ZmEREB180 modulates waterlogging tolerance in maize by regulating root development and antioxidant gene expression

With climate change increasing the frequency of extreme weather events, waterlogging has become a significant threat to agricultural production, especially in maize-growing regions. Waterlogging induces hypoxic conditions in the root zone, limiting maize growth and yield (Liang et al., 2020; Pedersen et al., 2017). Plants have evolved adaptive mechanisms, such as adventitious root (AR) formation and enhanced antioxidant activity, to cope with waterlogging stress (Pedersen et al., 2021; Yamauchi et al., 2018). However, the regulatory mechanisms in maize remain poorly understood.

Group VII ethylene response factor proteins (ERFVIIs) are key regulators of waterlogging tolerance in model plants (Hartman et al., 2021). Our previous work showed that ZmEREB180, a maize ERFVII, promotes waterlogging tolerance by enhancing AR formation and modulating antioxidant levels (Yu et al., 2019). In this study, we cloned the full-length coding sequence of ZmEREB180 and inserted it into the pM999 vector. The recombinants and empty vector were transiently expressed in isolated B73 leaf protoplasts, followed by a transient and simplified cleavage under targets and tag-mentation (tsCUT&Tag) assay (Liang et al., 2024). A total of 4720 confident peaks corresponding to 3335 genes were identified (Table S1). Notably, 70.15% of these peaks were located in promoter regions, with 68.67% found in promoters less than 1 kb upstream (Figure 1a). The highest enrichment was observed at the transcription start site (Figure 1b). Motif analysis revealed the GCC-box (GCCGCC) as the highest scoring motif (E-value = 5.7 × 10⁻¹⁰). Compared with RNA-Seq data (Yu et al., 2019) identified 421 genes that were differentially expressed in the ZmEREB180 overexpression lines, under waterlogged conditions, and were directly bound by ZmEREB180 (Figure 1c; Table S2). We focused on genes involved in root development and antioxidant pathways. Lateral organ boundaries domain (LBD) proteins play pivotal roles in organ development. Two LBD genes, ZmLBD5 and ZmLBD38 (Table S2), were up-regulated in an overexpression line and under waterlogging conditions, in which ZmLBD5 has been shown to promote AR formation (Feng et al., 2022). Four antioxidant genes, including two glutathione-S-transferases (GST, ZmGST8 and ZmGST31) and two peroxidases (POD, ZmPOD12 and ZmPOD55), exhibited similar expression profiles (Table S2). The tsCUT&Tag data revealed significant peaks in the promoter of these genes (Figure 1d). Additionally, GCC-box motifs were located in these regions, suggesting direct regulation by ZmEREB180 under waterlogging conditions.

Figure 1

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The regulatory networks of ZmEREB180 involved in maize waterlogging tolerance. (a) Distribution of identified peaks from ZmEREB180 tsCut&Tag. Two independent experiments were conducted and the overlapping genes were used for further analysis. (b) Peak profile flanking the 3-kb gene body. TSS, transcript start site; TTS, transcription termination site. (c) Overlap of genes identified by ZmEREB180 tsCut&Tag and differentially expressed genes in ZmEREB180 overexpression lines and in waterlogging stress. (d) Distribution of ZmEREB180 binding sites in six target genes. (e) Design for gene editing of ZmEREB180. (f) Expression levels of ZmEREB180 and its target genes in zmereb180 mutants and B104 roots under normal (0 h) and waterlogging conditions (4 h). The gene expression values represent the mean derived from three independent biological replicates. (g) Phenotypes of zmereb180 mutants and B104 under normal conditions and after 6 days of waterlogging, along with leaf and root characterization (h) and corresponding physiological responses, including POD and GST activity (i). (j, k) Luciferase activity of ZmEREB180 effector and its targets in maize protoplasts. (l) Electrophoretic mobility shift assay demonstrating the binding of ZmEREB180 to the promoters of its target genes. (m) Yeast two-hybrid assay to validate the interaction between ZmEREB180 and ZmMPK1/ZmMPK3. (n) In vitro pull-down assay confirming the interaction between ZmEREB180 and ZmMPK1/ZmMPK3. (o) Split luciferase assay to further validate the interaction between ZmEREB180 and ZmMPK1/ZmMPK3. (p) Luciferase activity of ZmEREB180 effector combined with ZmMPK1, ZmMPK3, and their target genes in maize protoplasts. (q) Phenotypes of ZmLBD5 overexpression lines and wild-type KN5585 under normal and 6 days of waterlogging conditions, along with root characterization (r). (s) Phenotypes of F1 hybrids derived from KN5585 and ZmLBD5#OE1 crossed with four inbred lines, under normal and 10 days of waterlogging conditions, along with leaf and root characterization (t). (u) Regulatory networks mediated by ZmEREB180.

Two CRISPR/Cas9-generated mutants, zmereb180-1 and zmereb180-2, in maize line B104 were analysed (Figures 1e and S1). The expression level of ZmLBD5, ZmLBD38 and the antioxidant genes was downregulated in both two mutants under waterlogging (Figure 1f; Table S3), indicating ZmEREB180 regulates these genes. No differences were observed between B104, zmereb180-1 and zmereb180-2 at the second leaf stage, but significant phenotypic differences were evident after 6 days of waterlogging treatment (Figures 1g and S2). Both mutants showed greater leaf injury, reduced root length, AR number, and AR length compared to B104 (Figure 1h). Increased POD and GST activity in B104 roots under waterlogging was diminished in the mutants (Figure 1i), supporting the role of ZmEREB180 in waterlogging tolerance.

To confirm the binding of ZmEREB180 to these targets, we performed dual luciferase reporter assays using 1.5-kb promoter fragments of ZmLBD5, ZmLBD38, ZmGST8, ZmGST31, ZmPOD12 and ZmPOD55 (Figure 1j). Co-transfection with ZmEREB180 in maize leaf protoplasts significantly enhanced the expression of all six genes, especially ZmLBD5, ZmLBD38 and ZmGST8 (Figures 1k and S3). Electrophoretic mobility shift assays (EMSAs) confirmed that ZmEREB180 binds directly to the GCC motifs in the promoters of these genes (Figures 1l and S4).

A time-course transcriptome analysis of waterlogged roots in B73 seedlings revealed a significant enrichment of mitogen-activated protein kinase (MPK) signalling under stress (Yu et al., 2020). Using a yeast two-hybrid assay, we identified two MPKs, ZmMPK1 and ZmMPK3, that interacted with ZmEREB180 (Figure 1m). GST pull-down assays confirmed that ZmEREB180 interacts with ZmMPK1 and ZmMPK3 in vitro (Figure 1n). A split luciferase assay further validated these interactions in plata (Figure 1o). Co-transfection of ZmMPK1 or ZmMPK3 with ZmEREB180 in maize leaf protoplasts significantly enhanced the activation of ZmLBD5, ZmLBD38, ZmGST8, ZmGST31, ZmPOD12 and ZmPOD55 promoters (Figure 1p), suggesting that ZmMPK1 and ZmMPK3 enhance ZmEREB180-mediated transcriptional activation.

To access the functional role of ZmEREB180 target genes under waterlogging, we subjected ZmLBD5 overexpression lines to waterlogging treatment. Overexpression lines, ZmLBD5#OE1 and ZmLBD5#OE2, showed significantly enhanced AR formation, seedling growth, and waterlogging tolerance after 6 days of stress, compared with wild-type KN5585 (Figures 1q, r, S5 and S6). F1 hybrids from a cross of ZmLBD5#OE1 and four maize inbred lines (B73, Huangzao4, Ye478 and ZHB12) also displayed improved waterlogging tolerance after 10 days of treatment (Figure 1s, t), with reduced leaf injury and enhanced root and seedling growth.

Our findings highlight the critical role of ZmEREB180 in regulating maize tolerance to waterlogging stress (Figure 1u). We demonstrate that ZmEREB180 directly interacts with key genes such as ZmLBD5, ZmLBD38, ZmGST8, ZmGST31, ZmPOD12 and ZmPOD55, positively modulating their expression under waterlogging. We also show that interactions with ZmMPK1 and ZmMPK3 enhance the activation of downstream target genes. Overexpression of ZmLBD5 improves AR formation and waterlogging tolerance across different genetic backgrounds, making it a promising target for developing maize varieties with improved resilience to waterlogging, major abiotic stress affecting global maize production.