ZmHB127 confers salt tolerance and yield stability in maize by transcriptionally activating ZmNQO1-mediated ROS detoxification

ZmHB127 confers salt tolerance and yield stability in maize by transcriptionally activating ZmNQO1-mediated ROS detoxification | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 23 February 2026 V1 Latest version Share on ZmHB127 confers salt tolerance and yield stability in maize by transcriptionally activating ZmNQO1-mediated ROS detoxification Authors : DongQing Zhang , Guangming zheng , Xiong Li , Fei Ban , Xiaofei He , Zhilong Liu , Lu Tian , … Show All … , Tongwen Shang , Ruichao Qiao , Zhaoyang Wang , Yajie Zhao , Xian Sheng Zhang , Chao Zhou , and Xiang-Yu Zhao 0000-0001-7259-8468 [email protected] Show Fewer Authors Info & Affiliations https://doi.org/10.22541/au.177182855.56071566/v1 114 views 62 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract With the continuous expansion of saline-alkaline land worldwide, salt stress has emerged as one of the most severe abiotic constraints on plant growth and development. However, maize (Zea mays L.), one of the most important staple crops globally, exhibits inherently low salt tolerance. In this study, we identified ZmHB127, which encodes a member of the maize HD-ZIP I transcription factor subfamily, as a key regulator of salt stress responses. Under salt stress conditions, overexpression of ZmHB127 significantly enhanced salt tolerance, whereas ZmHB127 knockout lines exhibited pronounced sensitivity. Analysis of reactive oxygen species (ROS) levels in leaves demonstrated that overexpression of ZmHB127 markedly enhanced the ROS-scavenging capacity of maize plants. By integrating DAP-seq and RNA-seq analyses, ZmNQO1 was identified as a putative downstream target of ZmHB127. Functional analyses further demonstrated that the activity of NAD(P)H:quinone oxidoreductase 1 was markedly enhanced, thereby promoting ROS detoxification in maize, in response to ZmHB127. Furthermore, the overexpression of ZmHB127 or ZmNQO1 significantly enhanced grain yield under salt stress conditions, highlighting their promise as candidate genes for salt-tolerant maize germplasm improvement. ZmHB127 confers salt tolerance and yield stability in maize by transcriptionally activating ZmNQO1 -mediated ROS detoxification DongQing Zhang 1 , Guangming zheng 1 , Xiong Li 1 , Fei Ban 1 , Xiaofei He 1 , Zhilong Liu 1 , Lu Tian 1 , Tongwen Shang 1 , Ruichao Qiao 1 , Zhaoyang Wang 1 , Yajie Zhao 1 , Xiansheng Zhang 1 , Chao Zhou 1, *, Xiangyu Zhao 1,2, * 1 State Key Laboratory of Crop Biology, College of Life Sciences, Shandong Agricultural University, Tai’an, Shandong, China 2 National Center of Technology Innovation for Comprehensive Utilization of Saline-Alkali Land Summary With the continuous expansion of saline-alkaline land worldwide, salt stress has emerged as one of the most severe abiotic constraints on plant growth and development. However, maize ( Zea mays L.), one of the most important staple crops globally, exhibits inherently low salt tolerance. In this study, we identified ZmHB127 , which encodes a member of the maize HD-ZIP I transcription factor subfamily, as a key regulator of salt stress responses. Under salt stress conditions, overexpression of ZmHB127 significantly enhanced salt tolerance, whereas ZmHB127 knockout lines exhibited pronounced sensitivity . Analysis of reactive oxygen species (ROS) levels in leaves demonstrated that overexpression of ZmHB127 markedly enhanced the ROS-scavenging capacity of maize plants. By integrating DAP-seq and RNA-seq analyses, ZmNQO1 was identified as a putative downstream target of ZmHB127 . Functional analyses further demonstrated that the activity of NAD(P)H:quinone oxidoreductase 1 was markedly enhanced , thereby promoting ROS detoxification in maize, in response to ZmHB127. Furthermore, the overexpression of ZmHB127 or ZmNQO1 significantly enhanced grain yield under salt stress conditions, highlighting their promise as candidate genes for salt-tolerant maize germplasm improvement. Keywords: maize, salt tolerance, ZmHB127 , ROS detoxification, grain yield Introduction Saline-alkaline stress is one of the most severe abiotic stresses limiting agricultural sustainability and crop productivity worldwide20142008(Cui et al., ; Munns and Tester, ). It is estimated that the global population will approach 10 billion by 2050, necessitating an increase of approximately 57% in global food production to meet future demand 20042006(Abrol, ; Rengasamy, ). However, the expansion of saline and alkaline soils, together with increasing climate variability, poses a major challenge to achieving this goal by severely constraining crop growth and yield2015(Munns and Gilliham, ). Maize ( Zea mays L.) is one of the most important cereal crops globally, serving not only as a primary food source for humans but also as a major component of animal feed and a key raw material for industrial applications2010(H. Charles J. Godfray, ). Nevertheless, maize is relatively sensitive to salinity, and its productivity is markedly reduced under salt stress conditions. With the increasing frequency and intensity of abiotic stresses driven by environmental degradation and climate change, improving stress tolerance in maize has become a critical objective for sustainable agricultural production2019(Bailey-Serres et al., ). Salt stress is tightly coupled with oxidative stress, primarily as a consequence of the excessive accumulation of reactive oxygen species (ROS) 2021(Sachdev et al.,). Under adverse environmental conditions, disruption of cellular metabolic homeostasis leads to impaired electron transport processes and enhanced electron leakage, resulting in the overproduction of ROS20202024(Zhao et al., ; Zhou et al., ). In animals, mitochondrial dysfunction reduces the efficiency of the electron transport chain, thereby promoting electron leakage and excessive ROS generation, which in turn causes oxidative damage to cellular membranes, proteins, and nucleic acids2019(Peoples et al., ). Such oxidative damage further exacerbates mitochondrial impairment, forming a detrimental feedback loop that compromises cellular viability2022(Wang et al., ). A comparable imbalance in ROS homeostasis also occurs in plants subjected to salt stress. High salinity perturbs photosynthetic and respiratory electron transport chains in chloroplasts and mitochondria, leading to excessive ROS accumulation2024(Selinski et al., ). If not efficiently scavenged, elevated ROS levels can disrupt membrane integrity, inactivate enzymes, and induce DNA damage, ultimately impairing plant growth and stress tolerance20132022(Baxter et al., ; Mittler et al., ). Therefore, maintaining ROS homeostasis through coordinated antioxidant systems is essential for plant adaptation to saline environments. NAD(P)H:quinone oxidoreductase 1 (NQO1) functions as a key component of cellular redox regulation by catalyzing the two-electron reduction of quinones, thereby preventing the formation of semiquinone radicals and subsequent reactive free radicals 20252010(Guo et al.,; Jung and Kwak, ). Through this mechanism, NQO1 contributes to the stabilization of intracellular redox balance, limits oxidative damage to macromolecules, and protects genomic integrity under environmental stress conditions. Consequently, NQO1-mediated redox regulation represents an important protective strategy against oxidative stress induced by salinity20242022(Liu et al., ; Preethi et al., ). Under natural and agricultural conditions, plants are frequently exposed to diverse biotic and abiotic stresses. In response to salt stress, transcription factors play central roles as molecular bridges linking upstream stress perception to downstream gene expression. By recognizing and binding specific cis-regulatory elements, transcription factors coordinate complex transcriptional networks that regulate plant stress adaptation. Among them, the homeodomain-leucine zipper (HD-ZIP) proteins represent a class of transcription factors unique to higher plants and belong to the homeobox gene family. HD-ZIP transcription factors have been extensively implicated in plant growth, development, and responses to environmental stresses 200720212013(Ariel et al., ; Perotti et al., ; Peterson et al., ). Structurally, HD-ZIP proteins contain a highly conserved homeodomain (HD) responsible for sequence-specific DNA binding, tightly linked to a leucine zipper (LZ) domain that mediates protein dimerization20091991(Elhiti and Stasolla, ; Ida Ruberti, ). Proper spatial arrangement between the HD and LZ domains is essential for DNA binding and transcriptional regulation by HD-ZIP proteins 20041993(Adriana E. Tron, ; Giovanna Sessa, ). In this study, we identified ZmHB127 , a member of the HD-ZIP I transcription factor family, as a positive regulator of salt tolerance in maize. We demonstrate that ZmHB127 directly regulates the expression of ZmNQO1 , leading to enhanced ROS-scavenging capacity and improved salt tolerance. Moreover, overexpression of ZmHB127 or ZmNQO1 significantly increased ear formation rate and grain yield under salt stress conditions. These findings expand the current understanding of the molecular mechanisms underlying salt stress tolerance in maize and provide promising candidate genes for the development of salt-tolerant maize germplasm. Salt stress modulates the expression of the HD-ZIP transcription factor ZmHB127 Previous studies have reported that the HD-ZIP transcription factor OsHOX22 ( Os04t0541700 ) in rice is induced by salt stress and negatively regulates salt tolerance2012(Zhang et al., ). Based on homology analysis, we identified ZmHB127 as the maize ortholog of OsHOX22 (Figure 1A). To gain insights into its structural features, a three-dimensional model of the ZmHB127 protein was predicted using AlphaFold2021(Jumper et al., ) . The model revealed a conserved homeodomain-leucine zipper (HD-ZIP) domain spanning amino acids 66-126. The hydrophobic residues within this domain form a compact and stable core, whereas the hydrophilic surface is likely involved in specific interactions with DNA, proteins, ligands, or signaling molecules (Figure 1B). To examine the transcriptional response of ZmHB127 to salt stress, maize seedlings were subjected to mixed salt treatment (150 mM NaCl:Na₂SO₄ = 9:1). Quantitative RT-PCR analysis showed that ZmHB127 expression exhibited a transient decrease followed by a pronounced increase within 0-6 h after salt treatment (Figure 1C), indicating that ZmHB127 is dynamically regulated in response to salt stress. To further investigate the cellular localization of ZmHB127, a ZmHB127-eGFP fusion construct and an empty vector control (pM999-eGFP) were transiently expressed in maize leaf protoplasts. Confocal microscopy revealed strong GFP fluorescence signals in both the nucleus and cytoplasm , suggesting that ZmHB127 localizes to these compartments (Figure 1D). Figure 1 Salt stress induces the expression of the maize HD-ZIP transcription factor ZmHB127 . (A) Phylogenetic analysis of ZmHB127 and its homologous HD-ZIP proteins from different plant species. (B) Predicted structural features of the ZmHB127 protein. Left, predicted secondary/tertiary structure model, with the conserved homeobox domain highlighted as purple helices. Right, predicted surface representation of ZmHB127, showing the distribution of polar atoms (red/blue) predominantly on the protein surface and hydrophobic carbon chains (light blue-gray) enriched in the interior. (C) Relative expression levels of ZmHB127 in maize seedlings during 0-6 h of salt stress treatment. (D) Subcellular localization of ZmHB127. Transient expression of 35S::eGFP (control) and 35S::ZmHB127-eGFP in maize leaf protoplasts. GFP fluorescence was observed using confocal microscopy. Scale bar = 5 μm. (E) Transcriptional activation assay of ZmHB127 in yeast. The recombinant construct pGBKT7–ZmHB127 and the empty vector control ( pGBKT7 ) were transformed into the Y2HGold yeast strain. Transcriptional activation activity was assessed based on yeast growth on SD/-Trp and SD/-Trp supplemented with 200 ng mL⁻¹ aureobasidin A (AbA). Yeast cultures were adjusted to an initial OD₆₀₀ of 0.5 and serially diluted (1, 1:5, 1:5², 1:5³, and 1:5⁴) prior to spotting. To determine whether ZmHB127 possesses transcriptional activation activity, the full-length ZmHB127 CDS was fused to the GAL4 DNA-binding domain in the pGBKT7 vector and transformed into the Y2HGold yeast strain. Both the empty vector control and pGBKT7-ZmHB127 transformants grew normally on SD/-Trp medium. However, only yeast cells harboring pGBKT7-ZmHB127 were able to grow on SD/-Trp medium supplemented with 200 ng mL⁻¹ aureobasidin A (AbA) and exhibited detectable β-galactosidase activity , whereas the empty vector control failed to grow under these conditions. These results demonstrate that ZmHB127 exhibits intrinsic transcriptional activation activity in yeast , consistent with its role as a functional transcription factor (Figure 1E). ZmHB127 positively regulates salt tolerance in maize To further elucidate the biological function of ZmHB127 , we generated ZmHB127 knockout and overexpression maize lines. Two independent CRISPR/Cas9-edited knockout lines with distinct mutation types were obtained, both of which were stably inherited and free of the Cas9 vector backbone. These lines were designated ZmHB127 KO-1 and ZmHB127 KO-2 , respectively. Compared with the wild type (WT), the ZmHB127 KO-1 line contained a single thymine (T) insertion near the protospacer-adjacent motif (PAM) site (TGG), resulting in a premature termination of protein translation at the 41st amino acid (Figure S1A). In contrast, the ZmHB127 KO-2 line harbored a 7-bp deletion, which also led to premature termination of ZmHB127 protein translation (Figure S1B). These mutations caused structural alterations and loss of function of the ZmHB127 protein in both knockout lines. For overexpression analysis, the coding sequence of ZmHB127 was cloned into an overexpression vector driven by the Ubiquitin promoter and introduced into maize immature embryos via Agrobacterium tumefaciens-mediated transformation. Two independent overexpression lines were obtained and designated ZmHB127 OE-1 and ZmHB127 OE-2 (Figure. S1C-D). To evaluate the role of ZmHB127 under salt stress, WT plants, knockout lines, and overexpression lines were grown under both normal and salt stress conditions. Under normal conditions, no obvious differences in growth were observed among WT and transgenic lines. However, under salt stress, ZmHB127 OE-1 and ZmHB127 OE-2 exhibited markedly improved growth performance compared with WT and knockout lines (Figure 2A). Plant height in the overexpression lines increased by 10.74% and 9.26%, respectively, relative to WT (Figure 2B), while aboveground biomass increased by 39.51% and 39.11%, respectively (Figure 2C). Taken together, these findings indicate that overexpression of ZmHB127 plays a positive role in enhancing maize tolerance to salt stress. Figure 2 ZmHB127 enhances salt tolerance in maize (A) Phenotypic comparison of salt tolerance among wild-type (WT), ZmHB127 knockout, and overexpression plants. WT and transgenic plants were grown under normal and salt stress conditions for 10 days. Similar results were observed in three independent experiments. Scale bar = 10 cm. (B–C) Quantitative analysis of plant height and aboveground biomass of WT and ZmHB127 knockout/overexpression plants under salt stress conditions. (D–H) Contents of hydrogen peroxide (H₂O₂), malondialdehyde (MDA), catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) in WT and ZmHB127 knockout/overexpression plants under normal and salt stress conditions. (I–J) DAB and NBT staining of leaf tips under normal and salt stress conditions. Leaf tips were observed using an Olympus microscope. Similar results were obtained from three independent experiments. Scale bar = 1 cm. Data are presented as means ± SD of biological replicates ( n = 15 ). Statistical significance was determined using a two-tailed t -test. Different asterisks indicate significant differences ( P < 0.05; * P < 0.01; ** P < 0.001). ZmHB127 alleviates ROS accumulation under salt stress Salt stress is known to induce the excessive production of reactive oxygen species (ROS) in plant cells 20202020(Anjum et al., ; Jia et al., ). To investigate whether ZmHB127 participates in ROS homeostasis under salt stress, we examined ROS accumulation in wild-type, ZmHB127 knockout, and overexpression lines. Under salt stress conditions, the contents of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) were significantly higher in the ZmHB127 knockout lines than in WT plants, whereas markedly lower levels were detected in the overexpression lines (Figure 2D-E). In addition, the activities of major ROS-scavenging enzymes, including catalase (CAT) , peroxidase (POD) , and superoxide dismutase (SOD) , were assessed (Figure 2F–H). Under salt stress, the activities of these antioxidant enzymes were significantly enhanced in the ZmHB127 overexpression lines, while reduced enzyme activities were observed in the knockout lines. These results suggest that ZmHB127 may regulate maize salt tolerance by promoting ROS scavenging . To further visualize ROS accumulation in leaves, 3,3′-diaminobenzidine (DAB) staining for H₂O₂ and nitroblue tetrazolium (NBT) staining for superoxide anion (O₂⁻) were performed in WT, ZmHB127 knockout, and overexpression plants under normal and salt stress conditions. Under salt stress, all genotypes exhibited increased accumulation of H₂O₂ and O₂⁻. However, compared with WT, ZmHB127 OE-1 and ZmHB127 OE-2 leaves showed markedly weaker DAB and NBT staining, whereas ZmHB127 KO-1 and ZmHB127 KO-2 leaves displayed stronger staining signals (Figure 2I–J), indicating higher ROS accumulation. Identification of downstream target genes of ZmHB127 by integrated RNA-seq and DAP-seq analyses To gain further insight into the molecular mechanisms by which ZmHB127 regulates salt tolerance in maize, transcriptome profiling was performed using roots of wild-type and ZmHB127 overexpression plants ( ZmHB127 OE-1 ) grown under normal and salt stress conditions. Differentially expressed genes (DEGs) were identified by comparing WT and ZmHB127 OE-1 under both conditions to screen for downstream salt stress–responsive genes regulated by ZmHB127 . Under salt stress, a total of 1,696 upregulated and 1,941 downregulated genes were identified in WT plants compared with normal conditions (Figure S2A)(Table S2). Similarly, 1,491 upregulated and 1,476 downregulated genes were detected in ZmHB127 OE-1 plants under salt stress (Figure S2B)(Table S3). Venn diagram analysis revealed that 1,329 DEGs were commonly involved in salt stress responses (Figure 3A)(Table S4). To further characterize the biological processes associated with ZmHB127 , Gene Ontology (GO) enrichment analysis was performed on these DEGs. Genes related to response to salt stress , autophagy , cellular response to nitrogen starvation , monooxygenase activity , oxidoreductase activity , and cell wall organization were significantly enriched (Figure 3B). These results indicate that ZmHB127 may regulate multiple biological processes involved in maize responses to salt stress . To dissect the downstream regulatory network of the ZmHB127 transcription factor, DNA affinity purification sequencing (DAP-seq) was conducted. In total, 29,724 ZmHB127 binding sites were identified across the maize genome. Genomic distribution analysis showed that the majority of binding sites were located in intergenic regions (59.20%) , followed by promoter regions (35.80%) , while a smaller proportion was found within introns (2.70%) and exons (0.80%) (Figure S3)(Table S5). The distribution of all binding sites within 2.5 kb upstream and downstream of transcription start sites (TSSs) is shown in Figure 3C. By mapping binding sites to gene features, 20,173 genes were identified as potential direct targets of ZmHB127 . Motif analysis of the ZmHB127-bound regions revealed CAATAAT as the most highly enriched core binding motif (Figure 3E). To identify key downstream genes regulated by ZmHB127 during salt stress, we integrated the DAP-seq identified putative target genes with salt stress-responsive DEGs from RNA-seq analysis , as well as genes highly expressed in roots . This integrative analysis identified two upregulated genes: Zm00001d039238 and Zm00001d047441 . Notably, Zm00001d047441 ( NADPH:quinone oxidoreductase 1 , ZmNQO1 ), was significantly upregulated in ZmHB127 OE-1 plants and displayed a prominent ZmHB127 binding peak in its non-coding region, indicating that ZmNQO1 is a likely direct downstream target of ZmHB127 (Figure 3F) Figure 3 Integrated RNA-seq and DAP-seq analyses reveal ZmHB127 downstream target genes. (A) Volcano plot of differentially expressed genes (DEGs) between WT and ZmHB127 OE-1 plants under salt stress conditions. DEGs were identified using the criteria of P < 0.05 and |fold change| (B) Gene Ontology (GO) enrichment analysis of DEGs between WT and ZmHB127 OE-1 plants under salt stress. (C) Distribution of ZmHB127 binding sites relative to transcription start sites (TSSs). (D) Venn diagram showing the overlap between ZmHB127 downstream putative target genes identified by DAP-seq and salt stress-responsive DEGs identified by RNA-seq. (E) The significantly enriched binding motif ( CAATAAT ) identified in ZmHB127-bound regions. (F) RNA-seq expression profile and DAP-seq binding peaks of ZmNQO1 . (G) Dual-luciferase (LUC) reporter assay showing the transcriptional activation of ZmNQO1 by ZmHB127 in Nicotiana benthamiana leaves. The empty vector was used as a negative control. (H) Electrophoretic mobility shift assay (EMSA) showing that GST-ZmHB127 binds to the non-coding region of ZmNQO1 containing the core motif CAATAAT . Binding was abolished when the core motif was mutated. (I) Yeast one-hybrid (Y1H) assay demonstrating the interaction between ZmHB127 and the ZmNQO1 . Yeast cells co-transformed with the prey vector ( pGADT7-HB127 ) and the bait vector ( pHIS2-NQO1 ) or control vectors ( pGADT7 and pHIS2-NQO1 ) were grown on SD/-His/-Leu/-Trp medium supplemented with 30 mM 3-amino-1,2,4-triazole (3-AT) . ZmHB127 directly binds to ZmNQO1 and activates its transcription A dual-luciferase (LUC) reporter assay was performed in Nicotiana benthamiana leaves to examine whether ZmHB127 activates ZmNQO1 transcription. A strong LUC signal was detected only when 35S::ZmHB127 was co-expressed with pNQO1-LUC , whereas no obvious signal was observed in the control combinations (Figure 3G). Consistently, the LUC/REN ratio was significantly higher in leaves co-expressing pNQO1-LUC and 35S::ZmHB127 compared with the control treatments (Figure S4A). To determine whether ZmHB127 directly binds to the ZmNQO1 regulatory region, an electrophoretic mobility shift assay (EMSA) was conducted. Incubation of biotin-labeled ZmNQO1 probes with GST-ZmHB127 protein resulted in a clear DNA-protein complex. The binding signal was progressively weakened by increasing concentrations of unlabeled competitor probes, whereas probes carrying mutations in the core motif failed to effectively compete for binding (Figure 3H). These results demonstrate that ZmHB127 specifically recognizes the CAATAATT motif within the non-coding region of ZmNQO1 . Furthermore, a yeast one-hybrid (Y1H) assay was performed to validate the interaction in vivo . Yeast cells co-transformed with pGADT7-HB127 and pHIS2-NQO1 were able to grow on SD/-His/-Leu/-Trp medium supplemented with 30 mM 3-amino-1,2,4-triazole (3-AT) even after serial dilutions (10¹-10⁴), whereas yeast cells carrying the empty prey vector ( pGADT7 + pHIS2-NQO1 ) exhibited markedly reduced growth (Figure 3I). Consistent with these findings, quantitative real-time PCR (qRT–PCR) analysis showed that the relative expression level of ZmNQO1 was significantly upregulated in ZmHB127 overexpression plants ( ZmHB127 OE-1 ) compared with WT plants (Figure S4B). Collectively, these results provide direct evidence that ZmHB127 binds to the non-coding region of ZmNQO1 and activates its transcription . ZmNQO1 positively regulates salt tolerance in maize To investigate the biological function of ZmNQO1 , maize immature embryos were genetically transformed via Agrobacterium tumefaciens –mediated transformation, resulting in two independent ZmNQO1 knockout lines. Compared with WT plants, the ZmNQO1 KO-1 line exhibited an 11-bp deletion at the first PAM site and a 2-bp deletion at the second PAM site, leading to amino acid substitutions from residues 59 to 100 and a premature termination of translation at amino acid 100. In the ZmNQO1 KO-2 mutant, a 2-bp deletion at the first PAM site and a 2-bp insertion at the second PAM site resulted in amino acid substitutions from residues 61 to 103 and premature termination at amino acid 103 (Figure S5A–B). These mutations are predicted to cause severe disruption of ZmNQO1 protein function. In parallel, ZmNQO1 overexpression lines were also generated (Figure S5C-D). Under normal growth conditions, no obvious differences in growth were observed between WT and ZmNQO1 transgenic plants. However, under salt stress conditions, WT plants exhibited reduced growth, whereas the overexpression lines showed markedly improved growth performance compared with both WT and knockout lines (Figure 4A). Specifically, plant height in the overexpression lines increased by 12.28% and 11.53% , respectively, relative to WT, whereas plant height in the knockout lines decreased by 18.73% and 18.83% compared with WT (Figure 4B). Moreover, under salt stress, aboveground biomass increased by 38.87% and 38.98% in the overexpression lines, while biomass in the knockout lines decreased by 46.94% and 42.73% , respectively, relative to WT (Figure 4C). Together, these results demonstrate that overexpression of ZmNQO1 significantly enhances salt tolerance in maize , whereas loss of ZmNQO1 function compromises plant performance under salt stress. Figure 4 ZmNQO1 positively regulates salt tolerance in maize (A) Phenotypes of WT, ZmNQO1 knockout, and overexpression plants grown under normal and salt stress conditions for 10 days. Similar results were observed in three independent experiments. Scale bar = 10 cm. (B-C) Statistical analysis of plant height and aboveground biomass of WT and ZmNQO1 knockout/overexpression plants under salt stress conditions. (D-E) Quinone reductase activity in WT and ZmHB127 and ZmNQO1 knockout/overexpression plants under normal and salt stress conditions. Menadione was used as the electron acceptor substrate, and WST-8 was used as the chromogenic reagent. (F) Relative expression levels of ZmNQO1 during 0-6 h of salt stress treatment. (G) In situ hybridization analysis of ZmNQO1 expression. The antisense probe was used to detect ZmNQO1 transcripts, while the sense probe served as a negative control. Scale bar = 100 μm. Data are presented as means ± SD. Statistical significance was determined using a two-tailed t -test. Different asterisks indicate significant differences ( P < 0.05; * P < 0.01; ** P < 0.001). ZmNQO1 alleviates ROS accumulation under salt stress Following salt stress treatment, the contents of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) were significantly increased in wild-type plants. In comparison, ZmNQO1 knockout lines exhibited even higher levels of H₂O₂ and MDA (Figure S6A-B). In contrast, ZmNQO1 overexpression lines showed significantly lower H₂O₂ and MDA contents than both WT and knockout plants, indicating that ROS accumulation was effectively suppressed in the overexpression lines. Correspondingly, under salt stress conditions, the activities of major antioxidant enzymes, including catalase (CAT) , peroxidase (POD) , and superoxide dismutase (SOD) , were markedly higher in the ZmNQO1 overexpression lines than in WT and knockout plants (Figure S6C-E). Consistent with these biochemical measurements, DAB staining for H₂O₂ and NBT staining for superoxide anion (O₂⁻) revealed that, under salt stress, leaves of ZmNQO1 overexpression plants accumulated substantially lower levels of ROS than those of WT and knockout plants (Figure S7A-B). Although ROS accumulation was observed in all genotypes under salt stress, knockout plants displayed more severe ROS accumulation, whereas overexpression plants exhibited reduced ROS levels compared with WT. These results suggest that ZmNQO1 enhances maize salt tolerance by promoting ROS scavenging under salt stress . In animals, NQO1 (NAD(P)H:quinone oxidoreductase 1) functions as a cytoprotective enzyme that catalyzes the two-electron reduction of quinones using NAD(P)H as an electron donor, generating non-toxic hydroquinones. This two-electron reduction mechanism effectively prevents the formation of reactive oxygen species, such as superoxide anions, during quinone redox cycling, thereby mitigating oxidative stress at its source. To examine whether a similar mechanism operates in maize, we performed assays to measure NQO1 enzymatic activity . In this assay, NQO1 catalyzes the reduction of menadione using NAD(P)H as the electron donor, converting oxidized menadione to its reduced form while generating NAD(P)⁺. Through electron coupling, WST-8 is subsequently reduced to a water-soluble orange formazan, enabling quantitative measurement of NQO1 activity. Our results showed that, under salt stress, NQO1 enzymatic activity was significantly increased in both ZmHB127 and ZmNQO1 overexpression lines , whereas no significant changes were detected in WT plants or in the knockout lines (Figure 4D-E). Moreover, the expression of ZmNQO1 exhibited a dynamic response to salt stress: transcript levels initially decreased during the first 0-2 h of treatment, followed by a gradual increase from 2 to 6 h (Figure 4F), a pattern resembling that observed for ZmHB127 . In situ hybridization analysis further revealed that ZmNQO1 transcripts were predominantly localized in the endodermal cells of the root (Figure 4G). Phenotypic analysis of mature ZmHB127 and ZmNQO1 transgenic plants Under normal growth conditions, no significant differences in plant height were observed among wild-type, ZmHB127 , and ZmNQO1 transgenic lines. All genotypes exhibited healthy growth, with intact leaf morphology, no visible wilting or damage, and comparable overall growth performance (Figure 5A). However, after continuous salt stress treatment for four weeks , plant height in WT plants was markedly reduced. This reduction was further exacerbated in the ZmHB127 and ZmNQO1 knockout lines , indicating enhanced sensitivity to salt stress. In contrast, ZmHB127 and ZmNQO1 overexpression lines maintained significantly greater plant height than both WT and knockout plants under salt stress (Figure 5B-C). A similar trend was observed for stem diameter at the third internode from the plant base , which closely mirrored the changes in plant height (Figure 5D). Leaf phenotypic analysis further supported these observations. Under salt stress, WT plants exhibited pronounced leaf tip curling and wilting. These symptoms were more severe in ZmNQO1 KO-1 plants, which displayed extensive leaf tip yellowing and increased leaf shrinkage. In contrast, ZmNQO1 OE-1 plants showed only mild leaf tip curling, with leaves remaining relatively expanded and exhibiting significantly less damage than those of WT and knockout plants (Figure 5E). Together, these results indicate that salt stress markedly inhibits maize growth and development, whereas overexpression of ZmHB127 or ZmNQO1 effectively alleviates salt-induced growth inhibition in mature plants . Figure 5 Assessment of salt tolerance in mature ZmHB127 and ZmNQO1 transgenic plants (A) Phenotypes of mature wild-type, ZmHB127 , and ZmNQO1 knockout and overexpression plants under normal growth conditions in the greenhouse. Control plants were grown with regular watering for 75 days. (B) Phenotypes of mature WT, ZmHB127 , and ZmNQO1 knockout and overexpression plants after salt stress treatment. Plants were grown under normal watering for 30 days, followed by weekly application of 2.5 L of mixed salt solution for four consecutive weeks. Scale bar = 10 cm. (C) Plant height of WT and transgenic lines at maturity under salt stress conditions. (D) Stem diameter at the third internode from the base of WT and transgenic plants at maturity under salt stress conditions. (E) Leaf tip wilting phenotypes of ear leaves from wild-type, ZmHB127 and ZmNQO1 knockout and overexpression plants. Scale bar = 10 cm. Data are presented as means ± SD. Statistical significance was determined using a two-tailed t -test. Different asterisks indicate significant differences ( P < 0.05; * P < 0.01; ** P < 0.001). Overexpression of ZmHB127 and ZmNQO1 increases grain yield under salt stress Salt stress severely reduces crop productivity. Under normal growth conditions, no significant differences were observed among wild-type, ZmHB127 , and ZmNQO1 knockout and overexpression lines with respect to ear traits, grain yield, or developmental timing. All genotypes exhibited comparable ear length, and grain yield per plant, as well as synchronized flowering time, silking time, and anthesis-silking interval (ASI), indicating normal reproductive development. Under salt stress conditions, both flowering and silking were significantly delayed in WT plants, accompanied by a marked extension of ASI, reflecting impaired synchrony between male and female inflorescence development. This phenotype was further exacerbated in ZmHB127 and ZmNQO1 knockout lines, which exhibited more pronounced delays in flowering and silking, substantially prolonged ASI, and severe disruption of reproductive coordination. In contrast, ZmHB127 and ZmNQO1 overexpression lines displayed flowering and silking times that were much closer to those observed under control conditions, with ASI maintained within a relatively narrow range, indicating improved synchrony of reproductive development under salt stress (Figure 6B). Consistent with these developmental responses, salt stress led to a significant reduction in ear length, seed-setting rate, and grain yield per plant in WT plants, accompanied by pronounced ear tip barrenness. These yield penalties were even more severe in ZmHB127 and ZmNQO1 knockout lines, which showed further reductions in sparse grain filling, near-complete loss of kernel set, and extremely low yield per plant. By contrast, the ZmHB127 OE-1 and ZmNQO1 OE-1 overexpression lines maintained significantly longer ears and higher seed-setting rates than WT and knockout lines, resulting in a markedly attenuated reduction in grain yield under salt stress (Figure 6A、C). Figure 6 Overexpression of ZmHB127 and ZmNQO1 enhances maize yield under salt stress. (A) Representative ear phenotypes of wild-type, ZmHB127 and ZmNQO1 knockout and overexpression plants under normal irrigation and salt stress conditions. Scale bar = 2 cm. (B) Anthesis–silking interval of WT, ZmHB127 and ZmNQO1 knockout and overexpression plants at the mature stage. (C) Grain yield per plant of WT, ZmHB127 and ZmNQO1 knockout and overexpression plants. (D) Proposed working model illustrating that ZmHB127 positively regulates ZmNQO1 transcription , thereby promoting ROS scavenging, alleviating oxidative damage, and enhancing maize salt tolerance. Loss of ZmHB127 function disrupts this regulatory pathway, leading to excessive ROS accumulation, increased salt sensitivity, and growth inhibition. Data are presented as mean ± SD. Statistical significance was determined by two-tailed t -tests ( P < 0.05; P < 0.01; P < 0.001). Overall, these results demonstrate that overexpression of ZmHB127 and ZmNQO1 effectively alleviates the inhibitory effects of salt stress on ear development, yield formation, and reproductive-stage coordination in maize, whereas loss of ZmHB127 or ZmNQO1 function increases plant sensitivity to salinity. Therefore, ZmHB127 and ZmNQO1 act as positive regulators of yield stability and reproductive development under salt stress conditions. Discussion Enhancing crop tolerance to abiotic stresses remains a central challenge in global agricultural production. Maize is inherently sensitive to salinity, and the progressive expansion of soil salinization worldwide poses an increasingly severe threat to maize yield stability and productivity2015(Farooq et al., ). Consequently, deciphering the molecular mechanisms governing maize responses to salt stress, improving crop performance under saline conditions, and breeding salt-tolerant maize cultivars are of paramount importance2021(Zhang et al., ). Over the past decades, genome-editing technologies have undergone rapid development 20202012(Broeders et al., ; Gasiunas et al., ). High-precision targeted genome editing has emerged as a powerful strategy for crop genetic improvement, enabling the acceleration of breeding programs aimed at enhancing abiotic stress tolerance and ensuring global food security 20202013(Ahmar et al., ; Gaj et al., ). In this study, we demonstrate that ZmHB127 plays an essential role in regulating maize responses to salt stress. ZmHB127 belongs to the maize HD-ZIP transcription factor family, a group of plant-specific regulators that are widely involved in plant development and abiotic stress responses 20072005(Ariel et al., ; Henriksson et al., ). Extensive studies have revealed that different HD-ZIP subfamilies exhibit distinct biological functions. Increasing evidence indicates that the expression of HD-ZIP I subfamily members is predominantly regulated by environmental stresses such as drought and salinity 2019202220162017(Gong et al., ; Qiu et al., ; Romani et al., ; Yang et al., ). For example, overexpression of ZmHB53 enhances drought tolerance in maize by interacting with PYL4 to form an ABA-dependent complex 2025(Zhong et al., ). Zmhdz9, a member of the HD-Zip transcription factor family, enhances drought stress tolerance in maize through the regulation of ABA biosynthesis and lignin accumulation 2024(Jiao et al.,).Mutation of ZmOCL1 , an HD-Zip IV transcription factor in maize, renders plants sensitive to drought stress 2026(Yang et al.,). In apple, the HD-ZIP transcription factor MdHB7-like positively regulates salt tolerance by directly activating MdATG18a and MdCCX1 , thereby promoting autophagy and Na⁺ efflux under salt stress 2023(Yang et al., ). The rice homolog of ZmHB127 , OsHOX22 ( Os04t0541700 ) , has been functionally characterized2012(Zhang et al., ). OsHOX22 expression is strongly induced by salt stress and polyethylene glycol treatment, and T-DNA insertion mutants display reduced ABA sensitivity but enhanced tolerance to drought and salt stress at the seedling stage. OsHOX22 functions through ABA-mediated signaling pathways to regulate ABA biosynthesis and stress responses. However, whether ZmHB127 is directly induced by ABA remains unclear and warrants further investigation. By integrating transcriptomic analysis and DNA affinity purification sequencing (DAP-seq), we identified three candidate downstream target genes of ZmHB127, which were significantly enriched in oxidoreductase-related functional categories. Among these, Zm00001eb394340 , encoding NAD(P)H:quinone oxidoreductase 1 (ZmNQO1) , was identified as a direct target of ZmHB127. We further demonstrated that ZmHB127 directly binds to the non-coding region of ZmNQO1 and activates its transcription. In plants, the biological functions of NQO1 have been scarcely reported. In contrast, animal NQO1 is well known as a quinone reductase involved in detoxification pathways, where it catalyzes the two-electron reduction of quinones to hydroquinones, thereby protecting cells from oxidative stress 2020(Ahmed Atia,). Quinones are aromatic organic compounds characterized by a six-carbon ring with two conjugated double bonds and diketone groups2024(Dong et al., ). In plants, quinone compounds have been implicated in parasitic plant development, such as haustorium formation in Striga , where quinone production is dependent on ROS signaling 2020(Laohavisit et al., ). Moreover, most abiotic stresses induce excessive ROS accumulation in plant cells, leading to oxidative damage and stress responses 2024(Wang et al., ). Our functional analyses revealed that ZmNQO1 enhances the detoxification of oxidized quinone compounds (e.g., menadione, vitamin K3) and reduces ROS accumulation2021(Ross and Siegel, ), thereby mitigating the detrimental effects of salt stress. Nevertheless, the precise relationship between quinone metabolism, ROS homeostasis, and salt tolerance in plants remains to be further elucidated. Upon salt stress, ZmHB127 expression is strongly induced in maize . When ZmHB127 is disrupted , it fails to transcriptionally activate its downstream target ZmNQO1 , resulting in impaired quinone detoxification and ineffective ROS scavenging. Consequently, excessive ROS accumulate beyond the buffering capacity of the antioxidant enzyme system, including SOD, CAT, and POD , leading to severe oxidative damage. Under these conditions, maize plants exhibit pronounced salt-sensitive phenotypes, characterized by reduced plant height, leaf chlorosis and wilting, and markedly inhibited growth. In contrast, overexpression of ZmHB127 robustly activates ZmNQO1 transcription , ensuring efficient functioning of this regulatory pathway. Elevated ZmNQO1 levels markedly enhance ROS detoxification, maintaining cellular ROS homeostasis at non-toxic levels. In coordination with the enzymatic antioxidant machinery (SOD/CAT/POD), oxidative damage is effectively suppressed, thereby conferring substantially improved salt tolerance in maize. Collectively, these findings support a model in which ZmHB127 functions as a molecular “salt tolerance switch” , activating the ZmNQO1-mediated ROS detoxification pathway and enabling maize plants to sustain growth and development under saline conditions (Figure 6D). Previous studies have shown that enhanced antioxidant capacity often correlates with improved stress tolerance and yield performance. For instance, overexpression of OsSDG721 in rice confers salt-alkaline tolerance by reducing ROS levels, increasing survival rates, and accelerating heading and maturity 2021(Liu et al.,). In wheat, TaASRI-D overexpression enhances antioxidant capacity and ABA sensitivity, leading to improved salt tolerance and increased yield2021(Qiu et al., ). In maize, knockout of ZmDapF1 results in yield advantages under normal conditions and mitigates yield loss under drought stress 2025(Lian et al., ). Natural variation in ZmSRO1d-R enhances drought tolerance and increases yield under drought stress but causes yield penalties under normal conditions, highlighting the challenge of balancing stress tolerance and productivity 2022(Gao et al.,). Notably, allelic effects on stress tolerance and yield are highly dependent on genetic background, as exemplified by zmiceb lines showing differential yield performance across maize backgrounds 2025(Zhou et al., ). Compared with drought stress, the relationship between salt tolerance and yield in maize remains poorly understood. Although ZmASR6 has been reported to positively regulate salt tolerance, wild-type plants exhibit higher kernel weight and grain number than ZmASR6 knockout lines under normal conditions, suggesting potential trade-offs 2025(Li et al.,). In this study, we provide compelling genetic and molecular evidence that ZmHB127-ZmNQO1 constitutes a previously uncharacterized regulatory module governing maize salt tolerance. We demonstrate that ZmHB127 directly binds to the noncoding region of ZmNQO1 and transcriptionally activates its expression , thereby enhancing quinone detoxification capacity, promoting ROS homeostasis, and ultimately conferring improved salt stress tolerance. Notably, overexpression of ZmHB127 or ZmNQO1 significantly enhances stress resilience without incurring a yield penalty under saline conditions , highlighting a favorable balance between stress tolerance and productivity. Compared with previously reported salt-tolerance regulators in maize, which often improve stress resistance at the expense of growth or yield, the ZmHB127-ZmNQO1 module represents a distinct regulatory mechanism that integrates oxidative stress mitigation with agronomic trait stability . Given the limited number of functionally validated genes and molecular markers currently available for molecular breeding of salt-tolerant maize, ZmHB127 and ZmNQO1 emerge as promising genetic targets . Their deployment in elite inbred lines, either through transgenic approaches or precision genome editing, holds substantial potential for accelerating the development of high-yielding, salt-tolerant maize cultivars, thereby providing valuable genetic resources for sustainable maize production in saline and salt-affected soils. Conclusion In summary, ZmHB127 functions as a key HD-ZIP transcription factor that positively regulates maize salt tolerance by directly activating ZmNQO1 expression. We demonstrate that the ZmHB127-ZmNQO1 module plays a central role in maintaining redox homeostasis under salt stress by promoting efficient ROS detoxification in coordination with the antioxidant enzyme system. Genetic disruption of either ZmHB127 or ZmNQO1 leads to excessive ROS accumulation, severe oxidative damage, and pronounced salt sensitivity, whereas overexpression of these genes markedly enhances stress tolerance. Importantly, enhanced salt tolerance is achieved without compromising yield performance and is accompanied by improved agronomic traits at the reproductive stage under saline conditions. These findings uncover a previously unrecognized HD-ZIP-mediated redox regulatory pathway in maize and highlight ZmHB127 and ZmNQO1 as promising molecular targets for breeding high-yield, salt-tolerant maize cultivars suitable for saline soils. Methods Plant materials and treatment conditions Maize inbred line B104 was used as the recipient material. CRISPR/Cas9 knockout vectors for ZmHB127 and ZmNQO1 genes were constructed using pBUE411-2gR as the vector backbone. Agrobacterium-mediated genetic transformation of maize immature embryos was performed to obtain gene-edited knockout lines of ZmHB127 and ZmNQO1 . The CDS sequences of ZmHB127 and ZmNQO1 were respectively cloned into overexpression vectors driven by the Ubiquitin promoter. These vectors were introduced into immature embryos of maize inbred line B104 via Agrobacterium-mediated transformation to generate overexpression lines for both genes. Maize seeds with plump kernels and uniform size were germinated and evenly planted in plastic pots containing equal weights of mixed substrate. The seedlings were cultured in an artificial climate chamber for 10 days under controlled conditions: long-day photoperiod (16 h light/8 h dark), temperature of 27.5℃, and relative humidity of 55%. For salt stress simulation, the treatment group was irrigated with 150 mM mixed salt solution (NaCl:Na₂SO₄ = 9:1, m/m) until the soil was saturated, while the control group was watered with an equal volume of distilled water. All experiments were conducted with three independent biological replicates. qRT-PCR analysis Ten-day-old B104 seedlings were treated with mixed salt solution. Roots were collected at 0, 1, 2, 3, 4, 5, and 6 h after treatment. Total RNA was extracted using a RNA extraction kit (Cwbio), and cDNA was synthesized by reverse transcription followed by quantitative real‑time PCR (qRT‑qPCR) analysis. Subcellular localization The ZmHB127-eGFP fusion protein expression vector and empty pM999-eGFP vector were transformed into maize leaf protoplasts for transient expression. The subcellular localization of eGFP fluorescence signals was observed under a laser confocal microscope(Zeiss LSM880). Yeast transcriptional activation assay The CDS sequence of ZmHB127 was ligated into the pGBKT7 yeast expression vector to construct pGBKT7-ZmHB127 . The empty pGBKT7 vector and pGBKT7-ZmHB127 were separately transformed into Y2HGold yeast strain, and the transformed cells were spread on SD/-Trp deficient medium. After single colonies emerged, individual colonies were picked and streaked onto SD/-Trp deficient medium and SD/-Trp medium supplemented with 200 ng/mL Aureobasidin A (AbA). The plates were incubated upside down at 30 °C for 3-5 days, and yeast growth was observed. Determination of H₂O₂, MDA contents and CAT, POD, SOD activities Leaves were collected from 10-day-old plants after 12 h of salt treatment, and ground into powder in liquid nitrogen. The contents of hydrogen peroxide (H₂O₂) and malondialdehyde (MDA), as well as the activities of catalase (CAT), peroxidase (POD), and superoxide dismutase (SOD) in leaves, were measured using corresponding commercial kits (Suzhou Grace Biotechnology Co.,Ltd.) following the manufacturer’s instructions. NQO1 enzyme activity assay Leaves were collected from 10-day-old plants after 12 h of salt treatment, and ground into powder in liquid nitrogen. Using menadione as the substrate, the reaction system contained NAD(P)H, leaf cell lysate (with NQO1 enzyme), reaction buffer, and WST-8. After adding the substrate to initiate the reaction, the absorbance (OD value) at 450 nm was monitored continuously.Enzyme activity was defined as follows: one unit (U) represents the amount of enzyme that increases the absorbance at 450 nm by 1 unit per minute per gram of fresh leaf tissue. ROS staining DAB staining (for H₂O₂ detection):Leaves of 10-day-old plants were collected after 6 h of salt treatment, and immersed in 1 mg/mL DAB staining solution (prepared with 10 mM sodium phosphate buffer, pH 6.5). The leaves were incubated at room temperature in the dark for 8-12 h. Subsequently, 95% ethanol was used for boiling decolorization (to remove chlorophyll) until the leaves turned pale yellow. Brown precipitates (H₂O₂-DAB complexes) were observed under an optical microscope. NBT staining (for Superoxide anion detection):Leaves of 10-day-old plants were collected after 6 h of salt treatment, and immersed in 0.5 mg/mL NBT staining solution (prepared with 50 mM sodium phosphate buffer, pH 7.5). The leaves were incubated at room temperature in the dark for 8-12 h. Decolorization and observation procedures were the same as those for DAB staining. Blue formazan precipitates (superoxide anion-NBT complexes) were observed under an optical microscope. RNA-Seq analysis Roots of 10-day-old wild-type (WT) and ZmHB127 KO-1 lines were collected after 1 h of mixed salt solution treatment, rinsed with clean water, snap-frozen in liquid nitrogen, and stored. Each sample was a pool of roots from 3 independent plants (biological replicate), with 3 biological replicates set for all groups. Total RNA was extracted from roots using the TRIzol method, and library construction and transcriptome sequencing were performed by LC-Bio Technologies (Hangzhou) Co., Ltd.For sequencing data processing: clean reads were mapped to the maize reference genome using BWA and Hisat2 software; gene quantification was conducted via HTSseq and FeatureCounts software. After TMM normalization of the matrix data, low-abundance genes were filtered out, and differentially expressed genes (DEGs) between the treatment and control groups were identified using edgeR and DESeq2 software (filter criteria: |fold change| ≥ 1.5 and adjusted P < 0.05). DNA affinity purification sequencing (DAP-seq) and data analysis The full-length CDS of the target gene was recombined into a vector with an affinity tag to construct a protein expression vector, followed by in vitro protein expression to generate a fusion protein of the transcription factor and affinity tag.Genomic DNA (gDNA) was extracted from samples to construct a gDNA library. The in vitro-expressed affinity-tagged transcription factor was incubated with the gDNA library; DNA fragments bound to the transcription factor were eluted and subjected to high-throughput sequencing. Luciferase (LUC) reporter assay The sequence of ZmNQO1 was cloned into the pGreenII 0800-pLUC vector to generate pNQO1-LUC (reporter vector). The CDS of ZmHB127 was cloned into the pGreenII 62-SK vector to construct 35S:ZmHB127 (effector vector). Agrobacteria harboring pNQO1-LUC and 35S:ZmHB127 were mixed at an equal ratio and infiltrated into Nicotiana benthamiana leaves. After 1–2 days of incubation in the dark, leaf samples were treated with a dual-luciferase assay kit. Fluorescence activities of LUC (firefly luciferase) and REN (Renilla luciferase, internal control) were quantified using a chemiluminometer. The LUC/REN ratio was used to evaluate the activation activity . Yeast one-hybrid (Y1H) Assay The CDS sequence of ZmHB127 was cloned into the pGADT7 vector, and a 300-bp non-coding region of ZmNQO1 containing the CAATAATT motif was cloned into the p53-His2 vector. The two recombinant vectors were co-transformed into Y187 competent cells. For the control group, the empty pGADT7 vector and pHis2-NQO1 vector were co-transformed. All transformed cells were plated on SD/-His/-Leu/-Trp deficient medium supplemented with 30 mM 3AT and cultured for 3 days. Electrophoretic mobility shift assay (EMSA) The CDS sequence of ZmHB127 was cloned into a GST-tagged vector and transformed into BL21 competent cells; GST-fused ZmHB127 protein was then purified. Biotin-labeled probe (30 bp, containing the CAATAATT motif of ZmNQO1 ), unlabeled competitor probe, and mutant probe were synthesized. Following the manufacturer’s instructions for the Beyotime EMSA Chemiluminescent Kit, GST-ZmHB127 protein was incubated with probes, and non-denaturing polyacrylamide gel electrophoresis (PAGE) was performed in 0.5×TBE buffer at 100 V for 1 h. After membrane transfer, signals were detected via chemiluminescence using a Bio-Rad gel imaging system. In situ hybridization analysis Seeds of maize inbred line B104 were germinated for 5-6 days. Root segments (1-1.5 cm long, including root cap, meristematic, elongation, and maturation zones) were excised under a stereomicroscope, immediately immersed in ice-cold FAA fixative, vacuumed at 0.08 MPa for 15 min, and fixed at 4℃ for 16 h. After gradient ethanol dehydration, samples were embedded in paraffin. Paraffin sections (10µm thick, cross and longitudinal) were mounted on lysine-coated slides and dried in a 42℃ oven for 2 days. A 300-bp specific cDNA fragment of ZmNQO1 was used as the template. Primers with a T7 promoter sequence at the 5’ end were used to amplify sense and antisense probe templates. Digoxigenin-labeled RNA probes were synthesized via in vitro transcription with T7 RNA polymerase. After template digestion with RNase-free DNaseⅠ, probes were precipitated, resuspended in 50µL DEPC-H 2 O, and stored at -80℃ until use. Sections were subjected to dewaxing, rehydration, proteinase K digestion, PBS washing, FAA fixation, and ethanol dehydration. Hybridization buffer containing probes was added, and sections were incubated overnight at 55℃ in a humid chamber. Unbound probes were eluted with 2×SSC; sections were blocked with blocking solution (Blocking reagent + TBS-T) for 1 h, then incubated with anti-digoxigenin antibody solution at room temperature for 1.5 h. After TBS-T washing, NBT-BCIP chromogenic solution (1:50 diluted in TNM-50 buffer) was added, and sections were incubated overnight at room temperature in the dark. Once signals were clear, sections were observed and imaged using an Olympus BX51 microscope. Cultivation of mature maize plants All materials were grown in the greenhouse at the Science and Technology Innovation Park (South Campus, Shandong Agricultural University; 36°15′N, 117°16′E), with controlled conditions: 20-26°C, 16 h light/8 h dark.Plump, uniform-sized maize seeds were sown in pots (40 cm diameter × 48 cm height) filled with nutrient soil:vermiculite (1:1, v/v) plus sufficient base fertilizer (3 seeds/pot, ~5 cm depth). After 15 days of growth, one uniformly vigorous plant per pot was retained.Control plants received normal watering. For the treatment group, 2.5 L mixed salt solution (as above) was irrigated weekly from the 30-day-old stage for 4 weeks, followed by resuming normal watering. Agronomic traits were measured at maturity. Statistical methods All experimental data are presented as mean ± standard deviation (SD) from at least three independent biological replicates. Statistical significance between two groups was analyzed using two-tailed Student’s t-test (conducted via GraphPad Prism 9.0 software). Significance levels are indicated as follows: P < 0.05; * P < 0.01; ** P < 0.001. Acknowledgements This work was supported by the National Key Research and Development Program of China (2023YFD1200500), the National Natural Science Foundation of China (U25A6026), Key R&D Program of Shandong Province, China (2023LZGC001). Author contributions X.Y.Z. and C.Z. designed the study and conceived the project. D.Q.Z., Y.J.Z., and X.S.Z. provided constructive suggestions. D.Q.Z., G.M.Z., X.L., F.B., X.F.H., Z.L.L., Lu Tian., T.W.S., R.C.Q., and Z.Y.W. performed the experiments. G.M.Z., X.L., and F.B. analysed data. X.Y.Z and C.Z. drafted the manuscript. 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Effects of ZmNQO1 knockout and overexpression on ROS accumulation and antioxidant enzyme activities. Figure S7. ROS accumulation in leaves of ZmNQO1 knockout and overexpression lines. Table S1. List of the primers used in this study. Table S2. DEGs of Wild-type (Salt stress_VS_Control). Table S3. DEGs of ZmHB127 OE (Salt stress_VS_Control). Table S4. DEGs of ZmHB127 OE (Salt stress) VS Wild-type (Salt stress). Table S5. DAP-seq data for ZmHB127. Information & Authors Information Version history V1 Version 1 23 February 2026 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords grain yield growth maize ros detoxification salt tolerance transcriptome zmhb127 Authors Affiliations DongQing Zhang Shandong Agriculture University College of Life Sciences View all articles by this author Guangming zheng Shandong Agriculture University College of Life Sciences View all articles by this author Xiong Li Shandong Agriculture University College of Life Sciences View all articles by this author Fei Ban Shandong Agriculture University College of Life Sciences View all articles by this author Xiaofei He Shandong Agriculture University College of Life Sciences View all articles by this author Zhilong Liu Shandong Agriculture University College of Life Sciences View all articles by this author Lu Tian Shandong Agriculture University College of Life Sciences View all articles by this author Tongwen Shang Shandong Agriculture University College of Life Sciences View all articles by this author Ruichao Qiao Shandong Agriculture University College of Life Sciences View all articles by this author Zhaoyang Wang Shandong Agriculture University College of Life Sciences View all articles by this author Yajie Zhao Shandong Agriculture University College of Life Sciences View all articles by this author Xian Sheng Zhang Shandong Agriculture University College of Life Sciences View all articles by this author Chao Zhou Shandong Agriculture University College of Life Sciences View all articles by this author Xiang-Yu Zhao 0000-0001-7259-8468 [email protected] Shandong Agriculture University College of Life Sciences View all articles by this author Metrics & Citations Metrics Article Usage 114 views 62 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation DongQing Zhang, Guangming zheng, Xiong Li, et al. ZmHB127 confers salt tolerance and yield stability in maize by transcriptionally activating ZmNQO1-mediated ROS detoxification. Authorea . 23 February 2026. DOI: https://doi.org/10.22541/au.177182855.56071566/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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