Natural Allelic Variations in ZmDT1 Enhance Drought Resistance in Maize

Maize (Zea mays) is a primary global food source, major livestock feed and industrial raw material. Climate change-amplified water deficit restricts maize growth, with US data (1961–2021) showing that drought causes 17%–29% yield loss, threatening food security (Yang et al. 2023). Identifying maize drought response mechanisms, key tolerance genes and their roles in adaptive plasticity is vital for breeding adaptable varieties, yet remains a major unsolved challenge (Yang et al. 2023). To identify maize drought-tolerance genes, we evaluated the drought phenotypes and root architectures of 150 accessions in field. Two genetically stable inbred lines—drought-tolerant B7 with lower water loss and higher survival rates and drought-sensitive A4 with severe leaf wilting—were selected (Figure 1a, Figure S1a,b). Using F2 plants with extreme leaf wilting under drought and divergent root biomasses, we performed bulked segregant analysis (BSA) coupled with DNA sequencing. This analysis identified three quantitative trait loci (QTLs) on chromosome 2: drought tolerance 1 (DT1), DT2 and DT3 (Figure 1b, Table S1), laying the groundwork for uncovering key drought-tolerance genes in maize. To validate the candidate intervals and clarify their drought-tolerance functions, a BC6F3 population was developed by backcrossing F1 to drought-tolerant parent B7 (successive BC6) and subsequent selfing. Interval-specific markers maintained target QTL heterozygosity while fixing others, enabling near-isogenic line (NIL) construction. This key strategy isolates individual locus effects by minimising non-target genetic background interference. Drought assays revealed that NIL-DT1B7 exhibited significantly lower water loss and higher survival rates than NIL-DT1A4 (Figure 1c, Figure S2a,b), confirming the DT1 interval's key role in maize drought response. To pinpoint the DT1-mediated drought-tolerance gene, we fine-mapped 1995 BC6F3 and BC6F4 individuals, narrowing DT1 to a 150-kb interval containing five candidate genes (Figure 1d, Table S2). Expression analysis under drought revealed that only Zm00001d003377 was upregulated in NIL-DT1B7 (Figure 1e). This gene is designated ZmDT1, encoding a serine/threonine protein kinase, and is expressed in seeds, stems and roots (Figure S2c), suggesting potential roles in multiple tissues. Promoter analysis identified a single-base mutation within the ACGTG motif—a conserved core element of the ABA-responsive element (ABRE) (Figure 1f, Figure S3). Transcriptional activity assays demonstrated that ZmDT1B7 has higher promoter activity than ZmDT1A4 (Figure 1g, Figure S2d), indicating that this mutation may underlie the differential drought stress responses between B7 and A4. To further confirm ZmDT1's role in drought tolerance, we generated ZmDT1 knockout (ko, via gene editing) and overexpression (OE) lines in the B104 wild-type (WT) background (Figure S4a–c). Under well-watered conditions, WT, ko and OE lines showed no significant growth differences. When seedlings were subjected to drought stress by withholding water, ZmDT1ko lines wilted more severely, while ZmDT1-OE lines were more tolerant (Figure 1h,i). Following rehydration, OE lines had significantly higher survival rates and ko lines lower than WT (Figure 1h,i). Consistent with these phenotypes, OE lines displayed a lower water loss rate than WT, while ko lines exhibited a higher water loss rate (Figure S4d). Together, these results provide direct evidence that ZmDT1 positively regulates maize drought responses, reinforcing its key role in mediating drought tolerance. To further explore DT1's across-species role in drought responses, we analysed two Arabidopsis loss-of-function mutants (atdt1-1 and atdt1-2) (Figure S5a,b). Under drought stress, atdt1 mutants displayed greater sensitivity than wild-type Col-0, showing higher water loss rate and lower survival rates (Figure 1j,k). These findings confirmed that DT1 function is conserved between monocot maize and dicot Arabidopsis, underscoring its potential as a broad target for enhancing cross-species drought tolerance. To explore the link between DT1 and abscisic acid (ABA), a phytohormone well established to play a pivotal role in plant drought responses (Liu et al. 2022), we assessed the ABA responsiveness of the Arabidopsis mutants. Results showed that atdt1 exhibited a higher cotyledon greening rate under ABA treatment than Col-0 (Figure 1l), suggesting that DT1 may regulate plant drought tolerance via ABA signalling. To further explore how ZmDT1 mediates drought tolerance, we assessed ABA-induced stomatal movement in ZmDT1 ko and OE lines. All lines had fully open stomata under control conditions (Figure 1m,n). After ABA treatment, ko lines showed larger stomata apertures than WT, whereas OE lines exhibited more pronounced closure (Figure 1m,n). Furthermore, ZmDT1ko-1 had higher stomatal conductance and ZmDT1-OE1 had lower conductance (Figure 1o), with no differences in stomata density among all lines (Figure 1p). These findings clearly demonstrate that ZmDT1 regulates maize drought resistance via the ABA pathway, clarifying its role in modulating ABA-induced stomatal movement to improve drought tolerance. Given the ABRE motif difference in ZmDT1B7 and ZmDT1A4 promoters and their divergent drought responses, we explored the transcriptional regulators of ZmDT1. ZmABF2 was predominantly induced under drought conditions (Figure 1q), identifying it as a strong candidate regulator. Transient tobacco leaf assays showed ZmABF2 activates the luciferase (LUC) reporter driven by either promoter, with significantly stronger activation of the B7 haplotype than A4 (Figure 1r), consistent with their differential drought responsiveness. Consistently, electrophoretic mobility shift assay (EMSA) further confirmed that GST-ZmABF2 directly binds both ZmDT1 promoter alleles, with significantly stronger affinity for the B7 haplotype than A4 (Figure S5c). Based on these findings, we propose a working model for ZmDT1-mediated drought tolerance (Figure 1s): drought stress-induced ABA accumulation upregulates ZmABF2 expression, and ZmABF2 directly binds to the ZmDT1 promoter to enhance ZmDT1 transcription and promote its kinase activity, thereby facilitating phosphorylation of an unidentified substrate protein. ZmDT1 and its phosphorylated substrate act downstream of the ABA pathway to modulate stomatal movement, ultimately boosting drought tolerance in maize. In conclusion, our study identified DT1 as a conserved essential gene for drought tolerance in both monocot maize and dicot Arabidopsis, deepening understanding of plant drought adaptation and providing targets for molecular breeding to improve agricultural resilience under climate change. F.Y. and Q.X. designed the research. N.H., S.Z., X.S. Z.S. and C.L. performed the experiments. R.X., S.W., J.S., X.Q., G.B., F.Q. and X.Y. analysed data. F.Y., N.H. and S.Z. wrote the manuscript and revised the paper. All authors reviewed the manuscript. This work was supported by the National Natural Science Foundation of China grants (32222010, 32300283 and 32322008), Ningxia Hui Autonomous Region Key R&D Program (2024BBF02001), Beijing Nova Program (20240484588) and Pinduoduo-China Agricultural University Research Fund (PC2024B01004). The data that support the findings of this study are available on request from the corresponding author. The data are not publicly available due to privacy or ethical restrictions. Appendix S1: pbi70490-sup-0001-AppendixS1.doc. Figures S1–S5: pbi70490-sup-0002-FiguresS1-S5.pptx. Tables S1–S3: pbi70490-sup-0003-TablesS1-S3.doc. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.