The ZmbHLH47-ZmSnRK2.9 Module Promotes Drought Tolerance in Maize

Drought stress globally poses a significant threat to maize (Zea mays L.) productivity and the underlying molecular mechanisms of drought tolerance remain elusive. In this study, we characterized ZmbHLH47, a basic helix–loop–helix (bHLH) transcription factor, as a positive regulator of drought tolerance in maize. ZmbHLH47 expression was notably induced by both drought stress and abscisic acid (ABA). Transgenic plants overexpressing ZmbHLH47 displayed elevated drought tolerance and ABA responsiveness, while the zmbhlh47 mutant exhibited increased drought sensitivity and reduced ABA sensitivity. Mechanistically, it was revealed that ZmbHLH47 could directly bind to the promoter of ZmSnRK2.9 gene, a member of the subgroup III SnRK2 kinases, activating its expression. Furthermore, ZmSnRK2.9-overexpressing plants exhibited enhanced ABA sensitivity and drought tolerance, whereas the zmsnrk2.9 mutant displayed a decreased sensitivity to both. Notably, overexpressing ZmbHLH47 in the zmsnrk2.9 mutant closely resembled the zmsnrk2.9 mutant, indicating the importance of the ZmbHLH47-ZmSnRK2.9 module in ABA response and drought tolerance. These findings provided valuable insights and a potential genetic resource for enhancing the environmental adaptability of maize.


Introduction
Drought stress stands as a prominent environmental factor hindering agricultural progress and global crop productivity [1][2][3][4][5][6].Insufficient water availability limits plant growth and development, leading to reduced plant production and the substantial impairment of crop performance [7].In response to drought stress, plants undergo morphological and molecular alterations, manifesting as noticeable changes in photosynthesis, stomatal aperture, hormone level, leaf growth, stem extension, root proliferation, and hydraulic conductivity [7][8][9][10][11].Maize, a crucial food and industrial crop globally [12,13], faces an escalating threat in its cultivation due to climate changes and unpredictable rainfall patterns, contributing to the increased occurrence of severe drought stress [14].Furthermore, it is noteworthy that due to its shallow roots, maize exhibits insensitivity to drought and intolerance to soils with water deficiency [15].Hence, revealing the molecular mechanisms involved in conferring resistance to drought in maize holds significant promise for advancing the breeding and cultivation of drought-tolerant varieties.
The basic helix-loop-helix (bHLH) transcription factors (TFs) constitute the secondlargest gene family of TFs in plants [16].In this family, members exhibit a remarkably conserved bHLH domain, playing a pivotal role in DNA binding and facilitating proteinprotein interactions [17,18].In the maize genome, a total of 208 bHLH genes have been identified [19].However, only a limited number of bHLH TFs have been recognized for their involvement in conferring drought tolerance in maize.For instance, Gao et al. demonstrated that overexpressing ZmPIF3 in rice (Oryza sativa) enhanced tolerance to drought and salt stresses [20].Additionally, ZmPTF1, known for its roles in root development and lowphosphate stress tolerance in maize, was found to improve drought tolerance by promoting ABA synthesis [21].Moreover, ZmbHLH124 emerged as a positive regulator of drought tolerance in maize, partly due to the direct activation of ZmDREB2A, mediating plant responses to water deficit [22].Moreover, when overexpressed in rice (Oryza sativa), ZmPIF1 has demonstrated drought resistance, possibly linked to reduced stomatal aperture and transpiration rate [23].However, a more comprehensive characterization is needed to fully understand the functions and regulatory roles of bHLH TFs associated with maize drought tolerance.
In this contemporary investigation, it was attempted to delineate the affirmative contributions of the bHLH transcription factor ZmbHLH47 and subgroup III SnRK2 protein kinase ZmSnRK2.9 in the ABA response and drought tolerance in maize.Moreover, ZmbHLH47 has been recognized as a factor that could directly bind to the ZmSnRK2.9 promoter, leading to the activation of its expression and finely tuning the levels of ABAresponsive genes, particularly under a drought stress condition.The research contributes to a comprehensive comprehension of the mechanisms that would underlie drought stress tolerance in maize and provides a valuable genetic asset for the advancement of droughtresistant maize varieties.

Identifying and Analyzing the Sequence of ZmbHLH47
In the pursuit of identifying novel abiotic stress-tolerant genes, we conducted an analysis of publicly available expression data from a previously published study [43].
The investigation hinted at the potential candidacy of ZmbHLH47 (GRMZM2G133675) as a novel abiotic stress-responsive gene.The ZmbHLH47 gene (GRMZM2G133675) encompasses a complete open reading frame (ORF) spanning 825 base pairs, encoding a polypeptide of 274 amino acid residues.This polypeptide has a molecular mass of 29.36 kilodalton (kDa) and an isoelectric point (pI) of 5.65.A phylogenetic tree, constructed based on ZmbHLH47 and its homologs in maize, rice, and Arabidopsis, revealed a close relationship among ZmbHLH47, OsbHLH1, OsbHLH63, and AtPOPEYE (Figure S1A).A precise analysis involved aligning the amino acid sequences of ZmbHLH47 with those of its homologous proteins, as illustrated in Figure S1B.The results underscored that the ZmbHLH47 protein is a canonical bHLH transcription factor, which is characterized by a conserved bHLH domain known for its role in DNA binding and the formation of protein complexes [44][45][46].

ZmbHLH47 Functions as a Transcription Factor
To assess the transcription factor (TF) activity of ZmbHLH47 and pinpoint the region responsible for this activity, distinct segments of the coding sequence were autonomously linked to the GAL4 DNA-binding domain in the pGBKT7 vector.These segments encompassed the entire coding sequence (1-274 aa), the N-terminal and middle domain sequence (1-127 aa), the N-terminal sequence (1-67 aa), the middle domain and C-terminal sequence (68-274 aa), and the C-terminal sequence (128-274 aa).Yeast cells transformed with ZmbHLH47 (1-274 aa), ZmbHLH47 (68-274 aa), and ZmbHLH47 (128-274 aa) displayed a robust growth on SD/-Trp/-His/-Ade medium (Figure S2A), unequivocally indicating the transcriptional activation activity of ZmbHLH47.Notably, the C-terminal region (128-274 aa) was identified as a contributor to the transactivation activity of ZmbHLH47.
Furthermore, to delineate the subcellular localization of ZmbHLH47, we carried out transient co-expression assays by introducing a construct encoding GFP-tagged ZmbHLH47 and a nuclear marker (D53-mCherry) into maize protoplasts.Notably, intense green fluorescence emitted by the ZmbHLH47-GFP protein was distinctly confined to the nucleus, displaying accurate co-localization with the established nuclear marker D53-mCherry protein [47].In contrast, the GFP protein signal was discerned throughout the plasma membrane, cytoplasm, and nucleus, presenting a stark disparity, highlighting distinct cellular compartments (Figure S2B,C).These findings consistently supported the role of ZmbHLH47 as a transcription factor, as its protein predominantly localizes in the nucleus.

Expression Analysis of ZmbHLH47
To ascertain the tissue-specific transcript abundance of ZmbHLH47 in maize, we undertook quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis.The outcomes revealed a noticeable pattern of expression for ZmbHLH47, with the apex in leaves, succeeded by descending levels in roots, embryos, endosperms, pollens, and stems.Conversely, relatively diminished expression levels were noted in ears and silks (Figure S3A).Furthermore, it was attempted to carry out qRT-PCR to explore the relative expression level of ZmbHLH47 under abiotic stress and exogenous ABA.Intriguingly, treatments with NaCl, PEG, and exogenous ABA swiftly and significantly induced the expression level of ZmbHLH47 (Figure S3B-D).These outcomes strongly suggested that ZmbHLH47 could potentially play a notable function in mediating the maize response to diverse abiotic stresses.

ZmbHLH47 Functions as a Positive Regulator of Drought Tolerance in Maize
To elucidate the functional role of ZmbHLH47 in maize, it was attempted to engineer both the overexpression lines and loss-of-function mutants.Post-examining the transcript level and protein abundance, we judiciously selected two independent overexpression lines (ZmbHLH47-OE#3 and ZmbHLH47-OE#11) for detailed investigation (Figure 1A,B).Employing the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated 9 (CRISPR/Cas9) genome editing system [48,49], an attempt was made to establish two loss-of-function mutants of ZmbHLH47, designated as zmbhlh47-c1 and zmbhlh47-c2.zmbhlh47-c1 harbored a 1-bp insertion (42-bp downstream of ATG), while zmbhlh47-c2 carried a 5-bp deletion (from 44-to 50-bp downstream of ATG), inducing frameshifts in the ORF and the premature termination of translation (Figure 1C,D).The protein abundance of ZmbHLH47 in ZmbHLH47-OE transgenic seedlings (#3, #11).Anti-Flag antibody was utilized to detect ZmbHLH47.Actin served as a control.(C) Schematic diagrams of zmbhlh47-crispr mutants generated using CRISPR/Cas9-mediated genome editing.(D) The zmbhlh47-c1 and zmbhlh47-c2 mutations were identified using Sanger sequencing compared with WT. (E) Drought tolerance phenotypes of WT, ZmbHLH47-OE, and zmbhlh47-crispr plants.Twelve−day−old seedlings were subjected to drought stress by withholding water for 8 days, after which they were re-watered for 3 d (Re−watering).Scale bars = 5 cm.(F) Statistical analysis of survival rates after drought stress as shown in (E).At least 30 seedlings of each line per replicate were used for survival rate analysis.Data shown are means ± SD of three biological replicates.ns indicates no significant difference to the corresponding controls.**** indicates significant difference to the corresponding controls with p < 0.0001 (Student's t−test).(G) Relative water content (RWC) in WT, ZmbHLH47-OE, and zmbhlh47-crispr plants under well−watered and drought conditions.Data shown are means ± SD of three biological replicates.ns indicates no significant difference to the corresponding controls.Significant differences are indicated using Student's t−test: *** p < 0.001; **** p < 0.0001.(H,I) Malondialdehyde (MDA) content (H) and the percentage leakage of electrolyte (I) of WT, ZmbHLH47-OE, and zmbhlh47-crispr plants under well-watered and drought conditions.DW, dry weight.The values are presented as means ± SD of three biological replicates.ns indicates no significant difference to the corresponding controls.** p < 0.01; *** p < 0.001; **** p < 0.0001, indicating significant differences to the corresponding controls (Student's t−test).
The ZmbHLH47-OE lines, zmbhlh47-crispr mutants, and wild−type (WT) plants were cultivated until they reached a 12−day−old stage in soil.Subsequently, they underwent 8 days of drought stress without watering, followed by 3 days of rewatering.Notably, no discernible differences in growth phenotypes were discerned among WT, ZmbHLH47-OE, and zmbhlh47-crispr plants under well-watered conditions (Figure 1E).In contrast, under conditions of drought stress or rehydration, ZmbHLH47-OE plants exhibited less severe wilting, whereas zmbhlh47-crispr plants incurred more damage compared with WT plants (Figure 1E).In concurrence with these observations, after drought stress, ZmbHLH47-OE plants displayed a significantly higher survival rate, and their leaf relative water content was significantly greater, while zmbhlh47-crispr plants exhibited significantly lower values relative to WT plants (Figure 1F,G).
In addition, as malondialdehyde (MDA) content and electrolyte leakage serve as indicative markers of drought tolerance [50,51], it was attempted to calculate these parameters for WT, ZmbHLH47-OE, and zmbhlh47-crispr plants.Prior to drought stress, MDA content and electrolyte leakage exhibited similar levels among WT, OE, and Crispr lines (Figure 1H,I).However, subsequent to drought stress, compared with those of WT plants, the MDA content and electrolyte leakage were dramatically lower in ZmbHLH47-OE plants and significantly higher in zmbhlh47-crispr plants, respectively (Figure 1H,I).These outcomes strongly advocated for ZmbHLH47 as a positive regulator of drought tolerance in maize.
Furthermore, we extended our inquiry to assess the involvement of ZmbHLH47 in the response to salt stress.The seedlings of WT, ZmbHLH47-OE, and zmbhlh47-crispr were cultivated until they reached a two−week−old stage and were then subjected to either water (control) or 200 mM NaCl treatment.Intriguingly, in terms of growth performance, dry weight, and survival rate, no overt distinctions were discerned among WT, ZmbHLH47-OE, and zmbhlh47-crispr plants under both normal and salt stress conditions (200 mM NaCl) (Figure S4A-C).This unequivocally suggested that ZmbHLH47 does not play a role in the salt stress response in maize.Hence, based on the aforementioned findings, ZmbHLH47 emerges as a discernible positive regulator specifically implicated in maize drought tolerance, while remaining unrelated to salt tolerance.

ZmbHLH47 Promotes ABA Sensitivity in Maize
ABA is widely acknowledged for its pivotal role in plant-drought resistance [52][53][54].In an endeavor to further elucidate the involvement of ZmbHLH47 in ABA signaling, we compared the phenotypes of various genotypes during the seedling growth stage.The outcomes revealed that, concerning root length and fresh weight, no significant differences were discerned among the overexpression lines, WT, and the mutants under a normal condition.In contrast, when subjected to 10 µM ABA treatment, ZmbHLH47 overexpression lines exhibited elevated sensitivity to ABA, while the mutants displayed greater ABA insensitivity relative to the WT control (Figure 2A-C).2.6.ZmbHLH47 Specially Regulates the Expression of ZmSnRK2.9 To further unravel the molecular mechanisms underlying ZmbHLH47′s role as a pos- Moreover, qRT−PCR analysis was undertaken to quantify the transcript levels of selected ABA−responsive genes, namely ZmRAB18, ZmABF2, and ZmABI4 [55][56][57][58].Under normal conditions, all three genes exhibited similar expression levels across different genotypes.However, following ABA treatment, their expression levels were markedly elevated in the overexpression lines and significantly reduced in the mutants compared to WT plants, respectively (Figure 2D-F).Collectively, these findings robustly underscored that ZmbHLH47 exerts a positive influence on ABA response, which may likely contribute to its role in enhancing drought tolerance in maize.

ZmbHLH47 Specially Regulates the Expression of ZmSnRK2.9
To further unravel the molecular mechanisms underlying ZmbHLH47 ′ s role as a positive regulator in maize drought tolerance, we investigated whether it modulates the ABA biosynthesis or downstream signal transduction pathway under drought stress condition.Intriguingly, as depicted in Figure S5A-D, no significant differences were discerned among WT, ZmbHLH47-OE, and zmbhlh47-crispr plants under both normal and drought conditions, upon the evaluation of the ABA content and the expression levels of pivotal genes involved in ABA biosynthesis.It was unveiled that ABA biosynthesis was not intricately associated with ZmbHLH47.Furthermore, an assessment of the expression levels of genes related to ABA signal transduction, including ZmPYLs, ZmPP2C-As, and ZmSnRK2s [39][40][41], revealed through qRT−PCR results that ZmSnRK2.9, a member of subgroup III SnRK2 kinases [41], demonstrated an elevated level of induction in ZmbHLH47-overexpressing plants versus wild−type plants following drought stress treatment (Figures S6A-K, S7A-J and S8A-K).Conversely, the degree of induction in zmbhlh47-crispr plants was significantly lower than that in WT plants (Figures S6A-K, S7A-J and S8A-K).In concert, these outcomes suggested that ZmbHLH47 selectively regulates the expression of ZmSnRK2.9, contributing to its positive role in drought tolerance.

ZmSnRK2.9 Is a Direct Target of ZmbHLH47
To further figure out whether ZmbHLH47 directly modulates the expression of Zm-SnRK2.9,we attempted to evaluate ZmbHLH47 ′ s binding capacity to the ZmSnRK2.9 promoter using yeast one−hybrid (Y1H) assay.As depicted in Figure 3A, yeast cells, subjected to co−transfection with the pHis2.1-ZmSnRK2.9 pro and the pGADT7-ZmbHLH47 plasmids, demonstrated an unhampered growth on SD/−His−Leu−Trp medium containing 3-AT.This outcome highlighted the plausible interaction of ZmbHLH47 with the promoter region of ZmSnRK2.9 within the yeast system.
To assess drought tolerance, 12−day−old plants of WT, ZmSnRK2.9-OE, and zmsnrk2.9-crisprunderwent water deficit treatment, followed by a rehydration process, as previously described.As illustrated in Figure 4E, under well−watered conditions, the growth performance of WT, ZmSnRK2.9-OE, and zmsnrk2.9-crisprplants showed no discernible differences.Conversely, under water deficit and rehydration conditions, the The bHLH domain in bHLH-type transcription factors exhibited a preference for binding E-box (CANNTG) motifs in downstream target genes [45,59,60].Multiple E−boxes were identified in the ZmSnRK2.9 promoter region, hinting at ZmSnRK2.9 being a potential target of ZmbHLH47 (Figure 3B).To verify this hypothesis, we conducted chromatin immunoprecipitation (ChIP) assay using an anti−Flag antibody with 14−day−old ZmbHLH47-OE seedlings, both under normal conditions and following a 12 h drought treatment, followed using qPCR.Out of the identified five putative binding sites (P1-P5) within the ZmSnRK2.9 promoter, ChIP−qPCR analysis unveiled that ZmbHLH47-Flag exhibited specific binding affinity to the P3 and P5 sites in ZmbHLH47-Flag samples, highlighting a distinction absence in the WT ChIP samples (Figure 3B).The significant elevation in fold enrichment at the P3 and P5 sites following drought treatment is noteworthy, underscoring an augmented binding affinity of ZmbHLH47 to the ZmSnRK2.9 promoter in response to drought conditions (Figure 3B).Subsequent electrophoretic mobility shift assays (EMSAs) provided evidence that His−ZmbHLH47 could directly interact with the conserved P3 motif within the ZmSnRK2.9 promoter (Figure 3C).The incremental inclusion of an unlabeled wild−type probe effectively disrupted ZmbHLH47 binding to the biotin-labeled probe, while the mutated probe proved ineffective in competing for binding (Figure 3C).These outcomes unequivocally highlighted that ZmbHLH47 could directly interact with the ZmSnRK2.9 promoter.
Additionally, transitory transactivation assessment was undertaken employing the P3 promoter fragment of ZmSnRK2.9 in a dual luciferase (LUC) reporter system (Figure 3D).Maize leaf protoplasts underwent co−transformation with the ZmbHLH47-GFP effector construct and the ZmSnRK2.9P3 :LUC reporter construct.Consistent with expectations, drought treatment elicited the anticipated induction of ZmSnRK2.9 transcription.Furthermore, the co−transformation of the LUC reporter with ZmbHLH47-GFP led to the activation of ZmSnRK2.9 expression under normal conditions, and this activation was even more pronounced after drought treatment (Figure 3D).Hence, these outcomes suggested that ZmbHLH47 positively regulates ZmSnRK2.9 transcription by directly binding to its promoter.
To assess drought tolerance, 12−day−old plants of WT, ZmSnRK2.9-OE, and zmsnrk2.9crisprunderwent water deficit treatment, followed by a rehydration process, as previously described.As illustrated in Figure 4E, under well−watered conditions, the growth performance of WT, ZmSnRK2.9-OE, and zmsnrk2.9-crisprplants showed no discernible differences.Conversely, under water deficit and rehydration conditions, the Crispr plants exhibited more severe dehydration, whereas the OE plants displayed less rolling and wilting compared with the WT plants.As anticipated, after drought treatment, OE plants displayed an escalated survival rate and relative water content, whereas Crispr plants manifested a significantly diminished survival rate and relative water content versus WT plants (Figure 4F,G).Additionally, the MDA content and electrolyte leakage were correlated with the growth phenotype, survival rate, and relative water content.Water deficit treatment induced notable increases in the MDA content and electrolyte leakage compared with the well−watered condition in WT plants, and these effects were exacerbated in zmsnrk2.9crisprplants while alleviated in ZmSnRK2.9-OEplants (Figure 4H,I).In conclusion, these outcomes highlighted that ZmSnRK2.9 could function as an affirmative modulator of drought tolerance in maize, mirroring the role of ZmbHLH47.correlated with the growth phenotype, survival rate, and relative water content.Water deficit treatment induced notable increases in the MDA content and electrolyte leakage compared with the well−watered condition in WT plants, and these effects were exacerbated in zmsnrk2.9-crisprplants while alleviated in ZmSnRK2.9-OEplants (Figure 4H,I).In conclusion, these outcomes highlighted that ZmSnRK2.9 could function as an affirmative modulator of drought tolerance in maize, mirroring the role of ZmbHLH47.The protein abundance of ZmSnRK2.9 in ZmSnRK2.9-OEtransgenic seedlings (#2, #6).Anti-Flag antibody was utilized to detect ZmSnRK2.9.Actin served as a control.(C) Schematic diagrams of zmsnrk2.9-crisprmutants generated using CRISPR/Cas9−mediated genome editing.(D) The zmsnrk2.9-c1 and zmsnrk2.9-c2mutations were identified using Sanger sequencing compared with WT. (E) Drought tolerance phenotypes of WT, ZmSnRK2.9-OE, and zmsnrk2.9-crisprplants.Twelve−day−old seedlings were subjected to drought stress by withholding water for 8 days, after which they were re-watered for 3 d (Re−watering).Scale bars = 5 cm.(F) Statistical analysis of survival rates after drought stress as shown in (E).At least 30 seedlings of each line per replicate were used for survival rate analysis.Data shown are means ± SD of three biological replicates.ns indicates no significant difference to the corresponding controls.** p < 0.01; *** p < 0.001; **** p < 0.0001, indicating significant differences to the corresponding controls (Student's t−test).(G) Relative water content (RWC) in WT, ZmSnRK2.9-OE, and zmsnrk2.9crisprplants under well-watered and drought conditions.Data shown are means ± SD of three biological replicates.ns indicates no significant difference to the corresponding controls.Significant differences are indicated using Student's t−test: ** p < 0.01; *** p < 0.001; **** p < 0.0001.(H,I) Malondialdehyde (MDA) content (H) and percentage leakage of electrolyte (I) of WT, ZmSnRK2.9-OE, and zmsnrk2.9-crisprplants under well−watered and drought conditions.DW, dry weight.The values are presented as means ± SD of three biological replicates.ns indicates no significant difference to the corresponding controls.** p < 0.01; *** p < 0.001; **** p < 0.0001, indicating significant differences to the corresponding controls (Student's t−test).

ZmSnRK2.9 Positively Regulates the ABA Response
To determine the involvement of ZmSnRK2.9 in the ABA−response pathway, we assessed the responses of WT plants, ZmSnRK2.9-OE, and zmsnrk2.9-crisprplants to different concentrations of ABA (0 and 10 µM).Under the 10 µM ABA treatment, the zmsnrk2.9-crisprplants exhibited significantly increased root length and fresh weight compared with the WT, while the ZmSnRK2.9-OEplants displayed a shorter root length and smaller fresh weight (Figure 5A-C).Additionally, we examined the expression levels of ABA−responsive genes, including ZmRAB18, ZmABF2, and ZmABI4, in WT, ZmSnRK2.9-OE, and zmsnrk2.9-crisprplants under ABA treatment (50 µM).Following ABA treatment, the expression levels of all three genes were markedly higher in the OE lines and lower in the mutants compared with the WT (Figure 5D-F).Hence, these outcomes demonstrated that ZmSnRK2.9 positively regulates the ABA response in maize.

The ZmbHLH47-ZmSnRK2.9 Module Positively Regulates Drought Tolerance in Maize
In recent decades, the escalating threat of drought stress has posed a significant challenge to global agriculture sustainability.Consequently, it has become imperative to elucidate the underlying genetic and molecular mechanisms governing drought resistance in maize for effective crop enhancement.In this investigation, we recognized the ZmbHLH47-ZmSnRK2.9module as a prospective affirmative modulator of drought tolerance in maize.Initially, ZmbHLH47 was demonstrated to bestow both ABA sensitivity and resilience to drought in maize (Figure 1A-I and Figure 2A-F).Subsequent comprehensive assays revealed that ZmbHLH47 could activate the expression level of the ZmSnRK2.9 gene, a member of the subgroup III SnRK2 kinases, likely by directly binding to its promoter (Figure S8A-K and Figure 3A-D).Furthermore, the overexpression of ZmSnRK2.9 heightened ABA sensitivity and drought resistance, while the knockout of ZmSnRK2.9 in maize diminished ABA sensitivity and drought tolerance (Figure 4A-I and Figure 5A-F).Finally, our genetic evidence suggested that ZmbHLH47 could function as the upstream of ZmSnRK2.9, exerting a positive regulatory influence on ABA response and drought tolerance in maize (Figure 6A-F and Figure 7A-E).

ZmbHLH47 May Specially Respond to Osmotic Stress in Maize
The qRT−PCR analysis revealed a significant upregulation of the ZmbHLH47 gene in response to NaCl, PEG, and exogenous ABA treatments (Figure S3A-D).Notably, ZmbHLH47 exhibited a specific involvement in ABA signal transduction and drought response in maize, with no discernible role in salt response (Figure 1A-I, Figure 2A-F and Figure S4A-C).It was well−established that drought could induce osmotic stress in plant cells, leading to the rapid accumulation of ABA [6,29].Conversely, salinity could negatively impact plant growth and development through ionic stress, osmotic stress, and secondary stresses (e.g., oxidative stress) induced by the excess accumulation of reactive oxygen species [61][62][63][64][65]. Consequently, our speculation was that ZmbHLH47 could specifically respond to the osmotic stress triggered by drought in maize.However, a more in−depth understanding of the detailed molecular mechanism necessitates further investigations.
3.3.ZmSnRK2.9,One of Subclass III SnRK2s, Functions as a Positive Regulator of ABA Response and Drought Tolerance in Maize Notably, the subgroup III SnRK2 protein kinases have gained widespread recognition for their pivotal regulatory roles in the ABA signaling pathway and subsequent stress responses in plants [41,66].In Arabidopsis, the well−documented rapid and robust activation of three subclass III SnRK2s-SnRK2.2,SnRK2.3, and SnRK2.6-byABA could lead to the phosphorylation of downstream effectors, such as AREB/ABFs, significantly contributing to ABA responsiveness and drought tolerance in the plant [67,68].Similarly, in rice, members of the subclass III group of SnRK2, namely SAPK8, SAPK9, and SAPK10, have exhibited to play crucial roles in controlling ABA-mediated seed germination and seedling growth [69,70].Despite these insightful findings, the specific contributions of subclass III SnRK2 protein kinases to ABA response and drought tolerance in maize remain to be elucidated.A recent study demonstrated that ZmSnRK2.10, a member of the subgroup III SnRK2 protein kinases, could be activated by ABA, and its overexpression could partially complement the ABA−insensitive phenotype of the snrk2.2/2.3/2.6 mutant in Arabidopsis [41].In the present investigation, we identified ZmSnRK2.9, another member of the subgroup III SnRK2 kinases, as a positive regulator of ABA response and drought tolerance in maize (Figure 4A-I and Figure 5A-F).Interestingly, in contrast to Arabidopsis, where SnRK2.2,SnRK2.3, and SnRK2.6 function redundantly, the ZmSnRK2.9 single mutant exhibited strong ABA insensitivity and enhanced drought tolerance.We hypothesize that this phenomenon may arise from distinct temporal and spatial expression patterns of subclass III SnRK2 protein kinases in maize.Further evidence is required to validate this hypothesis in the future studies.

Botanical Specimens and Growth Conditions
All plants with overexpressed (OE) genes and those with CRISPR−Cas9−induced mutations were attained from Wimi Biotechnology (Jiangsu) Co., Ltd., situated in Changzhou, China.The respective maize seeds were planted and cultivated in a meticulously controlled growth chamber (Percival).The environment within the chamber was upheld at a 16 h photoperiod featuring 400 µmol m −2 s −1 light intensity of a temperature of 25 • C, followed by 8 h of darkness at 22 • C. Additionally, a relative humidity was set at 70%.
For the salt treatment, a well-established procedure was followed [71].Briefly, 2−week−old maize seedlings underwent irrigation with a 200 mM NaCl solution.After an interval of 12 days, comprehensive photographic documentation of the plants was undertaken.Following this, the dry weight of the aerial components of the plants was quantified post-exposure to 200 mM NaCl for a duration of 15 days.Additionally, the survival rate of the plants was intricately calculated after enduring a 35−day treatment with 200 mM NaCl.
The drought treatment protocol was implemented as outlined in [72].Maize seedlings at the age of 12 days underwent an 8−day water deprivation period, followed by a subsequent 3−day rehydration phase.Phenotypic observations were documented and the survival rate was computed.Relative water content was assessed following the procedure detailed in [73].About 0.3 g fresh leaf samples (L1-L3 from the top) were collected for malondialdehyde (MDA) content analysis, involving homogenization in 10% trichloroacetic acid (TCA) containing 0.65% 2-thiobarbituric acid (TBA), followed by heating at 100 • C for 15 min, as in [74].The percentage of electrolyte leakage was assessed following the methodology outlined in [75].
Seedlings at the tri−foliate stage underwent distinct treatments involving immersion in a solution with 200 mM NaCl, 10% PEG 4000, or 50 µM ABA for the designated duration.

Phylogenetic and Sequence Analysis
Utilizing default settings for pairwise and multiple alignments, MEGA 7.0 software was employed to align 13 bHLHs sourced from maize, rice, and Arabidopsis [76].The alignment data were then utilized to construct a phylogenetic tree using the neighbor-joining method [77].For the analysis of the bHLH domain in ZmbHLH47, the sequence of ZmbHLH47 and its closely related homologs were aligned via Genedoc software (Version 2.7) [78].

Subcellular Localization Assay
For the detailed analysis of the subcellular localization of the ZmbHLH47 protein, we engineered a Super:ZmbHLH47:GFP vector, leveraging the pSuper1300-GFP platform [79].In this experimental setup, we incorporated the nuclear localization protein D53 (DWARF53) [47].The co-transformation of maize protoplasts involved the Super:ZmbHLH47:GFP and Super:D53:mCherry vectors.As a negative control, maize protoplasts underwent a genetic modification process involving the co-transformation of Super:GFP and Super:D53:mCherry vectors.The subsequent visualization of fluorescent signals was undertaken utilizing the sophisticated LSM880 laser scanning confocal microscope provided by Carl Zeiss that was headquartered in Jena, Germany.Comprehensive primer details for the subcellular localization assay are summarized in Table S1.

qRT-PCR
In strict accordance with the manufacturer's protocols, maize tissues underwent a meticulous total RNA extraction process utilizing the FastPure ®® Universal Plant Total RNA Isolation Kit (Vazyme, Nanjing, China).Following this, 1 µg of the extracted total RNA underwent a sophisticated reverse transcription procedure using the HiScript ®® III RT SuperMix for qPCR (+gDNA wiper) kit provided by Vazyme.The subsequent qRT-PCR was conducted on a state-of-the-art Stratagene Mx3000P real-time system cycler (Agilent) employing the advanced ChamQ Universal SYBR qPCR Master Mix provided by Vazyme.
The chosen reference control for this meticulous process was actin1 (GRMZM2G126010).Each experiment was executed with three technical replicates, and a minimum of three independent biological experiments were carried out.Comprehensive details pertaining to the primers for qRT-PCR can be accessible in Table S1.

Y1H Assay
In the DNA binding assay, the promoter region (1.5 kb upstream of ATG) of the ZmSnRK2.9 gene underwent precise amplification and was seamlessly incorporated into the pHis2.1 reporter vector.The coding sequence of ZmbHLH47 was subsequently integrated in−frame with the GAL4 activation domain within the pGADT7 vector.The Y1H assay unfolded according to the manufacturer's meticulous instructions (Clontech).The evaluation of ZmbHLH47 ′ s DNA binding activity was predicated on the growth performance exhibited by the co−transformants on selective medium supplemented with 3−Amino−1,2,4-triazole (3−AT).Comprehensive details pertaining to the primers for Y1H assay can be accessible in Table S1.

ChIP−qPCR Analysis
Conducting ChIP−qPCR assay involved intricate steps adhering to established protocols [80,81].Specifically, four grams of maize seedlings aged three weeks underwent cross-linking in 1% formaldehyde under vacuum, followed by the sonication of the resulting chromatin complexes into fragments ranging from 200 to 500 bp.Immunoprecipitation, washing, and reverse cross−linking were performed using polyclonal anti−Flag M2 beads (Sigma-Aldrich, St. Louis, MO, USA) to capture the protein−DNA complex.The precipitated DNA underwent quantitative PCR analysis, with a minimum of three independent experiments conducted, and the presentation includes one representative dataset.Comprehensive details pertaining to the primers can be accessible in Table S1.

EMSA
EMSA was undertaken following established procedures [82].The coding sequence of ZmbHLH47 was inserted into the pET30a vector to produce the His-ZmbHLH47 fusion protein.Subsequently, the purification of the recombinant protein His-ZmbHLH47 was undertaken through Ni Sepharose High Performance (GE Healthcare, Chicago, IL, USA), following the prescribed procedures provided by the manufacturer.It was attempted to carry out EMSA utilizing a Chemiluminescent EMSA kit attained from Beyotime, China.Biotin-labeled probes for EMSA were synthesized by Sangon Biotech Co., Ltd.(Shanghai, China).The details of primers and probe sequences employed in the EMSA are summarized in Table S1.

Transcriptional Activity Assay
To delineate the transcriptional activation domain of ZmbHLH47, there was an attempt to amplify both the intact and truncated sequences of ZmbHLH47, followed by incorporating the in−frame with the GAL4 DNA binding domain within the pGBKT7 vector.These resultant constructs, alongside the pGBKT7 empty vector, were individually introduced into the yeast strain Y2HGold.The identification of the transcriptional activation domain of ZmbHLH47 was undertaken upon monitoring cell growth on SD/−Trp−His−Ade medium.
To interrogate the transcriptional activation of ZmbHLH47 on ZmSnRK2.9, we attempted to conduct co−transfections of effector and reporter constructs in protoplasts isolated from maize seedlings aged two weeks.Using the internal control 35S:REN, the measurement of the activities of LUC and REN was undertaken via the Trans Detect Double-Luciferase Reporter Assay kit provided by TransGen Biotech, utilizing a GloMax 20/20 luminometer attained from Promega for detection.Rigorous experimentation was upheld through a minimum of three independent biological replicates.Comprehensive details regarding primer specifications for the transcriptional activity assay are thoughtfully documented in Table S1.

ABA Content Analysis
Leaves from 12−day−old maize seedlings, subjected to either a 10−day drought treatment or maintained without drought stress, were selected for ABA measurement.About 0.5 g leaves (L1-L3 from the top) were finely ground in liquid nitrogen and then extracted with methanol containing 20% water (v/v).The purification and quantification of ABA followed the protocol outlined in [83,84].Three independent biological replicates were conducted, with each biological replicate comprising 10 plants from each line.

ABA Sensitivity Assay
The seeds of diverse genotypes underwent sterilization with 5% sodium hypochlorite for 10 min, followed by thrice rinsing with ultrapure water.Subsequently, the seeds were positioned on wet filter paper for a duration of 4 days until the primary roots attained an approximate length of 3 cm.Afterward, the seedlings were transferred and cultivated in Hoagland solution under greenhouse conditions for 7 days.Representative images were captured, and subsequently, the measurement and calculation of both root length and the fresh weight of each seedling were carried out.

Western Blot Analysis
Total protein extraction from maize seedlings was accomplished using radio immunoprecipitation assay (RIPA) buffer (Solarbio, Beijing, China) supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF).The ensuing proteins, subsequent to separation through 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), were translocated onto polyvinylidene fluoride (PVDF) membranes (Millipore, Burlington, MA, USA).These membranes underwent a blocking step in 1× TBST buffer (0.02% Tween in tris-buffered saline (TBS)) supplemented with 5% milk, particularly for 2 h at room temperature.This was followed by an overnight incubation with the primary antibody (4 • C).After extensive washing in 1× TBST buffer, the membranes experienced a 2 h incubation at room temperature with secondary antibodies conjugated to horseradish peroxidase.The detection of blots was undertaken utilizing BeyoECL plus (Beyotime).

Plant Transformation
The coding sequences of ZmbHLH47 and ZmSnRK2.9 were amplified and subsequently endowed with a Flag tag at their C termini.Following this, the resulting DNA fragments were integrated downstream of the ubiquitin promoter.The requisite target sites, which were strategically positioned in the inaugural exons of ZmbHLH47 and ZmPYL9 to facilitate the creation of CRISPR/Cas9 knockout lines, were sourced from CRISPR-P (http://crispr.hzau.edu.cn/CRISPR2/) (accessed on 10 June 2021).The acquisition of transgenic plants was executed through the meticulous process of Agrobacterium-mediated transformation.For a more precise exploration, the subsequent analyses were conducted exclusively on the homozygous T4 overexpression (OE) lines.Additionally, in the ensuing experiments, the homozygous CRISPR−Cas9 mutant lines-void of the Cas9 transgene-served as pivotal participants.It is imperative to underscore that this part of the investigation was adroitly executed by Wimi Biotechnology.Comprehensive details pertaining to the primers can be accessed in Table S1.

Statistical Analysis
The examination of datasets comprising two distinct groups entailed a thorough implementation of Student's t−test, where the designation "ns" indicates the absence of a statistically significant difference relative to the corresponding controls.The symbols "*", "**", "***", and "****" were employed to denote a significant difference from the respective controls, with p-values falling beneath the thresholds of 0.05, 0.01, 0.001, and 0.0001, respectively.A form of mean ± standard deviation (SD) was regarded to present data.For more intricate comparisons involving multiple groups, an all-encompassing approach was utilized, involving the application of one−way ANOVA followed by Tukey's test for post hoc analysis.Distinct letters were employed as indicators to denote statistically significant differences (p < 0.05) among groups.A proposed model for the function of ZmbHLH47-ZmSnRK2.9 module in drought tolerance in maize.Upon environmental drought stress, the transcript abundance and DNA binding affinity of ZmbHLH47 is significantly upregulated, which directly binds to the E−box elements of ZmSnRK2.9 promoter to activate its expression, leading to the upregulation of ABA−responsive genes.Eventually, the ABA response is promoted, and the drought tolerance is increased in maize.Arrows mean positive regulation.

Figure 1 .
Figure 1.ZmbHLH47 confers drought tolerance in maize.(A) The transcript abundance of ZmbHLH47 in WT and ZmbHLH47-OE seedlings.Expression in WT was set to 1.00.Data shown are means ± SD of three biological replicates.**** indicates significant difference to the corresponding controls with Int. J. Mol.Sci.2024, 25, 4957 7 of 24

Figure 2 .
Figure 2. ZmbHLH47 enhances ABA sensitivity and promotes ABA response in maize.(A) Growth performance of WT, ZmbHLH47-OE, and zmbhlh47-crispr plants grown in Hoagland solution with or without ABA.(B,C) The root lengths and fresh weights of WT, ZmbHLH47-OE, and zmbhlh47crispr plants treated with 0 or 10 µM ABA for 7 d.Data are means of three biological replicates means ± SD (n = 60 for (B,C)).ns indicates no significant difference to the corresponding controls.** p < 0.01; *** p < 0.001; **** p < 0.0001, indicating significant differences to the corresponding controls (Student's t−test).(D-F) Determination of ABA-responsive gene expression levels among WT, ZmbHLH47-OE, and zmbhlh47-crispr plants in response to treatment with 50 µM ABA.Expression in the untreated WT was set to 1.00.Data shown are means ± SD of three biological replicates.ns indicates no significant difference to the corresponding controls.Significant differences are indicated using Student's t−test: *** p < 0.001; **** p < 0.0001.

Figure 2 .
Figure 2. ZmbHLH47 enhances ABA sensitivity and promotes ABA response in maize.(A) Growth performance of WT, ZmbHLH47-OE, and zmbhlh47-crispr plants grown in Hoagland solution with or without ABA.(B,C) The root lengths and fresh weights of WT, ZmbHLH47-OE, and zmbhlh47-crispr plants treated with 0 or 10 µM ABA for 7 d.Data are means of three biological replicates means ± SD (n = 60 for (B,C)).ns indicates no significant difference to the corresponding controls.** p < 0.01; *** p < 0.001; **** p < 0.0001, indicating significant differences to the corresponding controls (Student's

Figure 3 .
Figure 3. ZmbHLH47 directly binds to the ZmSnRK2.9 promoter and activates its expression.(A) Y1H assay showing direct binding of ZmbHLH47 to the ZmSnRK2.9 promoter in yeast.AD: pGADT7, −WL: −Leu−Trp, −WLH + 3AT: −His−Leu−Trp + 3−Amino−1,2,4−triazole. (B) ChIP assay showing the binding of ZmbHLH47 to the ZmSnRK2.9 promoter in vivo.ZmbHLH47-Flag seedlings were conducted with sufficient water or drought for 12 h.Utilizing specific primers (P1−P5) within the ZmSnRK2.9 promoter region, qPCR was performed to quantify the immunoprecipitated DNA.Input (%) represents relative enrichment.Data shown are means ± SD of three biological replicates with one−way ANOVA and Tukey's test.Different letters indicate significant differences (p < 0.05).(C) EMSA assay showing ZmbHLH47 specifically binds to the E−box in the P3 fragment of ZmSnRK2.9 promoter.Wild-type and mutated competitor sequences are illustrated at the bottom.Arrowhead represents the shifted band.Three independent repeats showed similar results.(D) Dual−LUC assay showing that ZmbHLH47 positively regulates ZmSnRK2.9 transcription in maize protoplasts.Protoplasts co−transformed with ZmbHLH47-GFP and pZmSnRK2.9:LUCwere treated with or without 50 mM mannitol for 1 h.REN represents Renilla LUC, LUC represents firefly LUC.Data shown are means ± SD of three biological replicates with one−way ANOVA and Tukey's test.Different letters indicate significant differences (p < 0.05).

Figure 3 .
Figure 3. ZmbHLH47 directly binds to the ZmSnRK2.9 promoter and activates its expression.(A) Y1H assay showing direct binding of ZmbHLH47 to the ZmSnRK2.9 promoter in yeast.AD: pGADT7, −WL: −Leu−Trp, −WLH + 3AT: −His−Leu−Trp + 3−Amino−1,2,4−triazole. (B) ChIP assay showing the binding of ZmbHLH47 to the ZmSnRK2.9 promoter in vivo.ZmbHLH47-Flag seedlings were conducted with sufficient water or drought for 12 h.Utilizing specific primers (P1−P5) within the ZmSnRK2.9 promoter region, qPCR was performed to quantify the immunoprecipitated DNA.Input (%) represents relative enrichment.Data shown are means ± SD of three biological replicates with one−way ANOVA and Tukey's test.Different letters indicate significant differences (p < 0.05).(C) EMSA assay showing ZmbHLH47 specifically binds to the E−box in the P3 fragment of ZmSnRK2.9 promoter.Wild-type and mutated competitor sequences are illustrated at the bottom.Arrowhead represents the shifted band.Three independent repeats showed similar results.(D) Dual−LUC assay showing that ZmbHLH47 positively regulates ZmSnRK2.9 transcription in maize protoplasts.Protoplasts co−transformed with ZmbHLH47-GFP and pZmSnRK2.9:LUCwere treated with or without 50 mM mannitol for 1 h.REN represents Renilla LUC, LUC represents firefly LUC.Data shown are means ± SD of three biological replicates with one−way ANOVA and Tukey's test.Different letters indicate significant differences (p < 0.05).

Figure 8 .
Figure8.A proposed model for the function of ZmbHLH47-ZmSnRK2.9 module in drought tolerance in maize.Upon environmental drought stress, the transcript abundance and DNA binding affinity of ZmbHLH47 is significantly upregulated, which directly binds to the E−box elements of ZmSnRK2.9 promoter to activate its expression, leading to the upregulation of ABA−responsive genes.Eventually, the ABA response is promoted, and the drought tolerance is increased in maize.Arrows mean positive regulation.