayashi et al., 2015). Among them, AtMYB3R1 displays a unique dual function as both a transcriptional activator and repressor; consequently, AtMYB3R1 and AtMYB3R4 are classified as activator‐type MYB3Rs, whereas AtMYB3R1, AtMYB3R3 and AtMYB3R5 function as repressor‐type MYB3Rs (Chen et al., 2017; Kobayashi et al., 2015). AtMYB3R1 and AtMYB3R4 activate the transcription of numerous G2/M transition genes, including CYCB1;1, CYCB2;1 and CDKB2;1, by binding to the conserved mitosis‐specific activator (MSA) motif in their promoters (Haga et al., 2011; Kobayashi et al., 2015). These two transcription factors are also required for cytokinesis through direct regulation of the cytokinesis‐specific syntaxin gene KNOLLE (Haga et al., 2007; Lukowitz et al., 1996). By contrast, AtMYB3R3 and AtMYB3R5 act as transcriptional repressors, suppressing cell‐cycle–associated gene expression outside the G2/M phase in proliferating cells and repressing G2/M‐specific genes, such as CYCB1;1 and KNOLLE, in post‐mitotic cells during organ development. These repressors are also essential for inhibiting cell division in response to DNA damage (Chen et al., 2017; Kobayashi et al., 2015).

Beyond their established roles in cell‐cycle regulation, 3R‐MYB proteins also contribute to plant drought tolerance; however, functional evidence remains limited. To date, only five 3R‐MYB members from different plant species (rice OsMYB3R‐2, wheat TaMYB3R1, rice OsTCL1 and OsTCL2 and maize MYB3R) have been functionally characterised in the context of drought stress (Cai et al., 2015; Dai et al., 2007; Wu et al., 2019; Yi et al., 2023). Heterologous expression of OsMYB3R‐2 or TaMYB3R1 enhances drought tolerance in transgenic Arabidopsis (Cai et al., 2015; Dai et al., 2007), whereas loss‐of‐function mutations in OsTCL1 and OsTCL2 impair seed germination under drought conditions in rice (Yi et al., 2023). Four MYB3R genes were identified in the maize genome and MYB3R (Zm00001d042910) is phylogenetically closely related to rice OsMYB3R‐2 and wheat TaMYB3R1, and its full‐length amino acid sequence shares 68.4% identity and 79.1% similarity with OsMYB3R‐2 (Wu et al., 2019; Zheng et al., 2022). Our previous findings showed that the maize MYB3R was induced by drought and ABA, and its overexpression improved drought resistance in transgenic Arabidopsis (Wu et al., 2019).

Cyclins are central regulators of cell‐cycle progression in eukaryotes. By forming active kinase complexes with cyclin‐dependent kinases, cyclins ensure the largely unidirectional execution of cell‐cycle phases and regulate cell proliferation and differentiation (Inzé & De Veylder, 2006). Plant cyclins are classified into multiple subtypes, including A‐, B‐, C‐, D‐, H‐, L‐, T‐, U‐, SDS‐ and J18‐type cyclins (Menges et al., 2005; Wang et al., 2004). Among these, B‐type cyclins are specifically expressed during the late G2 and mitotic phases and play key roles in the G2/M transition and M‐phase progression, as well as in plant developmental regulation (Romeiro Motta et al., 2021). Arabidopsis CYCB1;1 and CYCB1;2 are essential for proper microtubule organisation during cell division, and the CYCB1;1 CYCB1;2 double mutant exhibits severe developmental defects, including reduced root elongation, impaired shoot growth, elevated seed abortion, delayed endosperm proliferation, pollen defects and aberrant microtubule arrays (Romeiro Motta et al., 2021). CYCB2;2 and CYCB1 also promote root growth by increasing cell number (Doerner et al., 1996; Lee et al., 2003). ZmCYCB1‐1 regulates maize embryo development and seed size (Zhao et al., 2022), and ectopic expression of AtCYCB1;2 induces multicellular trichome formation in Arabidopsis (Schnittger et al., 2002). Moreover, maize CYCB1;2 promotes cell proliferation and is regulated by glucose and sucrose during seed germination (Lara‐Núñez et al., 2021). Despite these advances, whether B‐type cyclins involved in plant drought responses is largely unknown.

As a globally important cereal crop, maize (Zea mays L.) provides essential resources for food, feed and fuel. However, maize yield is frequently constrained by drought stress because of its complex and often severe impacts on plant growth and productivity (Gupta et al., 2020). Dissecting the genetic basis of drought tolerance and improving drought resistance through biotechnological approaches are therefore critical for sustainable maize production. Our previous studies have indicated that overexpression of maize MYB3R confers drought tolerance in transgenic Arabidopsis (Wu et al., 2019). Nevertheless, its biological function and molecular regulatory mechanisms underlying drought adaptation in maize remain largely unclear. In this study, we demonstrate that maize MYB3R enhances drought tolerance at the seedling stage by coordinating root development, regulation of stomatal density and aperture and antioxidant defence systems. Overexpression of MYB3R significantly improves drought tolerance, whereas loss‐of‐function myb3r mutants exhibit pronounced drought hypersensitivity. MYB3R directly activates the B‐type cyclin gene CYCB1;2 by binding to the mitosis‐specific activator motif in its promoter and loss of CYCB1;2 similarly compromises drought tolerance in maize seedlings. Collectively, these findings identify a MYB3R–CYCB1;2 regulatory module that positively regulates drought tolerance in maize.

RESULTS

MYB3R positively regulates drought tolerance in maize seedlings

We previously confirmed that ectopic expression of maize MYB3R enhanced drought tolerance in transgenic Arabidopsis (Wu et al., 2019). To further explore its biological function in maize, we first analysed the expression profiles of MYB3R. Analysis using MaizeGDB showed that MYB3R is ubiquitously expressed, with relatively high expression in internodes, endosperm, roots and leaves (Figure S1a). qPCR analysis further confirmed high MYB3R transcript abundance in lateral roots, shoots and leaves of 3‐leaf‐stage seedlings (Figure S1b). Consistently, β‐glucuronidase activity driven by the MYB3R promoter in transgenic Arabidopsis revealed a similar expression pattern (Figure S1c). Subcellular localisation analysis revealed that MYB3R colocalises with the nuclear marker NLS‐mRFP (nuclear localisation signal‐monomeric red fluorescent protein) in maize protoplasts, confirming its nuclear localisation (Figure S1d).

To determine whether MYB3R is involved in drought responses, MYB3R expression under ABA and PEG treatments was analysed by qPCR. MYB3R transcript levels were markedly induced by both 100 μM ABA and 20% PEG, reaching peak levels at 3 and 6 h, respectively, before gradually declining (Figure S1e,f). To assess MYB3R function genetically, two independent CRISPR‐Cas9 knockout mutants (myb3r‐1 and myb3r‐2) and two overexpression lines (MYB3R‐OE3 and MYB3R‐OE4), driven by the ZmUbi promoter, were generated for subsequent analyses (Figure 1A; Figure S2a–c). The myb3r‐1 mutant harboured a 1‐bp deletion in target site 1, whereas myb3r‐2 carried a 1‐bp insertion in target site 1 and an 8‐bp deletion in target site 2 (Figure 1A), resulting in predicted truncated proteins of 272 and 266 amino acids, respectively (Table S1). Drought tolerance was evaluated by side‐by‐side cultivation under soil‐grown conditions. Under well‐watered conditions, myb3r mutants displayed a slight dwarf phenotype compared with WT plants, whereas MYB3R‐OE lines showed no obvious growth differences (Figure 1B,C). Under drought stress, however, myb3r mutants exhibited pronounced wilting and significantly reduced survival following rewatering, with approximately 80% survival in WT plants compared with only ~17% in myb3r mutants (Figure 1B,D). In contrast, MYB3R‐OE plants displayed enhanced drought tolerance and significantly higher survival rates than WT plants (Figure 1C,E). Consistent with these phenotypes, ion leakage, an indicator of membrane damage, was significantly lower in myb3r mutants and higher in MYB3R‐OE lines compared with WT plants under drought stress (Figure 1F). These results demonstrate that MYB3R positively regulates drought tolerance in maize. Additionally, to examine whether this function is conserved across species, MYB3R was overexpressed in rice. Under PEG treatment, both lines exhibited significantly higher survival rates than WT plants, indicating enhanced drought tolerance (Figure S3).

Figure 1.

MYB3R positively regulates drought tolerance in maize.

Construction of the myb3r‐1 and myb3r‐2 mutants by CRISPR/Cas9‐mediated genome editing is shown in (A), Representative photographs of WT, myb3r (B) and MYB3R‐overexpressing plants (C) after drought treatment for 8 or 10 days followed by rewatering for 2 days are shown. Survival rates of WT, myb3r and MYB3R‐overexpressing plants after 10 days of drought stress and 2 days of recovery are quantified in (D, E). Plants were cultivated in a greenhouse at 28 °C under a 14 h light/10 h dark photoperiod with about 40% relative humidity. For drought treatments, wild‐type and mutant seedlings were grown together in the same pots. Each genotype included five pots with four seedlings each. All experiments were performed in four independent biological replicates with reproducible results. Relative ion leakage in leaves of WT, myb3r and MYB3R‐overexpressing plants after 4 days of drought stress is shown in (F).

MYB3R is involved in root development in maize

Under both well‐watered and drought stress conditions, MYB3R‐overexpressing lines exhibited a more developed root system than the WT (KN5585), whereas myb3r mutants showed the opposite phenotype (Figure S4). Compared with the WT, MYB3R‐OE lines displayed significantly longer primary, seminal and crown roots, as well as an increased number of lateral roots along the primary root. In contrast, the numbers of seminal and crown roots did not differ significantly among genotypes (Figure 1A–G).

Lateral roots constitute a major component of the maize root system. To assess whether MYB3R affects lateral root primordia (LRP) formation, LRPs on the primary roots of 7‐day‐old seedlings were visualised and quantified using methylene blue staining. Compared with the WT, myb3r mutants exhibited markedly fewer LRPs, whereas MYB3R‐OE lines showed a significant increase in LRP number (Figure 2H,I), indicating that MYB3R positively regulates LRP development.

Figure 2.

MYB3R is involved in root development in maize.

Representative images of three‐leaf‐stage WT, myb3r and MYB3R‐overexpressing seedlings and their root systems grown under normal conditions are shown in (A). Quantification of seminal root number (B), crown root number (C), lateral root number on the primary root (D), seminal root length (E), crown root length (F) and primary root length (G) corresponding to (A) is presented. Methylene blue staining of primary roots from 5‐day‐old WT, myb3r and MYB3R‐overexpressing seedlings is shown in (H) (scale bar = 1 cm), and quantification of lateral root primordia density is shown in (I). EdU staining of lateral root primordia in primary roots from 4‐day‐old WT, myb3r and MYB3R‐overexpressing seedlings is shown in (J) (scale bar = 100 μm). *p < 0.05 and **p < 0.01.

Because cell proliferation underlies lateral root initiation and root growth, mitotic activity in LRPs was evaluated using EdU incorporation assays (Macheret & Halazonetis, 2019). Cross‐sectional analyses revealed no obvious morphological differences among genotypes (Figure 2J). However, the number of EdU‐positive cells was significantly reduced in myb3r LRPs and increased in MYB3R‐OE LRPs relative to the WT (Figure 2J), demonstrating that MYB3R promotes cell proliferation in maize roots. Consistent with these findings, at later developmental stages myb3r mutants exhibited reduced root development and plant height, whereas MYB3R‐OE lines showed enhanced root growth and increased plant height compared with the WT (Figure S5). These results indicate that MYB3R plays a critical role in maize root development, likely by regulating organ growth through modulation of cell proliferation.

MYB3R affects leaf stomatal density and aperture

Stomata regulate gas exchange and transpirational water loss and are therefore critical for plant adaptation to drought stress (Melotto et al., 2006). Measurements of detached leaves showed that water loss occurred more slowly in MYB3R‐OE lines than in WT plants, whereas myb3r mutants exhibited significantly higher rates of water loss over the same period (Figure 3A). Because leaf water loss is closely associated with stomatal density and aperture, these traits were examined in detail. Microscopic analysis revealed that myb3r‐1 mutants had a higher number of stomata per unit area than WT plants, although stomatal morphology was largely comparable between genotypes (Figure 3B,D). Consistently, the stomatal index was significantly higher in myb3r‐1 mutants than in WT and MYB3R‐OE plants (Figure 3C,E). The stomatal aperture was significantly reduced in MYB3R‐OE lines but increased in myb3r‐1 mutants relative to WT plants (Figure 3C,F). These results indicate that MYB3R modulates leaf stomatal density and aperture, thereby contributing to drought stress responses in maize.

Figure 3.

MYB3R affects leaf stomatal density and aperture.

Relative water loss from detached leaves of WT, myb3r‐1 and MYB3R‐OE4 plants is shown in (A). Representative images of stomatal patterns on the abaxial epidermis of WT, myb3r‐1 and MYB3R‐OE4 leaves are shown in (B) (scale bar = 100 μm), and representative images of stomatal apertures in detached leaves are shown in (C) (scale bar = 10 μm). Quantification of stomatal density (D), stomatal index (E) and stomatal aperture (F).

MYB3R activates antioxidant defence under drought stress

Drought stress is known to activate antioxidant defence mechanisms in plants (Gao et al., 2022). To determine whether MYB3R is involved in regulating oxidative stress responses, DAB and NBT staining were performed to assess ROS accumulation in WT, myb3r and MYB3R‐OE plants. Under well‐watered conditions, no obvious differences in staining intensity or area were observed among genotypes (Figure 4A,B). In contrast, under drought stress, myb3r leaves exhibited markedly stronger staining and larger stained areas than WT leaves, indicating elevated ROS accumulation (Figure 4A,B). Conversely, MYB3R‐OE lines displayed weaker staining and reduced stained areas compared with WT plants (Figure 4A,B).

Figure 4.

MYB3R activates antioxidant defence under drought stress.

Histochemical detection of H₂O₂ by DAB staining (A) and superoxide radicals by NBT staining (B) in leaves of WT, myb3r and MYB3R‐overexpressing plants is shown. Quantification of H₂O₂ content (C) and MDA content (D) in the same genotypes is presented, together with measurements of POD (E), CAT (F) and SOD (G) activities.

To further quantify oxidative stress and antioxidant capacity, H₂O₂ content, MDA levels and the activities of POD, CAT and SOD were measured (Figure 4C–G). After drought treatment, myb3r mutants accumulated significantly higher levels of H₂O₂ (Figure 4C) and MDA (Figure 4D) than WT plants, whereas the activities of POD (Figure 4E), CAT (Figure 4F) and SOD (Figure 4G) were markedly reduced. In contrast, MYB3R‐OE lines showed reduced H₂O₂ and MDA accumulation and significantly enhanced antioxidant enzyme activities relative to WT plants (Figure 4C–G). These results demonstrate that MYB3R enhances drought tolerance in maize by activating antioxidant defence systems and maintaining redox homeostasis under drought stress.

MYB3R governs the transcription of multiple genes implicated in cell‐cycle regulation, plant development and stress response

To further elucidate the molecular basis of MYB3R‐mediated drought tolerance in maize, transcriptome profiling was performed in WT and MYB3R‐OE plants under control and drought stress conditions. Principal component analysis (PCA) revealed that the first two principal components accounted for 88.3% of the total variance, and biological replicates clustered tightly, indicating high reproducibility of the RNA‐seq data (Figure S6a). A large number of differentially expressed genes (DEGs, fold change (FC) > 1.5, False discovery rate (FDR) < 0.05) were identified across four pairwise comparisons: WT_D versus WT_C, OE_D versus OE_C, OE_C versus WT_C and OE_D versus WT_D (Figure 5A–C, Figure S6b, Data Set S1).

Figure 5.

Genome‐wide identification of MYB3R binding motifs and target genes by DAP‐seq.

A Venn diagram showing the overlap of MYB3R binding peaks identified from two independent DAP‐seq replicates is shown in (A). The genomic distribution of MYB3R binding peaks is shown in (B) and enrichment of binding peaks within the 2‐kb region upstream of transcription start sites is shown in (C). Gene Ontology enrichment analysis of MYB3R‐bound target genes is shown in (D). Overlap between MYB3R DAP‐seq targets and MYB3R‐regulated DEGs is shown in (E), with the distribution of activated and repressed genes shown in (F) and a heatmap of representative target gene expression shown in (G). Enriched MYB3R binding motifs identified by DAP‐seq, including the MSA motif, are shown in (H).

Overlap analysis revealed that 31.5% (793/2520) of DEGs in OE_C versus WT_C and 29.4% (995/3383) in OE_D versus WT_D overlapped with DEGs identified in WT_D versus WT_C, which were considered drought‐responsive genes. This substantial overlap indicates that MYB3R overexpression markedly reshapes the drought‐responsive transcriptional network in maize (Figure S6c,d). Among drought‐induced genes, 220 were constitutively upregulated in MYB3R‐OE plants relative to WT plants. Moreover, even under control conditions, 1035 genes were constitutively upregulated and 1485 genes were constitutively downregulated in MYB3R‐OE plants (Figure S7b,c). GO enrichment analysis showed that both upregulated and downregulated gene sets were significantly enriched in categories related to the cell cycle, plant development, metabolic processes, oxidation–reduction processes and stress responses (Figure S7d). Overall, these transcriptomic changes are consistent with the physiological and developmental phenotypes observed in myb3r mutants and MYB3R‐OE lines (Figures 1, 2, 3, 4), underscoring a central role for MYB3R in coordinating drought‐responsive gene expression in maize.

CYCB1;2 is a MYB3R target and is transactivated by MYB3R

In Arabidopsis and wheat, 3R‐MYB subfamily proteins preferentially associate with promoters containing the mitosis‐specific activator (MSA; core sequence AACGG) cis‐element (Bourbousse et al., 2018; Cai et al., 2015). To identify MYB3R binding motifs and target genes in maize, DNA affinity purification sequencing (DAP‐seq) was performed by incubating recombinant MYB3R–HaloTag protein with a genomic DNA library from the maize inbred line B73, followed by sequencing of MYB3R‐bound fragments. Two independent biological replicates identified 24 544 and 32 844 binding peaks, respectively, with an overlap of 10 429 peaks corresponding to 4337 putative target genes (Figure 5A; Supplemental Data Set S2). Genomic distribution analysis revealed that 14.11% of peaks were located in promoter regions, 3.39% in 5′ UTRs, 5.24% in 3′ UTRs, 2.59% in exons, 13.27% in introns and 7.11% in downstream regions (≤500 bp), whereas the remaining 53.76% were located in distal intergenic regions (Figure 5B). Among genic peaks, MYB3R showed a strong preference for binding proximal promoter regions near transcription start sites (Figure 5C). GO enrichment analysis indicated that putative MYB3R target genes were predominantly associated with cellular and metabolic processes, responses to stimuli, cell‐cycle regulation and developmental processes (Figure 5D).

Integration of DAP‐seq data with transcriptome analyses revealed that 319 MYB3R‐bound genes were transcriptionally regulated by MYB3R under drought stress, whereas 269 were regulated under control conditions, with 104 high‐confidence targets shared between both conditions (Figure 5E). MYB3R‐activated and MYB3R‐repressed genes were present in approximately equal proportions, suggesting that MYB3R may function as both a transcriptional activator and repressor in maize drought responses (Figure 5F,G). Motif enrichment analysis of MYB3R‐bound regions identified four significantly enriched core motifs, including the canonical MSA motif (AACGGCT) and three previously unreported motifs (motif 1: TTTACCGTT; motif 2: CGGTAAANAGT; motif 3: TACNGTNACT) (Figure 5H; Figure S8). Several high‐confidence target genes involved in plant development and drought tolerance were found to harbour these motifs (Figure S8).

To determine whether cyclin genes are direct targets of MYB3R, CYCB1;2 (Zm00001d010656) was selected for further analysis. DAP‐seq signals were clearly enriched in the CYCB1;2 promoter region (Figure 6A). Yeast one‐hybrid assays were then performed using full‐length and truncated promoter fragments of CYCB1;2, as well as versions carrying motif mutations (Figure 6B–D). Only the full‐length, P2P3 and P3 promoter fragments, all of which contained the P3 region, interacted with MYB3R, whereas mutation of the MSA motif within the P3 fragment abolished this interaction (Figure 6C,D). Consistently, dual‐LUC assays showed that co‐expression of 35S:MYB3R significantly enhanced luciferase activity driven by the P3 promoter fragment, whereas mutation of the MSA motif eliminated this activation (Figure 6E,F). These results were further confirmed in N. benthamiana leaves (Figure 6G). In addition, an electrophoretic mobility shift assay (EMSA) showed that recombinant MYB3R protein (His‐MYB3R) tagged with His specifically bound to the probes containing MSA motif of the CYCB1;2 promoter (Figure 6H and Figure S9). Moreover, RT‐qPCR analysis demonstrated that CYCB1;2 expression was markedly upregulated in MYB3R‐OE plants and downregulated in myb3r‐1 mutants (Figure 6I). These results demonstrate that CYCB1;2 is a direct transcriptional target of MYB3R, which binds to the MSA motif in the CYCB1;2 promoter to activate its expression.

Figure 6.

MYB3R directly targets CYCB1;2 and activates its expression.

Genome browser visualisation of MYB3R binding to the CYCB1;2 promoter region is shown in (A). Schematic diagrams of the CYCB1;2 promoter and its truncated or mutated versions containing alterations in the MSA motif are shown in (B). Yeast one‐hybrid analysis is shown in (C), and liquid β‐galactosidase assays of yeast are shown in (D). Schematic diagrams of effector and reporter constructs are shown in (E), and activation of CYCB1;2 promoter reporters by MYB3R in tobacco leaves is shown in (F, G). EMSA assays are shown in (H). Relative LUC/REN activities are indicated, with the empty 62SK vector used as a negative control. Relative expression levels of CYCB1;2 by RT‐qPCR are shown in (I). **p < 0.01.

CYCB1;2 regulates drought tolerance in maize

To investigate the biological function of CYCB1;2 in maize, two independent CRISPR‐Cas9 knockout mutants (cycb1;2–1 and cycb1;2–2) were generated (Figure 7A; Table S1). Phenotypic analyses were subsequently conducted to compare the CYCB1;2 mutants with WT plants. After 20 days of water withholding, both CYCB1;2 mutant lines exhibited enhanced drought sensitivity, characterised by more severe wilting symptoms and significantly lower survival rates than WT plants following drought stress (Figure 7B,C), similar to the phenotypes of myb3r mutants (Figures 1, 2, 3, 4).

Figure 7.

Loss of CYCB1;2 reduces drought tolerance in maize.

Construction of the cycb1;2–1 and cycb1;2–2 mutants by CRISPR/Cas9‐mediated genome editing is shown in (A). Representative photographs of WT and cycb1;2 plants after 20 days of drought stress are shown in (B), and survival rates following drought stress and rewatering are shown in (C). Plants were cultivated in a greenhouse at 28 °C under a 14 h light/10 h dark photoperiod with about 40% relative humidity. For drought treatments, wild‐type and mutant seedlings were grown together in the same pots. Each genotype included five pots with four seedlings each. All experiments were performed in four independent biological replicates with reproducible results. Quantification of seminal root and crown root length in three‐leaf‐stage WT and cycb1;2 seedlings is shown in (D). EdU staining of lateral root primordia in primary roots from 4‐day‐old WT and cycb1;2 seedlings is shown in (E) (scale bar = 100 μm). Quantification of stomatal aperture is shown in (F), and POD, CAT and SOD activities are shown in (G).

Consistent with these drought‐sensitive phenotypes, cycb1;2 mutants displayed markedly reduced root length and decreased mitotic activity in LRPs compared with WT plants (Figure 7D,E; Figure S9). In addition, the mutants exhibited increased leaf stomatal density and larger stomatal apertures (Figure 7F; Figure S10), together with significantly reduced activities of POD, CAT and SOD under drought stress (Figure 7G). These results demonstrate that CYCB1;2 positively regulates drought tolerance in maize by coordinating root development, stomatal density and aperture and antioxidant defence. These phenotypic similarities further support the conclusion that CYCB1;2 functions downstream of MYB3R in mediating drought resistance.

DISCUSSION

The roles of 3R‐MYB TFs in regulating the plant cell cycle and DNA damage responses during organ development have been extensively investigated, and their molecular mechanisms are relatively well defined (Bourbousse et al., 2018; Chen et al., 2017; Kobayashi et al., 2015; Zhang et al., 2025). In contrast, other biological functions of 3R‐MYB TFs, particularly their involvement in stress responses in crops, remain poorly understood. In this study, we demonstrate that the maize 3R‐MYB TF, MYB3R, targets CYCB1;2 and positively regulates drought tolerance in maize seedlings by coordinately modulating root development, leaf stomatal density and aperture and antioxidant defence.

Across plant species, MYB3R orthologues display a degree of functional conservation, although their regulatory roles are not identical. Phylogenetic and sequence analyses indicate that maize MYB3R is closely related to rice OsMYB3R‐2, wheat TaMYB3R1 and Arabidopsis AtMYB3R3 and AtMYB3R5 (Wu et al., 2019). In Arabidopsis, AtMYB3R3 and AtMYB3R5 are essential for organ development and for suppressing cell division in response to DNA damage (Chen et al., 2017; Kobayashi et al., 2015). In contrast, overexpression of OsMYB3R‐2, TaMYB3R1 or maize MYB3R in Arabidopsis confers enhanced tolerance to drought and other abiotic stresses (Cai et al., 2015; Dai et al., 2007; Wu et al., 2019). OsMYB3R‐2‐overexpressing plants show improved tolerance to cold, drought and salt stress, accompanied by induction of stress‐responsive genes such as DREB2A and COR15A (Dai et al., 2007), and OsMYB3R‐2 also regulates chilling tolerance in rice (Ma et al., 2009). Similarly, TaMYB3R1 enhances drought and salt tolerance in Arabidopsis by promoting stomatal closure, reducing water loss and modulating stress‐responsive gene expression (Cai et al., 2015). Overexpression of maize MYB3R in Arabidopsis confers comparable stress tolerance (Wu et al., 2019). Here, we extend these findings by demonstrating that increased MYB3R expression enhances drought tolerance in both maize and rice, whereas myb3r mutants exhibit strong drought hypersensitivity in maize seedlings (Figure 1; Figure S3). These phenotypes are associated with improved root system architecture (Figure 2), reduced stomatal aperture (Figure 3), enhanced antioxidant capacity (Figure 4) and widespread transcriptional reprogramming of genes involved in cell‐cycle regulation, development, redox processes and stress responses (Figure S7).

MYB3R orthologues appear to exert dual regulatory functions as transcriptional activators or repressors depending on species and context. In Arabidopsis, AtMYB3R3 and AtMYB3R5 act as MSA‐binding transcriptional repressors that suppress G2/M‐specific genes such as CYCB1;2 and KNOLLE in post‐mitotic cells and during DNA damage responses (Chen et al., 2017; Kobayashi et al., 2015). By contrast, OsMYB3R‐2 and TaMYB3R1 function predominantly as MSA‐binding transcriptional activators (Cai et al., 2015; Ma et al., 2009); for example, OsMYB3R‐2 directly activates OsCYCB1;1 to regulate cell‐cycle progression during chilling stress (Ma et al., 2009). In this study, we show that maize MYB3R binds the MSA motif in the CYCB1;2 promoter and activates its transcription, thereby promoting drought tolerance in maize seedlings (Figure 5). These findings highlight a conserved yet flexible regulatory role of MYB3R proteins in linking cell‐cycle control with stress adaptation across plant species. The plant cell cycle is a tightly coordinated process that underpins plant growth and development and relies on cyclins as phase‐specific regulators (Inzé & De Veylder, 2006). B‐type cyclins act primarily at the G2/M transition and during M‐phase progression and play evolutionarily conserved roles in microtubule organisation and plant organ development (Doerner et al., 1996; Lara‐Núñez et al., 2021; Schnittger & De Veylder, 2018; Zhao et al., 2022). In mammals, loss‐of‐CYCB1 function leads to very early embryonic lethality, highlighting its essential role in cell‐cycle control (Brandeis et al., 1998). In Arabidopsis, CYCB1;2 is required for free nuclear divisions during endosperm development and functions redundantly with CYCB1;1 and CYCB1;3; accordingly, cycb1;1 cycb1;2 double mutants exhibit markedly reduced root elongation (Romeiro Motta et al., 2021). Consistent with these observations, we found that loss‐of‐function cycb1;2 maize mutants display significantly reduced root length and diminished mitotic activity in LRPs (Figure 7). Despite the well‐established roles of B‐type cyclins in development, their functions in plant stress responses remain largely unexplored. Arabidopsis CYCB1;1 is strongly induced in cells experiencing DNA damage, suggesting a role in DNA repair (Schnittger & De Veylder, 2018). Here, we demonstrate that CYCB1;2 positively regulates drought tolerance in maize, likely through coordinated effects on root development, stomatal closure and the activities of CAT, POD and SOD (Figure 7). While the exact molecular cascade connecting CYCB1;2 to stomatal function and ROS scavenging is unclarified, cumulative work demonstrates crosstalk between cyclin–CYCLIN‐DEPENDENT KINASEs (CDK) modules and abiotic stress signalling. Such interplay orchestrates stomatal morphogenesis, systemic stress responses, ROS homeostasis and antioxidant gene transcription (Qi & Zhang, 2020; Zhao et al., 2017). A representative case is that loss of Arabidopsis CDKC;2 reshapes cell cycle and stomatal gene expression to strengthen drought tolerance (Zhao et al., 2017). Contrastingly, overexpression of maize MYB3R confers improved drought resilience (Figure 1). These opposite phenotypic outputs under drought suggest the two cell‐cycle regulators act via separate downstream signalling modules. Our findings provide new insights into the contribution of B‐type cyclins to drought stress tolerance in plants. Elucidating how the cell‐cycle regulator regulates stomatal closure and antioxidant enzyme activity merits further exploration. Additionally, exploring the functions of MYB3R and the MYB3R‐CYCB1;2 module in various abiotic stress responses and clarifying their genetic interaction is of great value.

Moreover, multiple high‐confidence MYB3R target genes implicated in root development, cell‐cycle regulation, DNA repair, stomatal closure, antioxidant defence and drought resistance, including KNOLLE, CPK9/13 and RBOHC (Figure S8; Data Set S2), were identified. For example, the calcium‐dependent protein kinase OsCPK9 enhances stomatal closure and root elongation, increases catalase activity and maintains H₂O₂ homeostasis, thereby improving drought tolerance in rice (Shi et al., 2025). Similarly, the plasma membrane‐localised NADPH oxidase ZmRBOHC promotes ROS accumulation in guard cells and induces stomatal closure, contributing to enhanced drought tolerance in maize (Gao et al., 2022). Future identification and functional characterisation of additional MYB3R target genes, together with dissection of the regulatory networks mediated by the MYB3R–CYCB1;2 module, will provide deeper mechanistic insights into drought tolerance in maize.

In summary, our results demonstrate that MYB3R and the MYB3R–CYCB1;2 regulatory module positively regulate drought tolerance in maize seedlings by coordinately controlling root development, stomatal density and aperture, antioxidant defence and the expression of a broad set of drought‐responsive genes. These findings provide mechanistic insight into the integration of cell‐cycle regulation with drought adaptation and offer a valuable molecular framework for improving drought tolerance in maize through targeted breeding strategies.

MATERIALS AND METHODS

Plant materials and growth conditions

The maize (Zea mays L.) inbred line KN5585 was used throughout this study. Plants were grown in a controlled growth chamber or greenhouse maintained at 28 °C with a 14 h light/10 h dark photoperiod. For hormone and osmotic stress treatments, plants were exposed to 20% PEG or 100 μM ABA for the specified durations.

For drought treatment, 3‐leaf‐stage maize plants were subjected to water withholding for 0, 6, 8, 10, 15, 20 or 25 days. At each time point, soil was weighed to calculate relative soil moisture content. Drought treatments were conducted by subjecting plants to natural soil drying under greenhouse conditions. Approximately 80 seedlings per genotype were assayed. wild‐type and mutant seedlings were grown together in the same pots. Each genotype included five pots with four seedlings each. All experiments were performed in four independent biological replicates with reproducible results. The second leaves were harvested and immediately frozen in liquid nitrogen for further analyses.

To generate CRISPR knockout constructs targeting MYB3R and CYCB1;2, a 20‐bp gene‐specific spacer sequence was inserted into the CRISPR/Cas9 expression vector. For overexpression analysis, the full‐length coding sequence of MYB3R was cloned into the WMV017 vector under the control of the ZmUbi promoter. Transgenic maize plants were produced in the KN5585 background by Jiangsu Weimi Biosciences Co., Ltd. (Changzhou, China). Gene‐specific primers are listed in Table S2.

RNA extraction and quantitative real‐time PCR analysis

Total RNA was extracted from frozen plant tissues using the Plant RNeasy Mini Kit (Tiangen Biotech, Beijing, China) according to the manufacturer's instructions. For RT‐qPCR analysis, 1 μg of total RNA was reverse‐transcribed into first‐strand cDNA using the Reverse Transcription System (Promega, Madison, WI, USA). Quantitative PCR was performed using Power SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) on an ABI 7500 Real‐Time PCR System (Applied Biosystems) following the manufacturer's protocols. Expression levels were normalised against maize ZmActin2, which served as the internal reference gene. Primer sequences used for RT‐qPCR are provided in Table S2.

Subcellular localisation of MYB3R

The pCAMBIA1300‐GFP vector was used to generate the MYB3R–GFP fusion construct. Maize mesophyll protoplasts were isolated from etiolated leaves of 12‐day‐old seedlings and transfected with the indicated constructs using a PEG‐mediated method as previously described (Chen et al., 2025); the 35S:GFP construct was used as a negative control. After incubation in the dark for 16–24 h, GFP fluorescence in transfected protoplasts was examined using a Zeiss LSM780 laser scanning confocal microscope (Zeiss, Oberkochen, Germany).

Transactivation activity assays

The coding sequence of MYB3R and its derivative variants were subcloned into the pGBKT7 vector using the ClonExpress II One Step Cloning Kit (Vazyme, Nanjing, China). Recombinant constructs were transformed into the yeast strain AH109 following the standard protocol (Clontech, Mountain View, CA, USA). Transformants were initially selected on SD/−Trp medium and subsequently transferred to SD/−Trp−Leu−His−Ade medium to assess transactivation activity.

DAB and NBT staining

DAB and NBT staining were performed as previously described (Li et al., 2025). Briefly, maize leaves were incubated in 1 mg mL⁻¹ 3,3′‐diaminobenzidine tetrahydrochloride (DAB) or nitroblue tetrazolium (NBT) solution for 8 h at room temperature. After staining, samples were transferred to a clearing solution consisting of ethanol:acetic acid:glycerol (3:1:1, v/v/v) and incubated at 95 °C until complete decolorisation.

Drought‐related physiological and biochemical indices

Malondialdehyde (MDA) content was measured using a Micro MDA Assay Kit (Solarbio Science & Technology Co., Ltd., Beijing, China) according to the manufacturer's instructions. The CAT, POD and SOD were determined using the corresponding enzyme activity assay kits from the same manufacturer.

Transverse sectioning, methylene blue staining and EdU staining

For transverse sectioning, maize roots were embedded in 4% (w/v) agarose and sectioned into 50 μm‐thick slices using a vibratome. Sections stained with safranin O were observed under a light microscope (CX23; Olympus, Tokyo, Japan). Lateral root primordia (LRP) were quantified following methylene blue staining. Briefly, 8‐cm‐long root segments were hydrolysed in 6 N HCl under vacuum for 15 min at room temperature, rinsed three times with distilled water and incubated in Schiff's solution (Sigma‐Aldrich, St. Louis, MO, USA) for 5 min prior to LRP counting.

For EdU staining, roots from 3‐day‐old seedlings were incubated in 50 μM EdU solution for 4 h, fixed in 4% (w/v) paraformaldehyde for 30 min and transversely sectioned using a vibratome. Sections were subsequently incubated with Apollo staining solution for 30 min. EdU‐labelled cells were visualised and quantified using a laser scanning confocal microscope (Olympus).

RNA‐seq analysis

RNA sequencing was performed as previously described (Chen et al., 2025). Three biological replicates of 3‐leaf‐stage WT and MYB3R‐overexpressing maize seedlings were analysed under control and drought stress conditions. Library construction and sequencing were conducted by BioMarker Technologies (Beijing, China). Clean reads were aligned to the maize B73 reference genome (RefGen version 4.47) using TopHat. Gene expression levels were quantified with Cufflinks (version 2.1.1) and normalised as fragments per kilobase of transcript per million mapped reads (FPKM). Differentially expressed genes (DEGs) were identified using the DESeq package in R software, with a false discovery rate (FDR) < 0.05 and fold change (FC) > 1.5 as significance thresholds. Principal component analysis (PCA) was performed using the R package GMODELS (http://www.r‐project.org). Gene Ontology enrichment analysis was conducted using agriGO v2.0 (http://systemsbiology.cau.edu.cn/agriGOv2/index.php). Heat maps were generated based on FPKM values using the OmicShare online platform (https://www.omicshare.com/tools).

DAP‐seq analysis

Recombinant MYB3R–HaloTag protein bound to HaloTag magnetic beads was incubated with a genomic DNA library prepared from the maize inbred line B73. DNA fragments bound by MYB3R were eluted from the beads and subjected to high‐throughput sequencing. Enriched binding motifs were identified using MEME‐ChIP (Machanick & Bailey, 2011).

Dual luciferase reporter (dual‐LUC) assay and yeast one‐hybrid (Y1H) assay

Dual‐LUC and Y1H assays were performed as previously described (Chen et al., 2025). Briefly, reporter and effector plasmids were co‐infiltrated into leaves of 5–6‐week‐old Nicotiana benthamiana plants using a needleless syringe. After 3 d of infiltration, luciferase signals were detected using a 5200 multichemiluminescent imaging system (Tanon Biomart, Beijing, China). LUC and REN activities were quantified using the Dual Luciferase Reporter Gene Assay Kit (Yeasen, Shanghai, China), and relative promoter activity was calculated as the LUC:REN ratio.

For the Y1H assay, the MYB3R coding sequence was inserted into the pGADT7‐Rec2 (AD‐rec) vector, while the CYCB1;2 promoter fragment was cloned into the pAbAi vector. Transformants were selected on SD/−Leu medium and subsequently screened on SD/−Leu medium supplemented with Aureobasidin A (Aba). Yeast cultures were adjusted to three serial dilutions, spotted onto selective plates and incubated at 30 °C for 2–3 days before imaging.

Electrophoretic mobility shift assay (EMSA)

An EMSA was performed as previously described (Li et al., 2024). Briefly, the recombinant protein used in the EMSA was expressed, the gene for which was prepared by cloning the coding sequence of MYB3R into the pCzn1 vector. Oligonucleotide probes of the CYCB1;2 promoter were synthesised and labelled with biotin at the 3′ end with a Biotin 3′ End DNA Labeling Kit (Thermo) according to the standard procedures. Each probe was mixed with purified recombinant protein at 25 °C for 20 min in reaction buffer (20 μL) containing 10× binding buffer, 50% (v/v) glycerol, 100 mM MgCl₂, 1 μg/μL poly(dI‐dC), 50 mM KCl and 1% (v/v) NP‐40. Biotin‐labelled DNA was detected according to the instructions of the LightShift Chemiluminescent EMSA Kit (Thermo). The luminescence was visualised on a Tanon‐5200 M imaging system.

Statistical analysis

All data were analysed using one‐way or two‐way analysis of variance (ANOVA) followed by Tukey's multiple comparison test or by unpaired t‐tests, as appropriate, using GraphPad Prism 7.0 (GraphPad Software, San Diego, CA, USA). All experiments were repeated three times with similar results. Data are means ± SD of three biological replicates. Different letters indicate significant differences at P < 0.05. For RNA‐seq analyses, P‐values and false discovery rates of differentially expressed genes were calculated using the DESeq2 package in R with default parameters.

AUTHOR CONTRIBUTIONS

KG, YJ and JW conceived and designed the research. CW performed the bioinformatics analyses. KG, YJ and YG performed the experiment, JL, LC, YY and CY assisted with the genotyping and phenotyping. CW, XL and JW analysed the data and prepared the manuscript. KG, YJ and YG contributed equally to this work. All authors read and approved the final manuscript.

CONFLICT OF INTEREST

The authors declare that the research was conducted without any known commercial or financial interests that would serve as a potential conflict of interest.

Supporting information

Figure S1. MYB3R is widely expressed and is induced by ABA and PEG treatments.

Figure S2. Characterisation of MYB3R‐overexpressing maize lines.

Figure S3. Overexpression of maize MYB3R enhances drought tolerance in transgenic rice.

Figure S4. Phenotypic analysis of WT, myb3r and MYB3R‐overexpressing maize seedlings under normal and drought conditions.

Figure S5. Morphological and root phenotypes of myb3r and MYB3R‐overexpressing plants in the field at the silking stage.

Figure S6. Identification of differentially expressed genes by RNA‐seq analysis.

Figure S7. Overexpression of MYB3R alters the expression of drought‐responsive genes.

Figure S8. Enriched MYB3R binding motifs and representative target genes identified by DAP‐seq.

Figure S9. Protein purification of His‐MYB3R fusion protein.

Figure S10. Phenotypic analysis of WT and cycb1;2 maize seedlings.

Table S1. Predicted amino acid sequences of maize MYB3R and CYCB1;2 in WT and CRISPR/Cas9‐generated mutant plants.

Table S2. Primer sequences used in this study.

Data Set S1. RNA ‐seq analysis of WT and MYB3R‐OE plants with or without drought stress.

Data Set S2. DAP‐seq analysis of MYB3R target genes.

DATA AVAILABILITY STATEMENT

The authors confirm that all experimental data are available and accessible via the main text and/or the supplemental data.