ZmELF6‐ZmPRR37 module regulates maize flowering and salt response

Summary

The control of flowering time in maize is crucial for reproductive success and yield, and it can be influenced by environmental stresses. Using the approaches of Ac/Ds transposon and transposable element amplicon sequencing techniques, we identified a Ds insertion mutant in the ZmPRR37 gene. The Ds insertion showed a significant correlation with days to anthesis. Further research indicated that ZmPRR37‐CR knockout mutants exhibited early flowering, whereas ZmPRR37‐overexpression lines displayed delayed flowering compared to WT under long‐day (LD) conditions. We demonstrated that ZmPRR37 repressed the expression of ZmNF‐YC2 and ZmNF‐YA3 to delay flowering. Association analysis revealed a significant correlation between flowering time and a SNP2071‐C/T located upstream of ZmPRR37. The SNP2071‐C/T impacted the binding capacity of ZmELF6 to the promoter of ZmPRR37. ZmELF6 also acted as a flowering suppressor in maize under LD conditions. Notably, our study unveiled that ZmPRR37 can enhance salt stress tolerance in maize by directly regulating the expression of ABA‐responsive gene ZmDhn1. ZmDhn1 negatively regulated maize salt stress resistance. In summary, our findings proposed a novel pathway for regulating photoperiodic flowering and responding to salt stress based on ZmPRR37 in maize, providing novel insights into the integration of abiotic stress signals into floral pathways.

Introduction

Maize (Zea mays L.), a short‐day C4 plant, was domesticated in southwestern Mexico from teosinte approximately 10 000 years ago (Matsuoka et al., 2002). Through natural and artificial selection, maize has achieved global cultivation (Buckler et al., 2009; Kuleshov, 1933; Swarts et al., 2017), becoming an economically significant crop for food, feed and fuel production (Xu and Crouch, 2008). The adaptation of maize to long‐day (LD) environments is facilitated by reduced photoperiod sensitivity (Huang et al., 2018; Hung et al., 2012; Yang et al., 2013). Several genes, including ZmCOL3, ZmCCT, ZmCCT9, ZCN8, ZmPhyB2, ZmNF‐YA3 and ZmNF‐YC2, have been reported to participate in the photoperiodic regulation of flowering in maize. CCT domain‐containing genes have been recognized for their crucial role in regulating flowering time, with a current knowledge of 53 CCT genes in the maize genome (Jin et al., 2018; Liu et al., 2020). ZmCOL3 was discovered to repress flowering by transactivating the transcription of ZmCCT or by interfering with the circadian clock (Jin et al., 2018). ZmCCT, identified as a flowering repressor under LD conditions, was investigated before (Hung et al., 2012; Yang et al., 2013). Another CCT gene, ZmCCT9, was found to inhibit the expression of ZCN8, consequently impeding flowering in maize under LD conditions (Huang et al., 2018). ZCN8, encoding maize florigen, acts as a floral activator and exhibits diurnal regulation in photoperiod‐sensitive tropical lines, distinguishing it from day‐neutral lines (Meng et al., 2011). ZmPhyB2, an active photoreceptor, accelerates flowering when its function is lost (Kumar et al., 2016; Sheehan et al., 2007). Through map‐based cloning, a nuclear transcription factor Y (NF‐Y) subunit coding gene, ZmNF‐YC2, was identified as a flowering activator under LD conditions (Su et al., 2021). ZmNF‐YC2 activates another NF‐Y gene, ZmNF‐YA3, which serves as a positive regulator of flowering time under LD conditions (Su et al., 2018).

The PRR subfamily, belonging to the CCT family, exhibits conserved protein sequences and functions across angiosperms (Linde et al., 2017; Murakami et al., 2007). In Arabidopsis, five PRRs (PRR9/7/5/3 and TOC1) are expressed from morning to night (Matsushika et al., 2000). Both PRR9, PRR7 and PRR5 have been found to accelerate flowering in Arabidopsis under LD conditions (Nakamichi et al., 2007; Yamamoto et al., 2003). An ortholog of PRR7, Photoperiod‐H1 (Ppd‐H1), was identified through positional cloning in barley grass grown under LD inductive conditions. Similar to Arabidopsis prr7 mutants, ppd‐H1 exhibited delayed flowering under LD conditions but not under SD conditions (Turner et al., 2005). A 2 kb deletion upstream of the coding region in wheat PRR gene Ppd‐D1, was associated with photoperiod insensitivity (Beales et al., 2007). In common wheat, a paralog of Ppd1 called TaPRR73 displayed circadian rhythm and was significantly associated with the heading date (Zhang et al., 2016). Koo et al. (2013) further identified a PRR gene, OsPRR37, responsible for the heading date2 (Hd2) QTL in rice. OsPRR37 was found to function as a suppressor by inhibiting the expression of Hd3a under LD conditions. Overexpression of the rice floral inhibitor OsPRR73 delayed the heading date under both LD and SD conditions, while OsPRR73 knockout mutants flowered early only under LD conditions (Liang et al., 2021). Under LD conditions, a PRR gene named SbPRR37 was identified by positional cloning as a major repressor of sorghum flowering (Murphy et al., 2011). Wang et al. (2020) identified a gene called GmPRR37 responsible for qFT12‐2 in soybean. GmPRR37 knockout mutants exhibited early flowering under LD conditions, while transgenic soybean plants overexpressing GmPRR37 displayed the opposite phenotype. Furthermore, two flowering genes, Tof11 and Tof12, were identified through whole‐genome resequencing combined with GWAS analysis and positional cloning (Lu et al., 2020). Tof11 and Tof12 encode PRR proteins that enhance the expression of the legume‐specific key photoperiod flowering suppressor gene E1, ultimately resulting in delayed flowering under LD conditions (Lu et al., 2020; Xia et al., 2012).

Flowering, a critical stage in the life cycle of plants, is susceptible to environmental stresses. In addition to its involvement in flowering time regulation, the PRRs have emerged as key regulators of cold, drought and salt stress responses. In Arabidopsis, the prr5prr7prr9 triple mutants exhibited enhanced tolerance to drought and cold treatments compared to the wild type (WT) (Liu et al., 2013). Transgenic Arabidopsis plants overexpressing PRR5‐VP demonstrated increased tolerance to cold, drought and salinity stresses by upregulating the expression of stress‐responsive genes (Nakamichi et al., 2016). Gol et al. (2021) demonstrated that Ppd‐H1 modulates spike development under drought stress. Under salt stress, OsPRR73 is induced and confers salt tolerance by repressing the expression of a Na⁺ transporter gene, OsHKT2.1, which negatively regulates salt tolerance in rice (Horie et al., 2007; Wei et al., 2021). Severe drought stress led to a significant reduction in the transcript levels of soybean PRRs GmPRR3/7/9‐LIKE (Marcolino‐Gomes et al., 2014). The influence of environmental stress on flowering timing varies among species and depends on multiple factors, as genetic pathways governing the trade‐off between flowering and stress responses have diverged to some extent in different plants (Chong et al., 2022; Jung and Müller, 2009). Consequently, further investigations are required to comprehend how maize integrates stress responses and flowering time.

In the current study, we identified a Ds insertion mutant in a pseudo‐response regulator protein‐coding gene, ZmPRR37, which exhibited a significant correlation with days to anthesis (DTA). Further genetic experiments indicated that ZmPRR37 acted as a suppressor of flowering time under LD but not SD conditions. Moreover, candidate gene association analysis revealed a significant correlation between flowering time and a SNP2071‐C/T within the ZmPRR37 promoter. Notably, the SNP2071‐C/T was found to influence the binding affinity of ZmELF6 to ZmPRR37 promoter. Mechanistically, ZmPRR37 delayed flowering by directly inhibiting the expression of ZmNF‐YA3 and ZmNF‐YC2. In summary, we have identified a novel pathway (ZmELF6‐ZmPRR37‐ZmNF‐Ys) that regulates photoperiodic flowering in maize. Intriguingly, ZmPRR37 not only regulates flowering but also enhances maize salt tolerance by modulating the expression of salt‐ and ABA‐responsive genes, with particular emphasis on the negative regulatory factor for salt stress, ZmDhn1. These findings highlight the crucial role of ZmPRR37 in the photoperiodic regulation of flowering and the response to salt stress in maize.

Results

ZmPRR37 delays flowering in maize under long‐day conditions

Previous studies have demonstrated the involvement of PRR37 proteins in the photoperiodic regulation of flowering and stress responses in other plants (Gol et al., 2021; Murphy et al., 2011). However, the biological function of maize PRR37 remains largely unknown. In this study, we identified a Zm00001d007240 (ZmPRR37) mutant (designated as zmprr37) using Ac/Ds transposon and TEA‐Seq techniques. The Ds transposon was inserted 26 639 bp downstream of ZmPRR37's ATG (Figure S1a). The insertion of Ds led to a sharp decrease in the expression level of ZmPRR37 in the zmprr37 mutant, almost undetectable (Figure S1b). ZmPRR37 encodes a protein consisting of 688 amino acids that contain a pseudo‐receiver domain and a CCT domain located at the N‐ and C‐terminal, respectively, which are present in all known plant PRR proteins. Phylogenetic analysis revealed that ZmPRR37 is most closely related to PRR37 proteins from Sorghum bicolor (Figure S1c).

To determine the role of ZmPRR37 in flowering time regulation, both WT and zmprr37 were planted in Zhengzhou (LD conditions) and Sanya (SD conditions). The results revealed that zmprr37 flowered 3–5 days earlier than WT under LD conditions, while the flowering time was similar under SD conditions (Figure 1a,b). There were no significant differences in plant height (Figure 1c), which was measured after flowering. To further investigate the function of ZmPRR37, we generated ZmPRR37‐CR knock‐out and ZmPRR37‐OE overexpression lines. Two target sites for Cas9 cleavage were identified within the fifth and seventh exons of ZmPRR37 (Figure S1d). Through PCR and sequencing analysis, we identified two independent lines with deletions in the target sites among 26 T₀ transgenic plants. Subsequently, these two mutants were self‐pollinated, leading to the generation of two homozygous knockout mutant lines (ZmPRR37‐CR1 and ZmPRR37‐CR2) that encoded a CCT domain‐deficient protein. Simultaneously, we obtained 10 ZmPRR37‐OE lines through genetic transformation in maize. RT‐qPCR analysis revealed that ZmPRR37 expression levels were highest in ZmPRR37‐OE2 and ZmPRR37‐OE5, which were utilized for subsequent experimental analyses (Figure S1e).

Figure 1.

ZmPRR37 delays flowering in maize under LD conditions. (a) Flowering phenotype of WT and zmprr37 Ds insertion mutant under LD and SD conditions. (b) Statistical analysis of days to anthesis in WT and zmprr37 Ds insertion mutant under LD and SD conditions. Data represent the mean ± SD of five plants for each line. (c) Plant height of WT and zmprr37 Ds insertion mutants under LD and SD conditions. Data represent the mean ± SD of five plants for each line. (d) Morphologies of WT, ZmPRR37‐CR1 (37‐CR1), ZmPRR37‐CR2 (37‐CR2), ZmPRR37‐OE2 (37‐OE2) and ZmPRR37‐OE5 (37‐OE5) lines under LD and SD conditions. (e) Statistical analysis of days to anthesis in WT, 37‐CR1, 37‐CR2, 37‐OE2 and 37‐OE5 lines under LD and SD conditions. Data represent the mean ± SD of five plants for each line. (f) Plant height of WT, 37‐CR1, 37‐CR2, 37‐OE2 and 37‐OE5 lines under LD and SD conditions. Data represent the mean ± SD of five plants for each line. (g, i) Microscopic photographs of tassels (g) from V6 to V14 and ears (i) from V8 to V14 of WT1, WT2, 37‐CR1, 37‐CR2, 37‐OE2 and 37‐OE5 growing under LD conditions. (h, j) The length of tassels (h) from V6 to V14 and ears (j) from V8 to V14 of WT1, WT2, 37‐CR1, 37‐CR2, 37‐OE2 and 37‐OE5 growing under LD conditions. Values represent mean ± SD from at least three individual plants in (h) and (j).

Under LD conditions, ZmPRR37‐CR exhibited earlier flowering, while ZmPRR37‐OE plants flowered later compared to the WT. However, no significant differences in flowering time were observed between ZmPRR37‐CR, ZmPRR37‐OE and WT under SD conditions (Figure 1d,e). Furthermore, there were no significant differences in plant height between these three lines (Figure 1f). Compared to SD conditions, the expression level of ZmPRR37 was higher under LD conditions (Figure S1f). These findings indicate that ZmPRR37 underlies photoperiod sensitivity and functions as a repressor of maize flowering time under LD conditions. To assess the developmental progress of tassels and ears, we compared WT, ZmPRR37‐OE and ZmPRR37‐CR plants grown in a greenhouse under LD conditions from the V6 to V14 stages. As depicted in Figure 1g–j, ZmPRR37‐CR displayed the earliest development, followed by the WT, while ZmPRR37‐OE exhibited the latest development (Figure S1g). However, there were no significant differences in plant height and yield among these three lines (Figure S1h–j), indicating that ZmPRR37 delays flowering by slowing the developmental process of maize under LD conditions.

ZmPRR37 improves salt resistance under long‐day conditions

Sequence analysis revealed that the promoter region of ZmPRR37 contains multiple light‐, stress‐ and abscisic acid‐responsiveness elements (Figure S2a). The expression of ZmPRR37 was effectively induced by salt and ABA treatments (Figure S2b). To investigate the involvement of ZmPRR37 in the regulation of stress response, WT and zmprr37 were subjected to drought and salt stresses under LD conditions. After approximately 10 days of water deprivation, no significant difference was observed between WT and zmprr37 plants (Figure S2c). However, under osmotic conditions induced by saturating the soil with a 250 mM NaCl solution, the zmprr37 plants showed increased sensitivity to salt stress compared to WT (Figure 2a). Furthermore, the zmprr37 plants exhibited a notable reduction in the activities of antioxidant enzymes, including superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), when exposed to salt stress, as compared to WT. The levels of soluble protein, carotenoids and total chlorophyll were significantly lower in zmprr37 plants than in WT under salt stress (Figure 2b). These findings reflect the diminished salt stress tolerance phenotype observed in the zmprr37 plants.

Figure 2.

ZmPRR37 improves salt tolerance in maize under LD conditions. (a) Salt resistance of WT and zmprr37 Ds insertion mutant under LD conditions. Pictures were taken before and after 10 days of salt treatment (250 mM NaCl). (b) Determination of enzymatic activity of POD, SOD and CAT, as well as the contents of soluble protein, carotenoids and total chlorophyll in WT and zmprr37 mutant under normal (N) and salt stress (S) conditions. Data represent means (n = 3; **P < 0.01). (c) Morphology of WT, ZmPRR37‐CR1 (37‐CR1), ZmPRR37‐CR2 (37‐CR2), ZmPRR37‐OE2 (37‐OE2) and ZmPRR37‐OE5 (37‐OE5) before and after salt stress. Pictures were taken under well‐watered conditions and after 20 days of salt treatment (250 mM NaCl). (d) Determination of enzymatic activity of POD, SOD, CAT and the contents of soluble protein, carotenoids and total chlorophyll in WT, 37‐CR1, 37‐CR2, 37‐OE2 and 37‐OE5 grown under normal (N) and salt stress (S) conditions. Data are reported as means (n = 3; *P < 0.05; **P < 0.01). (e) Statistical analysis of survival rates of WT, 37‐CR1, 37‐CR2, 37‐OE2 and 37‐OE5 grown under normal (N) and salt stress (S) conditions. Each sample was collected from three different plants with biological replicates. Data are reported as means (n = 3; *P < 0.05; **P < 0.01). (f) ABA contents in WT, 37‐CR1, 37‐CR2, 37‐OE2 and 37‐OE5 seedlings under normal (N) and salt stress (S) conditions. (g) Salt‐hypersensitive phenotype of ZmPRR37‐CR1 seedlings treated with exogenous ABA (100 μM). (h) Statistical analysis of survival rates of WT and ZmPRR37‐CR1 under salt treatment, and salt‐hypersensitive phenotype of ZmPRR37‐CR1 seedlings treated with ABA. For statistical analysis, each sample was collected from three random plants with biological replicates. Data are reported as means (n = 3; *P < 0.05; **P < 0.01). (i) Photographs were taken after 4 days of growth in water (Normal), 150 mM NaCl, or 10 μM ABA. Bar = 1 cm. (j, l) Microscopic photographs of tassels (j) from V6 to V14 and ears (l) from V8 to V14 of WT1, WT2, 37‐CR1, 37‐CR2, 37‐OE2 and 37‐OE5 under salt stress conditions. (k, m) The length of tassels (k) from V6 to V14 and ears (m) from V8 to V14 of WT1, WT2, 37‐CR1, 37‐CR2, 37‐OE2 and 37‐OE5 under salt stress conditions. Values represent mean ± SD from at least three individual plants in (k) and (m). Representative pictures (n) and statistical analysis (o) of the yield of WT1, WT2, 37‐CR1, 37‐CR2, 37‐OE2 and 37‐OE5 grown under salt stress conditions. Data are reported as means (n = 3; *P < 0.05; **P < 0.01).

To further confirm the role of ZmPRR37 in salt stress response, WT, ZmPRR37‐OE and ZmPRR37‐CR were subjected to salt stress under LD conditions. Similar to the zmprr37 Ds insertion mutant, the ZmPRR37‐CR mutants exhibited increased sensitivity to salt stress, while the ZmPRR37‐OE lines displayed enhanced resistance compared to WT (Figure 2c). Under salt stress, the activities of SOD, POD and CAT, were significantly reduced in ZmPRR37‐CR mutants, accompanied by decreased levels of soluble protein, carotenoids and total chlorophyll. Conversely, these measurements were increased in ZmPRR37‐OE lines (Figure 2d). Moreover, the survival rates of ZmPRR37‐CR lines were significantly lower than those of the WT plants, whereas the survival rates of ZmPRR37‐OE lines were significantly higher (Figure 2e). Abscisic acid (ABA) is known to play crucial roles in plant responses to various stresses, including salt stress (Lu et al., 2019). The concentration of ABA in the leaves of ZmPRR37‐OE significantly increased under salt stress (Figure 2f). Furthermore, spraying exogenous ABA reduced salt sensitivity in ZmPRR37‐CR and improved the survival rate (Figure 2g,h). On the contrary, the application of the ethylene donor ethephon (350 μM) did not show any improvement in the phenotype of ZmPRR37‐CR (Figure S2d). Under normal conditions, there were no significant differences in the root length among WT, ZmPRR37‐OE and ZmPRR37‐CR. However, under salt stress conditions, ZmPRR37‐OE displayed enhanced tolerance compared to WT and ZmPRR37‐CR. Additionally, ZmPRR37‐OE also exhibited increased sensitivity to ABA treatment during the seed germination stage (Figure 2i, Figure S2e). These findings suggest that ZmPRR37 may positively regulate salt stress tolerance through the ABA pathway.

We also evaluated the developmental progress of tassels and ears in WT, ZmPRR37‐OE and ZmPRR37‐CR plants under salt stress conditions during the V6‐V14 stages. As shown in Figure 2j–m, the development of WT, ZmPRR37‐OE and ZmPRR37‐CR plants was delayed under salt stress compared to normal conditions. The WT and ZmPRR37‐CR plants exhibited similar developmental patterns, while ZmPRR37‐OE plants showed slightly earlier development under salt stress (Figure S2f). Notably, compared to WT, ZmPRR37‐OE lines exhibited approximately a 40% increase in yield under salt stress, whereas ZmPRR37‐CR showed approximately a 50% yield loss (Figure 2n,o). Additionally, compared to WT, the plant height of ZmPRR37‐OE lines was significantly higher, while ZmPRR37‐CR was significantly lower under salt stress (Figure S2g). These results indicate that the growth and development of maize are negatively impacted by salt stress, but ZmPRR37 confers maize salt tolerance.

Combined omics analyses identified genes that are directly affected by ZmPRR37

Subcellular localization and transactivation analyses showed that ZmPRR37 is predominantly localized to the nucleus and exhibits transcriptional inhibitory activity in both yeast and Gal4‐LexA/UAS systems (Figure S3a–c). Then DAP‐seq assay was implemented to identify potential binding motifs and target genes of ZmPRR37. We identified 13 600 and 10 104 peaks from two biological replicates, with 7675 peaks overlapping (Figure S3d, Table S2). Three motifs, AAAWGGTC, GGCTAR and CAAWAATM, were identified (Figure 3a). Furthermore, the direct binding of ZmPRR37 to these motifs was confirmed through EMSA (Figure 3b). Analysis of their genomic distribution revealed that 45.4% (3485 peaks) were located in promoter regions, while the remaining 35.2% were found in intergenic regions (Figure S3e). Notably, the 3485 peaks in promoter regions corresponded to 2327 genes. Gene ontology (GO) enrichment analysis demonstrated that these genes are primarily involved in DNA damage repair, photoperiodic flowering regulation and cellular response to stimulation and stress (Figure 3c).

Figure 3.

Combined omics analysis of ZmPRR37. (a) Motifs identified using 200 bp regions surrounding ZmPRR37 binding sites by MEME‐CHIP online software. (b) Direct binding of ZmPRR37 to three probes containing each motif in panel (a). Competition for ZmPRR37 binding was conducted using 80 × cold probes (unlabeled probes). Mutant probe represents a labelled probe containing the mutated motif (AAATGGTC, GGCTAA and CAAAAATC mutated into CCACTACT). (c) Gene ontology (GO) enrichment analysis of the target genes of ZmPRR37. (d) Validation of expression differences observed by RNA‐Seq through RT‐qPCR of 20 randomly selected differentially expressed genes (DEGs). (e) Venn diagrams shows the expression patterns of DEGs (q < 0.01, fold change (FC) > 2) between ZmPRR37‐CR and WT under normal conditions and salt conditions. Red and dark numbers indicate the numbers of upregulated genes and downregulated genes, respectively. (f) Heatmap shows the gene expression levels in WT and ZmPRR37‐CR under normal conditions and salt treatment. The scale bar represents the log10 value of FPKM. Heatmap generated using TBtools.

To investigate target genes regulated by ZmPRR37, we collected leaves from WT and ZmPRR37‐CR under normal and salt conditions for RNA sequencing. Over 71% of the clean reads were successfully mapped to the maize V4 genome (Table S3). The reliability of the RNA‐seq data was verified by RT‐qPCR analysis of 20 randomly selected DEGs (Figure 3d). Under normal conditions, 1311 DEGs were identified (Figure 3e,f). GO enrichment analysis revealed that these genes are involved in oxidation–reduction processes, photosynthesis light reactions, circadian rhythms and photoperiodic flowering (Figure S3f). Additionally, a total of 3186 DEGs were detected under salt treatment (Figure 3e,f). These DEGs were found to be enriched in biological pathways related to reactive oxygen species, heat, abiotic stimuli, transmembrane and ion transport, oxidation–reduction processes, plant‐type cell wall biogenesis and response to hormones (Figure S3g).

ZmPRR37 directly binds to the promoters of ZmNF‐YA3 and ZmNF‐YC2 to repress their transcription

Based on the results of DAP‐Seq and RNA‐Seq analyses, we identified five genes (Table S4) associated with photoperiod, circadian clock, development and the transition from vegetative to reproductive phases of meristem as potential targets of ZmPRR37. Previous study has demonstrated the photoperiod‐responsive behaviour of ZmNF‐YA3 and ZmNF‐YC2, as well as their role in promoting flowering under long day conditions (Su et al., 2018, 2021). In the present study, the expression levels of ZmNF‐YA3 and ZmNF‐YC2 vary from LD to SD conditions and were repressed by ZmPRR37 (Figure 4a,d). DAP‐Seq results revealed the presence of ZmPRR37 binding peaks within the promoter regions of ZmNF‐YA3 and ZmNF‐YC2 (Figure 4b). And DAP‐qPCR results demonstrated the enrichment of 200‐bp promoter fragments containing ZmPRR37 binding sites in DAP products (Figure 4c), indicating the direct binding of ZmPRR37 to ZmNF‐YA3 and ZmNF‐YC2. Furthermore, RT‐qPCR analysis confirmed that the transcriptional levels of ZmNF‐YA3 and ZmNF‐YC2 were upregulated in ZmPRR37‐CR under LD conditions (Figure 4d). In addition, dual‐luciferase (dual‐luc) experiments were conducted using ZmPRR37 as effector and LUC reporter constructs driven by the promoters of ZmNF‐YA3 and ZmNF‐YC2 (Figure 4e). Dual‐luc assay demonstrated that ZmPRR37 repressed LUC activity controlled by the promoters of ZmNF‐YA3 and ZmNF‐YC2 (Figure 4f). These findings suggest that ZmPRR37 negatively regulates maize flowering under LD conditions through the direct inhibition of ZmNF‐YA3 and ZmNF‐YC2.

Figure 4.

Direct transcriptional repression of ZmNF‐YA3 and ZmNF‐YC2 by ZmPRR37. (a) The expression pattern of ZmNF‐YA3 and ZmNF‐YC2 in WT, ZmPRR37‐CR (CR) and ZmPRR37‐OE (OE) under LD and SD conditions. Data are presented as means (n = 3; **P < 0.01). (b) DAP‐seq results show the binding of ZmPRR37 to the promoters of ZmNF‐YA3 and ZmNF‐YC2. The red box indicates binding peaks, and the red arrow indicates the orientation of gene transcription. (c) DAP‐qPCR assay confirms the binding of ZmPRR37 to the promoters of ZmNF‐YA3 and ZmNF‐YC2. Data are presented as means ± SD (n = 2 biological replicates). (d) Expression analysis of ZmNF‐YA3 and ZmNF‐YC2 genes in WT and ZmPRR37‐CR during stages V5, V6 and V7 under LD conditions. Each sample was collected from three different plants with three biological replicates. (e) Schematic diagrams of the dual‐luc reporter and effector constructs. The LUC reporter driven by the ZmNF‐YA3 or ZmNF‐YC2 promoter was used, while the REN reporter gene was driven by the 35S promoter. T, terminator. (f) Transient gene expression assays in N. benthamiana. The LUC reporter was co‐transfected with different effectors as indicated. Data are presented as means (n = 3; **P < 0.01).

ZmPRR37 enhances salt tolerance by directly repressing the expression of ABA‐responsive gene ZmDhn1

We have identified 8 DEGs with ZmPRR37 binding peaks that are likely involved in ABA response, stress response and photosystem functions (Figure 5a, Figure S4a,b, Table S5). We measured their expression levels by RT‐qPCR in WT and ZmPRR37‐CR under normal and salt conditions, as well as ZmPRR37‐CR seedlings spraying with exogenous ABA (Figure 5b, Figure S4c). Among the 8 genes, ZmAIRP2, ZmHSF30, ZmDhn1 and ZmPSII‐6 were upregulated in ZmPRR37‐CR mutant under salt stress but exhibited downregulation after ABA spraying in ZmPRR37‐CR (Figure 5b). The expression patterns of ZmAIRP2, ZmHSF30, ZmDhn1 and ZmPSII‐6 were examined under normal and ABA treatments. RT‐qPCR analysis revealed that the expression levels of these four genes were altered upon treatment with salt and ABA (Figure S4d). Moreover, dual‐luc assays revealed that ZmPRR37 can repress LUC activity driven by the promoters of these 4 genes (Figure 5c). DAP‐qPCR confirmed the significant enrichment of 200‐bp promoter fragments containing ZmPRR37 binding sites of these four genes in the DAP products (Figure 5d). These results support the direct regulation of these 4 genes by ZmPRR37.

Figure 5.

Direct transcriptional repression of salt stress and ABA‐responsive target genes by ZmPRR37. (a) DAP‐seq results shows the binding of ZmPRR37 to the promoters of ZmAirp2, ZmHsf30, ZmDhn1 and ZmPSII‐6. The red box indicates binding peaks, and the red arrow indicates the orientation of gene transcription. (b) Expression analysis of ZmAirp2, ZmHsf30, ZmDhn1 and ZmPSII‐6 between WT and ZmPRR37‐CR under normal, salt stress and salt‐hypersensitive ZmPRR37‐CR seedlings treated with exogenous ABA. Each sample was collected from three different plants with three biological replicates. Data are presented as means (**P < 0.01). (c) Transient gene expression assays in N. benthamiana. The LUC reporter was co‐transfected with different effectors as indicated. Data are presented as means (n = 3; **P < 0.01). (d) DAP‐qPCR assay confirming the binding of ZmPRR37 to the promoters of the four genes mentioned above. Data are presented as means ± SD (n = 2 biological replicates). (e) The FPKM of Zm00001d008397 in WT and ZmPRR37‐CR under normal and salt conditions. (f) Expression analysis of Zm00001d008397 in WT, ZmPRR37‐OE and ZmPRR37‐CR under normal and salt conditions. Each sample was collected from three different plants with three biological replicates. Data are presented as means (*P < 0.05; **P < 0.01).

Notably, the expression of ZmDhn1 exhibited the most pronounced changes in response to salt and ABA treatments (Figure S4d). To further verify the role of ZmDhn1 in salt stress, we employed CRISPR/Cas9 technology to knockout ZmDhn1 in the maize inbred line B104. Two target sites for Cas9 cleavage were located within the first and second exons of ZmDhn1 (Figure 6a). Zmdhn1‐2, which exhibited low expression levels and lacked the conserved dehydrin domain, was chosen for subsequent experiments (Figure 6b,c). As expected, zmdhn1‐2 conferred enhanced salt stress tolerance in maize seedlings (Figure 6d), as evidenced by significantly higher survival rates and increased levels of POD, SOD, CAT, soluble protein, carotenoids and total chlorophyll contents compared to the WT under salt stress conditions (Figure 6e,f). The application of exogenous ABA resulted in a phenotypic similarity between WT and zmdhn1‐2 (Figure 6g). Under normal conditions, there was no significant difference in root length between WT and zmdhn1‐2. However, under ABA treatment, zmdhn1‐2 displayed longer root length than WT (Figure 6h,i). These observations suggest that zmdhn1‐2 exhibits decreased sensitivity to ABA. Taken together, these findings indicate that ZmPRR37 may play a role in salt stress response through the ABA pathway by directly regulating the expression of the ABA‐responsive gene ZmDhn1.

Figure 6.

ZmDhn1 as a negative regulator of maize salt tolerance. (a) Gene structure of ZmDhn1 and two target sites for CRISPR/Cas9 editing. Sequences of two homozygous knockout lines with deletions in target sites, resulting in zmdhn1‐1 and zmdhn1‐2. PAM, protospacer‐adjacent motif. Deletions are indicated by dashes. (b) The effect of gene editing on the structural alteration of the ZmDhn1 gene in knockout mutants. (c) Expression levels of ZmDhn1 were measured in both WT, zmdhn1‐1 and zmdhn1‐2 lines. Data represent the mean ± SD of three biological replicates (**P < 0.01). (d) Morphologies of WT and zmdhn1‐2 knockout lines under normal (N) and salt (S) conditions. Pictures were taken 14 days after salt treatment (250 mM NaCl). (e) Statistical analysis of survival rates of plants grown under normal (N) and salt stress (S) conditions. Each sample was collected from three different plants with biological replicates. Data are presented as means (**P < 0.01). (f) Determination of the enzymatic activity of POD, SOD, CAT and the contents of soluble protein, carotenoids and total chlorophyll in WT and zmdhn1‐2 mutants grown under normal (N) and salt stress (S) conditions. Data are presented as means (n = 3; **P < 0.01). Note: ‘dhn1’ represents zmdhn1‐2. (g) Morphologies of WT and zmdhn1‐2 knockout lines under well‐watered, salt and salt+ABA conditions. Pictures were taken 7 days after treatment. Bar = 5 cm. (h) Photographs were taken after 4 days of growth in water (Normal) or 10 μM ABA. Bar = 1 cm. (i) Statistical analysis of the root length of WT and zmdhn1‐2 plants under normal and ABA treatment conditions. Data represent the mean ± SD of three repeats for each line (**P < 0.01).

SNP2071‐C/T affects differential binding of ZmELF6 to the promoter of ZmPRR37

To further investigate the relationship between flowering time and ZmPRR37, we performed an association analysis using a genomic region spanning 3 kb upstream of TSS to 1 kb downstream of TTS of ZmPRR37 in an association panel containing 447 maize inbred lines assessed for DTA under LD conditions in Zhengzhou. A total of 89 insertion/deletions (InDels) and 767 single nucleotide polymorphisms (SNPs) were identified and tested for their association with flowering time using a general linear model (GLM). Three SNPs located in the promoter or introns of ZmPRR37 were found to be significantly associated with maize flowering time (Bonferroni threshold P < 3.2 × 10⁻⁴). SNP2071‐C/T, 2071 bp upstream of ZmPRR37's TSS, exhibited the strongest association with flowering time (P = 1.08 × 10⁻⁴; Figure 7a). Notably, SNP2071‐C/T was found to be adjacent to a putative C2H2‐binding motif (GGTGT/ACACC) (Figure 7b). C2H2 zinc‐finger proteins (C2H2‐ZFPs) have been implicated in flowering induction, and loss‐of‐function mutations in C2H2‐ZFPs have been shown to cause delayed or early flowering in various studies (Deng et al., 2017; Hu et al., 2013; Kozaki et al., 2004; Matsubara et al., 2008; Wu et al., 2008; Yokoo et al., 2014). Through Ac/Ds transposon and TEA‐Seq techniques, we identified a mutant of Zm00001d048404 (the maize orthologue of Arabidopsis ELF6, designated as ZmELF6; belonging to C2H2‐ZFPs family) (Figure S5a). RT‐qPCR assay showed that ZmELF6 exhibits responsiveness to changes in photoperiod (Figure 7c). The loss of ZmELF6 function resulted in early flowering under LD conditions (Figure 7d). ZmELF6 was found to possess a C2H2 domain and respond to flowering, and SNP2071‐C/T upstream of ZmPRR37 was located within the C2H2 binding motif. To investigate the binding affinity, EMSA was performed, revealing a stronger binding affinity of ZmELF6 to the probe from C‐haplotype lines containing the ACACC nucleotides compared to the probe from T‐haplotype lines containing the ACACT nucleotides (Figure 7e). Additionally, dual‐luc assays demonstrated that ZmELF6 significantly induced the expression of LUC driven by the ZmPRR37 promoter, with the promoter from C‐haplotype lines exhibiting stronger LUC expression compared to the promoter from T‐haplotype lines under LD conditions with salt treatment (Figure 7f). RT‐qPCR analysis also revealed down‐regulated expression of ZmPRR37 in the zmelf6 mutants under LD conditions (Figure 7g). Collectively, these results indicate that ZmELF6 influences the expression of ZmPRR37 by binding to its promoter, and the SNP2071‐C/T polymorphism affects the differential binding of ZmELF6 to the ZmPRR37 promoter.

Figure 7.

ZmELF6 delays flowering time and affects salt stress response in maize under LD conditions. (a) Association analysis of a region from 3 kb upstream of the transcription start site (TSS) to 1 kb downstream of the transcription termination site (TTS) of ZmPRR37 in a panel of 447 diverse maize inbred lines. The black arrow indicates the most significant association (SNP2071‐C/T). (b) SNP2071‐C/T flanks a putative C2H2‐binding motif (black box). (c) The expression pattern of ZmELF6 under LD and SD conditions. Data are presented as means (n = 3; *P < 0.05; **P < 0.01). (d) Morphology of WT and ZmELF6 plants grown under LD conditions. (e) Electrophoretic mobility shift assay (EMSA) shows the binding affinity of ZmELF6 to the C‐ or T‐haplotype C2H2 binding motif. a: probe from C‐haplotype lines containing the ACACC nucleotides. b: probe from T‐haplotype lines containing the ACACT nucleotides. c: mutated probe containing AAAAA. d: C‐haplotype free probe without ZmELF6 protein. (f) Transient gene expression assays in N. benthamiana. The luciferase (LUC) reporter was co‐transfected with different effectors as indicated. Data are presented as means (n = 3; *P < 0.05; **P < 0.01). (g) Relative mRNA level of ZmPRR37 in WT and zmelf6 under LD conditions treated with water or salt. Each sample was collected from three different plants with three biological replicates. (h) Morphology of WT and zmelf6 mutant treated with water or NaCl under LD conditions. Pictures were taken 10 days after salt treatment (250 mM NaCl). (i) Determination of the activity of POD, SOD and CAT, as well as the contents of soluble protein, carotenoids and total chlorophyll in WT and zmelf6 mutants grown under normal (N) or salt stress (S) conditions. Each sample was collected from three different plants with three biological replicates. Data are presented as means (*P < 0.05; **P < 0.01).

To assess the salt stress response of ZmELF6, WT and zmelf6 mutants were subjected to salt stress under LD conditions. As depicted in Figure 7h, zmelf6 mutants exhibited superior growth compared to the WT under salt stress. Furthermore, the activities of SOD, POD, CAT and the content of soluble protein, carotenoids and total chlorophyll were significantly higher in zmelf6 mutants than in the WT under salt stress (Figure 7i), consistent with the robust phenotype of zmelf6 mutants under salt conditions. To evaluate the developmental progression of tassels and ears in WT and zmelf6 mutants under normal and salt stress conditions, we observed their growth from the V6‐V15 stages under LD conditions. Plants subjected to salt stress exhibited delayed development compared to those grown under normal conditions (Figure S5b–e). Notably, zmelf6 exhibited accelerated development compared to WT. Moreover, the yield of zmelf6 was significantly higher than that of the WT (Figure S5f,g).

Discussion

ZmPRR37 delays maize flowering through directly repressing ZmNF‐YA3 and ZmNF‐YC2 under long‐day conditions

Photoperiod‐dependent flowering time is a crucial trait in maize breeding, subject to domestication and selection. In this study, we have confirmed that ZmPRR37 is responsive to photoperiod and acts as a suppressor of maize flowering time under LD conditions (Figure 1a,d). Our findings align with previous research in SD rice, sorghum and soybean, which also demonstrated the involvement of photoperiodic regulation in flowering (Koo et al., 2013; Murphy et al., 2011; Wang et al., 2020). However, the homologous genes of PRR37 in LD plants of Arabidopsis, grass barley and hexaploidy wheat play opposing roles (Beales et al., 2007; Nakamichi et al., 2007; Turner et al., 2005; Yamamoto et al., 2003). Specifically, PRR37 functions as an activator of flowering time under LD conditions in these LD plants. This discrepancy can be attributed to the genetic structural variations that occurred during the evolutionary process of flowering.

In rice, OsPRR37 inhibits the expression of Hd3a, resulting in delayed flowering under LD conditions (Koo et al., 2013). Similarly, in sorghum, SbPRR37 delays flowering under LD conditions by repressing the expression of flowering activators, namely SbEhd1, SbFT and SbZCN8 (Murphy et al., 2011). In soybean, GmPRR37 downregulates the expression of GmFT2a and GmFT5a while upregulating the expression of GmFT1a, thereby delaying flowering under LD conditions (Wang et al., 2020). In the present study, we identified that ZmPRR37 delays maize flowering by directly binding to the promoters of two NF‐Y family genes, ZmNF‐YA3 and ZmNF‐YC2, leading to the repression of their expression. The involvement of ZmNF‐YA3 and ZmNF‐YC2, flowering activators, in photoperiodic flowering regulation under LD conditions has been previously established (Su et al., 2018, 2021).

ZmPRR37 enhances salt tolerance through the ABA‐related pathway

PRRs have been recognized as key regulators in stress responsiveness in other species. OsPRR73 was found to be rapidly induced by NaCl treatment, and Osprr73 null mutants displayed hypersensitivity to sodium ion stress (Wei et al., 2021). In Arabidopsis, the triple mutant PRR5/7/9 exhibited increased salt stress tolerance, with an unclear mechanism (Nakamichi et al., 2009). As far as my knowledge, there have been no reports of PRR37 regulating salt stress response in other cereal crops. In our study, we discovered that ZmPRR37 positively regulates salt stress tolerance through the ABA‐related pathway in maize.

ZmPRR37‐OE lines strengthen salt stress tolerance with high ABA concentration. The salt‐sensitivity phenotype of ZmPRR37‐CR was rescued by exogenous ABA application. RNA‐seq analysis revealed that the expression of Zm00001d008397, a gene involved in the regulation of ABA biosynthetic process, was higher in WT compared to ZmPRR37‐CR (Figure 5e). Furthermore, RT‐qPCR analysis demonstrated a significant upregulation of Zm00001d008397 expression in ZmPRR37‐OE under salt conditions (Figure 5f). The promoter activity of Zm00001d008397 was not affected by ZmPRR37, suggesting that ZmPRR37 may participate in the ABA synthesis pathway by indirectly regulating the expression of Zm00001d008397. Besides, we identified 22 other DEGs related to ABA‐response or ABA signalling pathway, without ZmPRR37 binding peaks (Table S6).

We have discovered eight stress‐/ABA‐response DEGs with ZmPRR37 binding peaks. ZmPRR37 directly inhibits the promoter activity of these 8 genes. Four out of 8 genes, namely ZmAIRP2, ZmHSF30, ZmDhn1 and ZmPSII‐6 also respond to ABA. ZmDhn1 showed highly responsiveness to salt and ABA. Subsequent investigations revealed that zmdhn1‐2 strengthened maize salt stress tolerance and decreased ABA sensitivity, indicating its involvement in the ABA pathway. Previous studies have also shown that ZmDhn1 exhibits responses to salt and ABA (Kizis and Pagès, 2002; Nylander et al., 2001; Vendramin et al., 2020; Yang et al., 2012). Given that the promoter region of ZmDhn1 contains ABA‐responsive elements, it is expected that ZmDhn1 could respond to ABA. ZmDhn1 negatively regulates salt stress, consistent with a previous study reporting salt sensitivity in transgenic Arabidopsis lines overexpressing Caragana intermedia DHN1 (CiDHN1) (Wan et al., 2016). However, homologous of Dhn1 in rice and wheat play opposing roles. Overexpression of DHN in rice and wheat not only enhances salt stress tolerance but also improves drought stress tolerance (Habib et al., 2022; Kumar et al., 2014). This indicated that the mechanisms underlying DHN response to stress can vary among different species. In conclusion, our results suggest that ZmPRR37 enhances salt tolerance by directly repressing the expression of ZmDhn1.

Previous studies have reported that homologous genes of ZmAIRP2, ZmHSF30 and ZmPSII‐6 in other species also respond to salt and ABA treatments (Huang et al., 2016; Oh et al., 2017; Wang et al., 2021; Zang et al., 2019). In our study, we observed changes in the expression of ZmAIRP2, ZmPSII‐6 and ZmHSF30 in response to salt or ABA. Nevertheless, the functional roles of these target genes, regulated by ZmPRR37, in salt and ABA response have not been demonstrated through transgenic plants or mutants in maize. Further experiments are necessary to elucidate their specific contributions to salt stress and ABA response. These findings suggest that ZmPRR37 may participate in salt stress response through the ABA pathway, both directly and indirectly.

ZmELF6 directly binds to the promoter of ZmPRR37 to repress flowering in maize

Flower development involves intricate transcriptional regulation, and C2H2‐ZFPs represent the largest group of DNA‐binding domains in eukaryotic cells. C2H2‐ZFPs have the capability to interact with DNA, RNA and proteins, enabling them to perform diverse biological functions (Sommer et al., 1992). In Arabidopsis, C2H2‐ZFPs such as REF6, ELF6, CZS, SUF4 and LATE have been implicated in flowering induction. Loss‐of‐function mutations of these genes lead to delayed or early flowering (Kim et al., 2006; Krichevsky et al., 2007; Noh et al., 2004; Weingartner et al., 2011). For instance, ELF6 acts as a repressor in the photoperiodic pathway and loss of ELF6 function results in early flowering. In our study, zmelf6 Ds insertion mutant led to early flowering under LD conditions. Association analysis revealed that SNP2071‐C/T lying in ZmPRR37's promoter exhibited the strongest association with flowering time. EMSA and dual‐luc assays revealed that SNP2071‐C/T affected the binding of ZmELF6 to the promoter of ZmPRR37. Collectively, ZmELF6 functions as a repressor of the PRR37‐dependent photoperiodic flowering pathway. Moreover, we made an intriguing discovery that ELF6 in maize responds to salt stress, a phenomenon not yet reported in other species. Our study revealed that ZmELF6 acted as a negative regulator in salt stress. The expression level of ZmPRR37 was lower in zmelf6 than that in WT (Figure 7g), while the expression levels of those eight ZmPRR37 regulated salt‐stress‐responsive‐genes were higher in zmelf6 than those in WT (Figure S6a). After conducting an analysis, we speculate that ZmELF6 potentially acts as a negative regulator of salt stress due to the presence of a specific motif, motif2, within the ZmELF6 protein (Figure S6b). C2H2 genes, such as GhDi19‐3, GhDi19‐4 and GmDi19‐5, which share this motif2, also demonstrate negative regulation of salt stress (Feng et al., 2015; Zhao et al., 2022). While C2H2 genes that positively regulate salt stress response do not contain motif2. Evolutionary analysis reveals that ZmELF6 is most related to genes that negatively regulate salt tolerance, whereas it exhibits a more distant relationship with salt‐tolerant genes such as TaZNF, OsZFP213 and IbZFP1 (Figure S6c) (Ma et al., 2016; Wang et al., 2016; Zhang et al., 2018). It is possible that ZmELF6 is involved in salt stress not only through the ZmPRR37 pathway but also through an unknown regulatory pathway. It is worth noting that many genes involved in plant flowering time regulation also exhibit responses to environmental stress (Chong et al., 2022; Gol et al., 2021; Liu et al., 2013; Nakamichi et al., 2016). Therefore, more research is needed to elucidate the relationship between plant flowering time regulation and stress response.

In summary, our findings demonstrate that ZmPRR37 acts as a repressor of flowering under LD conditions by modulating the expression of ZmNF‐YA3 and ZmNF‐YC2. The SNP2071‐C/T variant affects the binding ability of ZmELF6 to the promoter of ZmPRR37. ZmELF6 functions as a repressor in the ZmPRR37‐dependent photoperiodic flowering pathway. Furthermore, ZmPRR37 plays a pivotal role in enhancing maize salt tolerance through ABA‐related pathway, particularly by directly suppressing ZmDhn1, a negative regulator of maize salt tolerance. Additionally, we found that ELF6 responds to salt stress, which has not been reported in other species yet. Based on these findings, we have proposed a novel pathway involving ZmPRR37 in regulating photoperiodic flowering and responding to salt stress in maize (Figure S7). Further efforts to identify superior haplotypes and uncover additional underlying pathways would undoubtedly contribute to the breeding of stress‐tolerant maize varieties.

Experimental procedures

Plant materials

Maize inbred line W22 carrying an Ac factor was used as a donor, while maize inbred line K17 derived from Tangsipingtou, a local Chinese germplasm, was used as a receptor. Pollen from W22 was used to cross‐pollinate K17, resulting in the generation of F₁ seeds. Seeds exhibiting purple dots from each generation were selected and backcrossed (BC) with K17. Seeds displaying purple dots from BC₄F₁ were then selected and test‐crossed (TC) with K17 for two additional generations, resulting in the production of TC₂F₁ seeds. Colourless seeds from TC₂F₁ were self‐pollinated, and mutants with stable Ds insertions were obtained. The Ds mutant library was constructed, and the Ds insertion sites were analysed using the Ac/Ds transposon technique and Transposable Element Amplicon Sequencing (TEA‐Seq) (Lyu et al., 2021).

In this study, through the Ac/Ds transposon technique and TEA‐Seq, we identified a Ds insertion mutant that exhibited a significant correlation with DTA. The Ds was inserted 26 639 bp downstream of the ATG start codon of the pseudo‐response regulator protein‐coding gene ZmPRR37 (designated as zmprr37). The Ds insertion was confirmed by PCR (Figure S1a). The ZmPRR37‐CRISPR/Cas9 (ZmPRR37‐CR) mutants were generated using the CRISPR/Cas9 technology (Xing et al., 2014), following the selection of guide RNAs (Table S1) through the online tool CRISPR‐PLANT (http://www.genome.arizona.edu/crispr/CRISPRsearch.html). The PCR fragment was amplified from pCBC‐MT1T2 and cloned into the pBUE411 vector. The coding sequence of ZmPRR37 was cloned into the pCAMBIA1304 vector. Subsequently, recombinant plasmids pBUE411‐PRR37 and pCAMBIA1304‐PRR37 were separately introduced into the maize inbred line ‘Yu808’ using Agrobacterium tumefaciens‐mediated transformation, resulting in the generation of ZmPRR37‐CR mutants and ZmPRR37 overexpression (ZmPRR37‐OE) lines from the T₂ plants.

Furthermore, through the Ac/Ds transposon technique and TEA‐Seq, we identified a mutant with a Ds inserted 4077 bp downstream of the ATG of ZmELF6 (designated as zmelf6) (Figure S5a). The zmdhn1 knockout mutants were generated using the CRISPR/Cas9 technology as previously described. The B104 and maize genetic transformation were conducted and the seeds of zmdhn1 and WT were provided by Beijing Bomeixingao Technology Company.

Plant growth, abiotic stress treatments

For the investigation of flowering time, the wild type (WT), ZmPRR37‐CR mutants and ZmPRR37‐OE lines were cultivated in the field under LD (Zhengzhou, Henan province, 34°48′N, 113°39′E, 14.3 h) and SD (Sanya, Hainan province, 18°37′N, 109°30′E, 10.3 h) conditions. For stress treatments, seedlings were grown in the growth chambers (15 h light/9 h dark, 28 °C day/22 °C night, 40% relative humidity) under well‐watered conditions. At the V3 stage, seedlings were subjected to various stress treatments under LD conditions. For drought treatment, water was withheld (Zhang et al., 2020). For salt treatment, seedlings were watered with a 250 mM NaCl solution. For Hormone treatments, 350 μM ethephon or 100 μM ABA was sprayed on leaves during salt treatment (Qiu et al., 2022; Zhu et al., 2020). Photos were taken at the indicated times. Leaf tissues were collected, immediately frozen in liquid nitrogen and subsequently stored at −80 °C.

ABA content of leaf samples was measured using an enzyme‐linked immunosorbent assay (ELISA) under normal and salt stress conditions, as previously described (Yang et al., 2001). Three replicates were performed and the data were presented as mean ± SD. Sterilized WT and transgenic maize seeds were germinated in water (as control) or in water supplemented with 200 mM NaCl or 10 μM ABA. The root length was measured at the indicated time.

Tassels, originating from the shoot apical meristem, and ears, developing from the axillary meristem, were dissected and examined under a stereomicroscope (Stemi508). Tassel samples were observed from the V6‐V14 stages, while ear samples were observed from the V8‐V14 stages for ZmPRR37 under normal and salt stress conditions in LD. For ZmELF6, tassel samples were observed from the V6‐V12 stages, and ear samples were observed from the V9‐V15 stages under normal and salt stress conditions in LD.

RNA sequencing and informatics analyses

For RNA sequencing analysis, seedlings at the 3‐leaf stage of WT and ZmPRR37‐CR were subjected to treatment with either 250 mM NaCl or water (control). Leaf tissues were collected at the V5 stage from both the control and treatment groups. Each sample was obtained from five randomly selected plants. The construction of cDNA libraries and high‐throughput sequencing were performed by Berry Genomics Corporation (Beijing, China).

The raw data underwent adapter and quality trimming using Trimmomatic. The resulting clean reads were aligned to the maize B73_V4_genome using TopHat2 (Kim et al., 2013). The reads mapped to the genome were assembled, and transcripts were reconstructed using Cufflinks (Trapnell et al., 2012). Cuffdiff was employed to generate expression data. The fragments per kilobase of transcript per million fragments mapped (FPKM) value was utilized to estimate the gene expression levels, FPKM >1 serving as the minimum expression standard. Genes exhibiting |log₂ fold‐change| > 1 and a q‐value <0.01 were considered significantly differentially expressed genes (DEGs). The DEGs were subsequently subjected to enrichment analysis of Gene Ontology (GO).

RNA isolation and RT‐qPCR analysis

Total RNA was extracted from the collected samples using Trizol (Invitrogen). A quantity of 2 μg of total RNA was reverse transcribed using the Hifair® III 1st Strand cDNA Synthesis Kit. Real‐time quantitative PCR (RT‐qPCR) was employed to quantify the expression of selected genes, utilizing the Hieff™ qPCR SYBR® Green Master Mix (YEASEN). The relative gene expression was calculated using the 2^−ΔΔCt method, with the transcript level of the ZmGAPDH gene (NM_001111943.1) used as the internal control. The primer sequences utilized in the RT‐qPCR analysis can be found in Table S1.

Association analysis

The genomic sequences of ZmPRR37, spanning 3 kb upstream of the transcription start site (TSS) to 1 kb downstream of the transcription termination site (TTS), were extracted from a maize association panel consisting of 447 diverse maize inbred lines. This candidate gene‐based approach was employed for the association analysis. The association population was phenotypically evaluated for DTA under both LD conditions in Zhengzhou and SD conditions in Sanya. To assess the association between genetic variants and DTA, the Fixed and random model Circulating Probability Unification (FarmCPU) method was applied, utilizing a Generalized Linear Model (GLM) framework (Liu et al., 2016). Bonferroni adjustment was performed to determine the significance threshold (P < 3.2 × 10⁻⁴) for identifying statistically significant associations.

Subcellular localization

The coding region of ZmPRR37 was cloned into the pCAMBIA1300‐GFP vector, downstream of the 35S promoter. Subsequently, the recombined plasmid or empty vector was separately introduced into Nicotiana benthamiana (N. benthamiana) leaves using Agrobacterium strain GV3101. The empty vector served as a control. Following a 2‐day incubation period in the dark, the GFP fluorescence was visualized using a confocal microscope (Zeiss LSM710). The fluorescence was excited at a wavelength of 488 nm and detected using a bandpass filter set at 505–530 nm.

Transcriptional activation assays

The transcriptional activation assay in yeast and the Gal4/UAS system was conducted following previously described methods (Su et al., 2021; Tiwari et al., 2001). The yeast strain AH109 (Stratagene), which carries the HIS3/ADE2/MEL1/lacZ reporter genes, was employed to assess the activation ability. The coding sequence of ZmPRR37 was cloned into a pGBKT7 vector. Subsequently, pGBKT7‐ZmPRR37 (PRR37), pGBKT7‐GAL4 (GAL4, positive control) or the empty vector pGBKT7 (BD, negative control) were individually transformed into AH109 competent cells. The yeast cells were grown on selective media SD/‐Trp, SD/‐Trp/‐His, or SD/‐Trp/‐His/‐Ade for 3–5 days at 28 °C to assess the transcriptional activation of ZmPRR37.

In the effector assays, the Gal4 DNA‐binding domain (DBD)‐tagged ZmPRR37, LexA DBD‐tagged VP16 and Gal4 DBD were employed as effectors. The reporter construct contained the luciferase reporter gene (LUC) as indicated. The activation ability assay was conducted in N. benthamiana leaves. Protoplasts were prepared from cells after 5 days of subculture. The cell walls were digested at 25 °C for 2 h using a solution containing 1% (w/v) cellulase Onozuka R‐10 (Serva), 0.5% (w/v), Macerozyme RS (Serva), 0.1% (w/v) pectinase (Sigma) and 0.25 M mannitol. The reporter and effector constructs or mock constructs were then introduced into the isolated protoplasts using the polyethylene glycol method. Luciferase activity was measured using the Dual‐Luciferase Reporter Assay System (Promega). The data presented represent the mean ± SD of three biological replicates.

DAP‐Seq experiment and data analysis

The DAP‐Seq assay was conducted following previously described methods (O'Malley et al., 2016). A genomic DNA (gDNA) library was constructed using the NEB Next® DNA Library Prep Master Mix set for Illumina kit (NEB #E6040S). In brief, gDNA from the Yu808 strain at the V6 stage was extracted, and 5–10 μL gDNA was fragmented to an average size of 200 bp. The DNA fragments were then subjected to end repair, A‐tailing and adapter ligation. The coding sequence of ZmPRR37 was cloned into the pFN19K HaloTag® T7 SP6 Flexi® vector. The Halotag‐ZmPRR37 fusion protein was expressed in a wheat germ master mix and purified using Magen HaloTag beads (Promega). The purified Halotag‐ZmPRR37 protein and the gDNA library were co‐incubated, followed by washing and elution. The DAP libraries were sequenced using the Illumina platform, generating 150 bp long paired‐end reads. Equal amounts of DAP libraries were used for real‐time quantitative PCR (DAP‐qPCR) using the appropriate DNA primers (Table S1).

The raw reads were subjected to adapter clipping and quality trimming using Trimmomatic. The resulting clean reads were mapped to the maize B73_V4_genome using Bowtie2 version 2.3.4.3 (Langmead and Salzberg, 2012). Peak calling was performed using MACS peak caller version 2.2.7.1 (Zhang et al., 2008). The targeted genes were identified if there were peaks within 3 kb upstream or downstream of each gene.

Dual‐luciferase assay

The promoters (~2.5 kb) of the target genes were cloned into the pGreenII0800‐LUC vector, generating pPromoter:LUC plasmids as reporters. The REN gene, driven by the 35S promoter, was used as a normalization control. The coding region of ZmPRR37 was cloned into the pCAMBIA1300 vector as an effector. The effector and reporter constructs were transformed into GV3101 and co‐transfected into N. benthamiana leaves. After 3 days of infiltration, total proteins were extracted for analysis. The luciferase signal was detected using the Dual‐Luciferase (dual‐luc) Reporter Assay System (Promega) following the manufacturer's instructions. The relative LUC activity was calculated by normalizing the LUC activity to the REN activity.

Electrophoretic mobility shift assay

The coding region of ZmPRR37 was cloned into the pGEX4T‐1 vector. pGEX4T‐1‐ZmPRR37 was transformed into the E. coli strain BL21 for the purification of ZmPRR37‐GST protein using corresponding affinity beads. DNA probes were synthesized and labelled according to the instructions provided in the DIG Gel Shift Kit (Roche). A total of 0.8 ng of DIG‐labelled probes were mixed with 25–75 ng of protein in a 20 μL binding buffer (20 mM Hepes, pH 7.6, 1 mM EDTA, 10 mM (NH4)₂SO₄, 1 mM DTT, 0.2% (w/v) Tween 20, 30 mM KCl, 1 μL poly(d(I‐C)), 0.1 μL poly L‐lysine). As a competition control, an 80‐fold excess of unlabeled probe was added to the reactions. The binding reactions were incubated at 25 °C for 15 min and then resolved on 6%–8% native polyacrylamide gels in 0.5× TBE buffer. Polyacrylamide gel electrophoresis was followed by blotting and chemiluminescent detection.

Author contributions

YC and LK designed the research. HS, LC, ZR, WS, BZ, SM, CS, DZ, ZL, HZ, WY, YL, LZ, YY and ZW performed the experiments. HS, LC and ZR analysed the data. HS, YZ, LC and LK wrote the manuscript. HS, LC and ZR contributed equally to this work.

Conflict of interest statement

The authors declare that they have no conflict of interest.

Supporting information

Figure S1 Character Analysis of ZmPRR37.

Figure S2 ZmPRR37 response to salt stress in maize under LD conditions.

Figure S3 DAP‐Seq and RNA‐Seq Analysis of ZmPRR37.

Figure S4 Target genes of ZmPRR37 in response to salt stress.

Figure S5 Zmelf6 mutants promote flowering and enhance salt tolerance in maize.

Figure S6 Analysis of C2H2 genes involved in salt stress response.

Figure S7 A hypothetical model illustrating the regulation of flowering time and salt stress response by ZmPRR37 in Maize under LD conditions.

Table S1 Primers used in this study.

Table S2 DAP‐seq results for ZmPRR37, first replicate. DAP‐seq results for ZmPRR37, second replicate.

Table S3 The summary of reads analysis from RNA‐Seq data.

Table S4 Flowering time‐related target genes and their functions.

Table S5 Stress response related target genes and their functions.

Table S6 DEGs related to ABA without ZmPRR37 binding peaks.