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Heat stress response factor ZmHSF10 positively regulates heat tolerance in maize

BMC Plant Biology volume 25, Article number: 1473 (2025) Cite this article

Abstract

High temperature stress poses a significant threat to the normal growth of maize seedlings, and key heat-resistant gene mining is the molecular basis for breeding new heat-resistant maize varieties. Through transcriptome sequencing of heat-tolerant hybrid ZD819 and its parental lines (ZD819-F, ZD819-M) under high-temperature stress, we identified 12 HSF (Heat shock transcription factors, HSFs) transcription factors from 12,442 differentially expressed genes. The results indicate that the maize hybrid ZD819 has stronger heat tolerance compared to its parent varieties (ZD819-F, ZD819-M). Transcriptome data identified 12 HSFs transcription factors, among which ZmHSF10 had the highest differential expression fold of 1279.40 before and after high-temperature treatment. The heat tolerance function of ZmHSF10 was studied by creating Arabidopsis thaliana materials overexpressing ZmHSF10 and obtaining ZmHSF10 silenced maize materials using VIGS technology. Genetic experiments have shown that overexpression of ZmHSF10 can stabilize the cell membrane stability of Arabidopsis plants under high temperature stress and improve their survival rate under high temperature treatment. Reducing the expression level of ZmHSF10 leads to a decrease in chlorophyll content, poor cell membrane stability, and lower relative water content in maize leaves under high temperature stress. These results preliminarily demonstrate that ZmHSF10 plays an important role in regulating heat tolerance in plants, providing genetic resources for enhancing heat tolerance in maize seedlings.

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Maize is an important food crop and plays an important role in ensuring food security [1]. In recent years, as global temperatures have risen, high temperatures and other extreme weather have occurred frequently, posing a serious threat to the safe and healthy production of maize. maize acts as a terrestrial plant that can adapt to the external environment by regulating physiological and biochemical activities in the body in order to maintain its growth and development. However, the mechanisms of action by which maize responds and adapts to high temperatures are not sufficiently clear, and more data are needed to explore the mechanisms of heat tolerance in maize.

Crop adaptation to high temperature stress is the result of the joint action of multiple regulatory pathways in plants. As the underlying code of life activities, gene expression is one of the important means to analyze the mechanism of high temperature tolerance in maize. RNA-seq is a sequencing analysis using high-throughput sequencing technology that reflects the level of gene expression and provides a means of obtaining simultaneous high and low levels of gene expression at the genome-wide level under different treatments. Increasingly, RNA-seq has been utilized to obtain the expression levels of genes in plants in response to different external stimuli, and to analyze the underlying logic of physiological activities of plants in response to external environmental stimuli. Cao et al. used transcriptomic data to mine multiple response pathways in plants to cope with drought stress, providing new insights into the molecular mechanisms of drought tolerance and new targets for mining maize drought resistance genetic resources [2]. Xue et al. used transcriptome data to identify ZmHSP18 from the ZmHSP20s gene family in response to heat stress and found that deletion of the ZmHSP18 gene enhances maize sensitivity to heat stress [3]. Cao et al. identified 15 drought-responsive ZmDOFs genes by RNA-seq data analysis and found that overexpression of ZmDOF22 enhanced drought tolerance in transgenic plants [4]. In conclusion, RNA-seq is a key tool for efficient mining of resistance gene resources.

Heat shock transcription factors (HSF) are widely found in eukaryotes and are one of the important families of transcription factors in plant resistance to heat stress [5]. Since the first plant HSF gene, LpHSF, was cloned [6], more and more HSFs genes have been identified in multiple species, with 25 HSFs genes identified in rice [7], 29 in maize [8], 18 in asparagus [9], and 78 in wheat [10]. HSF mostly binds to the heat stress element HSE as a homotrimer and regulates the expression of downstream genes [11]. Under normal growth conditions, HSF protein forms a complex with HSP90/HSP70 molecular chaperones and is in an inactive state; After signal transduction, the HSF protein separates from its partner protein and binds to the downstream promoter region to perform its function [12]. HSP proteins can prevent protein misfolding under heat stress and enhance plant tolerance to high temperature, and it was found that HSF can regulate HSP expression [13]. High-temperature-induced expression of AtHSFA2 enhances heat tolerance in plants by promoting the expression of genes such as APXs and HSPs [14]. Heterologous overexpression of the mountain grape HSF Gene VaHSFC1 promotes the massive expression of AtHSP21, AtHSP22, AtHSP26.5 and AtHSP70b under high temperature stress and enhances heat tolerance in transgenic Arabidopsis thaliana [15]. It was found that at normal temperature, SUMO-modified TaHsfA1 binds to TaHSPs and its transcriptional activity is inhibited; Upon heat stress, TaHsfA1 dissociates from the HSP protein complex and binds to the promoters of downstream target genes, activating the heat stress response pathway [16]. The role of HSFs in heat tolerance in plants has been extensively studied and validated.

In this study, the heat-tolerant maize hybrid ZD819 and its male parent material (ZD819-F) and female parent material (ZD819-M) were used as materials for maize seedling high-temperature treatment, and the HSF gene ZmHSF10, which responds to high-temperature stress, was identified from the RNA-seq data. Functional validation of the gene showed that overexpression of ZmHSF10 enhanced the heat tolerance of Arabidopsis thaliana, and lowering the expression of ZmHSF10 reduced the heat tolerance of the maize plants. ZmHSF10 expression reduced the heat tolerance of maize plants. This study provides genetic resources for breeding heat-tolerant maize and new ideas for mining key heat-tolerant genes.

Materials and methods

High-temperature treatment of maize seedlings and analysis of RNA-seq data

Seeds of the maize hybrid ZD819 and its parental (ZD819-F) and maternal (ZD819-M) materials were sown in soil and placed in an incubator for cultivation (25 ~ 28 °C, 16 h light/8 h dark). When it reaches the three leaf stage, it is subjected to a high temperature treatment of 37 ° C throughout the day, and samples were taken after 3 d of treatment for RNA-seq analysis and gene isolation. Determination of RNA-seq data and subsequent data analysis were performed by Guangdong Kidio Biological Co (https://www.omicshare.com/). Three maize seedlings were taken at each time and three replications were performed.

Acquisition of transgenic plants and identification of heat resistance

Specific primers were designed according to the B73 reference genome (Supplementary Table S1) ZmHSF10 open reading frame containing the cleavage site was amplified and the pEGC5941- ZmHSF10 fusion vector was constructed for obtaining ZmHSF10 overexpression plants. The pEGC5941-ZmHSF10 overexpression fusion vector constructed above was used to obtain transgenic Arabidopsis plants through inflorescence infection, and the T3 generation plants were used for high-temperature treatment. The high-temperature treatment was carried out in a light incubator (37 °C, 16 h light/8 h dark), and samples were taken after 3 d of treatment for the determination of relevant indexes.

Acquisition of ZmHSF10 VIGS Plants and Characterization of Heat Tolerance

A 167 bp specific fragment of the ORF region of ZmHSF10 was selected for primer design (Supplementary Table S1). Virus-induced gene silencing (VIGS) was used to silence ZmHSF10. BMV-GFP was the vector for transient silencing of gene expression. The ZmHSF10 gene was isolated from maize B73-329 and loaded into the BMV-GFP vector to obtain a BMV- ZmHSF10 fusion. BMV- ZmHSF10 and empty BMV-GFP were then introduced into Agrobacterium rhizogenic strain GV3101. Agrobacterium liquid junctions of BMV- ZmHSF10 and empty BMV-GFP were seeded onto the leaves of Benjamin’s tobacco plants, respectively, 24 h after infiltration of B73-329 maize seeds, as described previously. After three days, tobacco leaves were taken for virus extraction according to the previous method [17]. Extracted BMV- ZmHSF10 and BMV-GFP togaviruses’ were infected into the second leaf of B73-329 maize and incubated in a Light incubator at 20°C for 6 d. A Light-transmitting plastic cover was placed over the leaves to keep them moist, and the cover was subsequently removed and placed in a Light incubator at 22°C. BMV-GFP was used as a wild-type control, Hereafter referred to as WT. VIGS silencing at 7 d after viral infection was performed. VIGS silent material was subjected to 37 ° C for 24 h after virus infection for 7 days, and then samples were taken for the determination of relevant indicators.

Bioinformatics analysis of ZmHSFs

The RNA data analysis, differential expression gene analysis, VEN plot display of differential expression genes, GO enrichment analysis, KEGG pathway analysis, and statistical analysis of the number of transcription factors in differential genes of ZD819 and its parents before and after processing were all analysis and drawn by the omicsmart system of Gene Denovo Corporation (https://www.omicsmart.com). The differential expression gene screening tool is DESeq2. |FPKM values with a fold change| >1.5 and FDR < 0.05. PRdos (https://prdos.hgc.jp/cgi-bin/top.cgi) website was used to sequence the disordered sequence of ZmHSF10 protein, with parameters set to the website’s default values.

Design specific primers (Supplementary Table S1) to perform fluorescence quantitative expression analysis on the overexpression level of ZmHSF10 in Arabidopsis and the differentially expressed ZmHSFs in ZD819 and its parental samples before and after high-temperature treatment. Specifically, total RNA was extracted from the sample using Trizol and reverse transcribed into the first strand of cDNA as a template. Refer to Hieff ® Perform experiments using qPCR SYBR Green Master Mix (No Rox) instructions. Calculate the relative expression level of genes using the 2−∆∆ Ct method. Each sample is repeated 3 times.

Measurement of heat resistance related indexes

RWC (Relative water content, RWC) was calculated using the method of Parida et al. [18]. Leaf relative conductivity was determined with reference to the method of Zhang [19]. Chlorophyll was extracted from the leaves using 95% ethanol and the chlorophyll content was quantified by UV spectrophotometer [20].

Data processing and statistical analysis

Data were processed and graphed using Microsoft Excel 2016 software, between groups were evaluated using Student’s t-test. Statistical significance was set at P < 0.05, and all data were expressed as mean ± standard error.

Results

Maize hybrid ZD819 is more heat tolerant than its parents

In order to analyze the mechanism of heat tolerance in maize, the heat-tolerant maize inbred line ZD819 and its parental inbred lines ZD819-F, ZD819-M were used as experimental materials for high-temperature treatment. Before high-temperature treatment, the maize inbred line ZD819 and its parental three line had good growth, and the relative conductivity was at a low level (Fig. 1A). The level of chlorophyll content can reflect the strength of photosynthetic ability of the crop. The chlorophyll content of ZD819 was significantly higher than that of its parents under normal conditions, The chlorophyll content of ZD819 was 16.40% and 29.41% higher than that of ZD819-F and ZD819-M, respectively (Fig. 1C). After three days of high temperature treatment, the chlorophyll content of all three lines decreased, but the chlorophyll content of ZD819 was still higher than that of its parents (Fig. 1C). After high-temperature treatment, the plants of three line showed severe leaf curling,, but the growth of ZD819 was slightly better than that of its parents (Fig. 1A). The ion permeability of ZD819 was significantly lower than that of ZD819-F and ZD819-M (Fig. 1B). The results showed that ZD819 has higher high temperature resistance relative to its parents, can be used as a material to explore the heat resistance mechanism of maize.

Fig. 1
figure 1

Phenotypic analysis of ZD819, ZD819-F, and ZD891-M under high temperature stress. A: Phenotypic analysis of ZD819, ZD819-F, and ZD891-M under high temperature stress. B: Conductivity analysis of ZD819, ZD819-F, and ZD891-M before and after high-temperature treatment. n=30 C: Chl content analysis of ZD819, ZD819-F, and ZD891-M before and after high-temperature treatment n =30. ZD819 represents maize hybrid ZD819; ZD819-F represents the male parent material of maize hybrid ZD819; ZD819-M represents the parent material of maize hybrid ZD819. HT 3 d represents 3 days of high temperature stress treatment. Data are means ± SD from three biological replicates. **P < 0.01 by Student’s t-test

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Analysis of RNA-seq sequencing results of maize hybrid ZD819 and its parents under high temperature stress

In order to analyze the heat tolerance mechanism of maize inbred line ZD819, RNA-seq sequencing was performed. The results showed that ZD819-M, ZD819, ZD819-F had 18,806, 17,195 and 19,533 differential expressed genes were obtained after high-temperature treatment compared with normal growth conditions. Among them, there were 9611 up-regulated differential expressed genes and 9195 down-regulated differential expressed genes in ZD819-M; 8594 up-regulated differential genes and 9195 down-regulated expressed differential expressed genes in ZD819; and 8068 up-regulated differential expressed genes and 11,465 down-regulated differential expressed genes in ZD819-F (Fig. 2A). The Venn plots showed that the three Line had 11,124 differential expressed genes, which accounted for 59.15%, 64.69%, and 56.95% of the total number of differentials expressed genes in ZD819-M, ZD819, and ZD819-F, respectively (Fig. 2B). Interestingly, we identified 1318 differentially expressed genes unique to ZD891, which may have specific functions in heat stress(Fig. 2B).

Fig. 2
figure 2

Differential gene analysis of ZD819, ZD819-F, and ZD891-M under high temperature stress. A: Statistical analysis of the total number of differentially expressed genes between ZD819, ZD819-F, and ZD891-M before and after high temperature stress treatment. B: Differential gene Ven maps of ZD819, ZD819-F, and ZD891-M before and after high temperature stress treatment. ZD819-M-CK-vs-ZD819-M-HT represents the comparison of differentially expressed genes in ZD819 maternal material before and after high-temperature treatment. ZD819 CK vs ZD819 HT represents the comparison of differentially expressed genes in ZD819 material before and after high-temperature treatment. ZD819-F-CK-vs-ZD819-F-HT represents the comparison of differentially expressed genes in ZD819 paternal material before and after high-temperature treatment

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GO and KEGG analysis of differentially expressed genes

Combined GO and KEGG analysis of 11,124 differentially expressed genes shared by ZD819, ZD819-F, and ZD891-M and 1318 differentially expressed genes unique to ZD819 under high temperature stress, display the GO analysis and KEGG analysis data of the top 30 FDR values. The results showed that high temperature affected the expression of chloroplast, cytoplasmic and plastid-related genes. It also had an effect on the progression related to chloroplast-like vesicles, plastid-like vesicles, chloroplast-like vesicle membranes, photosynthetic membranes, and organelle envelope membranes. Interestingly, high temperature stress can also affect nucleotide binding (Fig. 3A). GO analysis of differentially expressed genes specific to ZD819 showed that they mainly focus on Carbohydrate metabolic process, Organelle sub compartment, Transferase complex, Sect. 61 transposon complex in terms of other aspects (Supplementary Fig. S1A). it suggests that some unique differential genes of ZD819 may endow ZD819 with more or stronger physiological activities to enhance its heat tolerance. Analysis of the KEGG data showed that high temperature stress affected the expression of genes related to Photosynthesis, Metabolic pathways, and carbon fixation by Calvin cycle. There were also effects on ribosome, glyoxylate and dicarboxylate metabolism, glycosylphosphatidylinositol (GPI)-anchor biosynthesis, Protein export and protein processing in endoplasmic reticulum(Fig. 3B). KEGG pathway analysis of differentially expressed genes specific to ZD819 showed that the MPK signaling pathway is involved in the response of ZD819 to high temperatures (Supplementary Fig. S1B; Fig. S2). The results indicate that high temperature stress generally affects the chloroplast, cytoplasmic homeostasis, photosynthesis, and synthesis and metabolism of cytoplasmic substances in maize, thereby affecting plant growth and development.

Fig. 3
figure 3

Combined GO and KEGG analysis of 11,124 differentially expressed genes shared by ZD819, ZD819-F, and ZD891-M and 1318 differentially expressed genes unique to ZD819 under high temperature stress. A: GO Term analysis. B: KEGG Pathay analysis. The image only displays GO enrichment and KEGG analysis data for the top 30 FDR values

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Analysis of the types and number of transcription factors in differentially expressed genes

High temperature can affect nucleotide binding (Fig. 3A), and transcription factors are important participants in the nucleotide binding process. Transcription factors play an important role in the life activities of plants as the transit stations of many signals in the plant, which are involved in regulating the expression of downstream genes. High temperature stress can lead to the production of reactive oxygen species in plants, which can be transmitted to HSFs transcription factors through direct or MPK signaling pathways. Subsequently, HSFs transcription factors enter the nucleus to initiate transcription of HSP and reactive oxygen species scavenging related enzymes [21]. As an important regulator of high temperature stress, HSF plays a crucial role in maize’s response to high temperature stress. A total of 617 transcription factors belonging to 52 transcription factor families were identified, including differentially expressed genes shared by ZD819 and its parents, as well as differentially expressed genes unique to ZD819. (Supplementary. Table S2; Table S3; Table S4; Table S5). The top 20 transcription factor families with the largest number of differentially expressed genes were selected for display. (Fig. 4). It was found that 22 out of 36 ARF (Auxin response factor, ARF) transcription factor genes in maize were differentially expressed before and after high temperature stress. 58.82% (10/17) of CONSTANS-like (CO-like, COL) transcription factor family genes showing differential expression before and after high temperature stress. It is worth noting that 41.38% (12/29) of HSF genes were differentially expressed before and after stress. As a key type of gene under high temperature stress, HSFs expression is often used as an indicator gene for plants under high temperature stress. It is interesting that the differentially expressed HSFs transcription factors after high temperature stress are not unique to ZD819, but are shared by all three maize line, implies that HSFs transcription factors are crucial and universal in maize response to high temperature stress.

Fig. 4
figure 4

Analysis of common differential transcription factors under high temperature stress in ZD819, ZD819-F, and ZD891-M. ZD819 represents maize hybrid ZD819; ZD819-F represents the male parent material of maize hybrid ZD819; ZD819-M represents the parent material of maize hybrid ZD819

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RNA-seq data analysis of ZmHSFs at high temperatures

A total of 12 differentially expressed genes were screened from within the HSF family genes (Supplementary Table S6). Presentation of the transcriptomic data of the screened ZmHSFs showed that the expression of ZmHSF5 was reduced in ZD819, ZD819-F and ZD891-M. Relative to normal growth conditions, the expression of ZmHSF5 decreased by 72.20% in ZD819-F, 81.11% in ZD819-M, and the largest decrease of 89.53% in ZD819 under high temperature treatment (Fig. 5A). The expression of all ZmHSFs except ZmHSF5 showed an increasing trend under high temperature treatment. Among them, ZmHSF10 showed the greatest fold difference in expression before and after high-temperature treatment (Fig. 5A). To verify the reliability of the results, we performed fluorescence quantitative expression analysis on the relevant ZmHSFs. The results showed that the fluorescence quantitative data was consistent with the transcriptome data. After high temperature stress, the expression level of ZmHSF5 showed a significant decrease in all three materials, and the largest decrease was observed in ZD819, at 86.80% (Fig. 5B). Other ZmHSFs showed a significant increase after high-temperature treatment. Moreover, the expression levels of ZmHSF10 and ZmHSF12 in ZD819 significantly increased by 1279.40 and 783.80 times, respectively, after high-temperature treatment. After high-temperature treatment, the expression of ZmHSF10 was 1279.40, 109.91, and 129.71fold higher than that under normal growth conditions in ZD819, ZD819-F, and ZD891-M materials, respectively. The greatest fold difference was observed in ZD819, implying that ZmHSF10 has an important role in the mechanism of ZD819 heat resistance. The results were validated using fluorescence quantification method, and the results were consistent with the trend of transcriptome data (Fig. 5B).

Fig. 5
figure 5

Analysis of ZmHSFs Response to High Temperature Stress. A: Displays the FPKM values of ZmHSFs from the RNA-seq data.B：The relative expression levels of ZmHSFs in ZD819, ZD819-F and ZD819-M before and after high temperature stress. ZD819-M-CK indicates that the female parent material of ZD819 grows normally. ZD819-M-HT represents the high-temperature treatment of the female parent material of ZD819. ZD819-CK represents normal growth of hybrid material ZD819. ZD819-HT indicates that the hybrid material ZD819 has undergone high-temperature treatment. ZD819-F-CK indicates that the male parent material of ZD819 grows normally. ZD819-F-HT represents the high-temperature treatment of the male parent material of ZD819. Data are means ± SD from three biological replicates. ** P< 0.01 by Student’s t-test

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ZmHSF10 improves heat tolerance in transgenic Arabidopsis thaliana

ZmHSF10 had a large fold difference before and after high-temperature treatment, suggesting that ZmHSF10 may have an important role in maize response to high-temperature stress. To investigate the function of ZmHSF10 in high-temperature stress, Arabidopsis plants overexpressing the ZmHSF10 gene were obtained, and OE-1, and OE-2 lines were selected for subsequent experiments (Supplementary Fig. S3). Before the high-temperature treatment, the WT, OE-1 and OE-2 line showed the same growth; after the high-temperature stress treatment, Arabidopsis plants showed wilting, yellow leaves, and some of the leaves even died, and the WT undesirable phenotype was significantly stronger than that of OE-1 and OE-2 (Fig. 6A). Relative conductivity measurements showed that before high temperature treatment, the relative conductivity levels of all lines were relatively low and there was no difference among the three lines; After high temperature treatment, the relative conductivity levels of all lines increased rapidly, but the relative conductivity of OE-1 and OE-2 was significantly lower than WT (Fig. 6B). The survival rate of the wild type after the high-temperature treatment was 42.05%, which was significantly lower than that of the overexpression plants of 67.13% and 65.24%, respectively (Fig. 6C). The results indicated that overexpression of ZmHSF10 could improve the heat tolerance of transgenic plants.

Fig. 6
figure 6

ZmHSF10 enhances heat tolerance in Arabidopsis thaliana. A：Phenotypic analysis of WT and ZmHSF10 overexpression material before and after high-temperature treatment. B：Measurement of ion permeability of WT and ZmHSF10 overexpression materials before and after high temperature stress treatment. C：Determination of survival rate of WT and ZmHSF10 overexpression materials after high temperature stress. 15 phenotypic data were collected for each material under each treatment. HT stands for high temperature stress treatment; 0 d represents before high-temperature treatment; 3 days for high-temperature treatment. Data are means ± SD from three biological replicates. **P < 0.01 by Student’s t-test

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Reduced expression of ZmHSF10 promotes sensitivity to high temperature in maize

In order to verify the mechanism of action of ZmHSF10 in response to high temperature in maize, the expression of ZmHSF10 was reduced in maize plants using VIGS technology, and VIGS line L-1 and L-2 were obtained and subjected to high temperature stress (Supplementary Fig. S4). Before the high-temperature treatment, the growth of WT, L-1 and L-2 line was consistent; After high temperature stress treatment, leaves of all line showed curling and yellowing phenomena, and the curling degree of leaves in L-1 and L-2 line was significantly higher than that in WT line (Fig. 7A). Before the high-temperature treatment, the chlorophyll content of each strain was at a high level, and after the high-temperature treatment, the chlorophyll content of each strain declined rapidly, and the L-1 and L-2 line’ chlorophyll content was significantly lower than that of WT (Fig. 7B). The Ion permeability of each strain was at a low level before high temperature treatment, and after high temperature treatment, the RWC content of L-1 and L-2 line increased rapidly and was significantly higher than the ion permeability of WT after high temperature stress (Fig. 7C). There was no difference in RWC among the line before high temperature treatment, and there was a decrease in RWC among the line after high temperature treatment, which was significantly lower in L-1 and L-2 than in WT (Fig. 7D). We obtained the same experimental results through three repeated experiments, the results showed that L-1 and L-2 line were significantly weaker in high temperature tolerance than WT.

Fig. 7
figure 7

The decrease in expression level of ZmHSF10 leads to a weakening of heat tolerance in maize plants. A: Phenotypic analysis of WT(BMV-GFP), L-1 and L-2 (BMV-ZmHSF10) before and after high-temperature treatment. B: Analysis of chlorophyll content in each strain before and after high-temperature treatment. C: Analysis of ion permeability of each strain before and after high-temperature treatment. D: RWC analysis of each strain before and after high-temperature treatment. Data are means ± SD from three biological replicates. **P < 0.01 by Student’s t-test

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Discussion

Maize as a solid organism, very susceptible to external adverse environmental impact on its own normal development, maize can be adapted to the external environment by changing the body’s physiological and biochemical reactions to maintain its own life activities [22]. High temperatures and other weather extremes lead to damage to the stability of maize cell membranes, triggering enzyme system disorders in the plant, affecting the development of maize, and even leading to the death of maize [23]. To cope with the adverse external environment, a large number of superior genes have been integrated through maize’s own long evolution and artificially accelerated improvement, and resistant maize varieties have been bred, thus increasing maize yields under adversity [24,25,26].

Wang identified the drought stress response gene ZmMYB12 from the maize drought-treated transcriptome, and heterologous overexpression of ZmMYB12 increased drought tolerance in transgenic Arabidopsis thaliana, while lowering the expression of ZmMYB12 significantly enhanced the sensitivity of the plants to drought stress [27]. Cao used maize drought rehydration transcriptome data to mine the key drought resistance gene ZmDOF22 and demonstrated its involvement in ABA signaling to positively regulate drought resistance in maize [4]. At present, the genetic resources for high temperature tolerance in maize are not sufficiently mined, so this paper utilizes the high temperature tolerant maize variety ZD819 and its parental materials for high temperature stress treatment, and the growth of ZD819 after high temperature treatment is superior to that of its parental material, and the membrane stabilization is stronger than that of its parental material. Elevated temperature will seriously inhibit plant photosynthesis and reduce carbohydrate synthesis, while at the same time, respiration will become strong, decomposing and consuming a large amount of organic nutrients [28]. The analysis of differentially expressed genes shared by GO and KEGG revealed that related processes such as chloroplasts, organelle membranes, and photosynthetic organisms were differentially expressed in each strain before and after high temperature stress, indicating a positive response to high temperature stress. We also noticed the presence of MPK signaling pathway members in the differentially expressed genes unique to ZD819 under high temperature stress. When plants face high temperatures, they produce ROS, which directly activate the transcriptional activity of HSFs, activate downstream HSP, and clear enzymes related to reactive oxygen species. Meanwhile, ROS also activates the MPK pathway, activating the downstream HSF transcription factor activity [21]. Cao et al. found that kinase AtMPK3 and phosphatase CPL1 regulate the phosphorylation status of HYL1, affecting its distribution in cells and thereby regulating plant heat tolerance [29]. In this article, the differential expression of MKK3, MKK7 and MPK13 specificity in ZD819 material before and after high-temperature treatment suggests that they play an important role in ZD819’s response to high-temperature stress. The mechanism by which the MPK signaling pathway participates in maize’s response to high temperature stress needs to be clarified and analyzed.

Transcription factors as regulatory switches of downstream gene expression have important roles in plant growth and development [30], among them, heat stress transcription factors are an important class of transcription factors present in eukaryotic organisms that regulate heat stress response [31]. Under high temperature stress, HSFs transcription factors participate in response downstream of MPKs [21]. Among the 12,142 differential genes containing 12 HSFs transcription factors, and these 12 HSFs were co enriched in three maize strains. ZmHSF10 was differentially expressed at a greater fold in different materials after high temperature treatment and was the most differentially expressed in the heat-tolerant ZD819, at 1279.40-fold. The expression profile of a gene under different conditions of treatment may reflect the mechanism of action in which the gene may be involved [32]. The high expression of ZmHSF10 in the heat-tolerant material ZD819 implies that it has an important role in participating in the response to high temperature stress in maize.

Numerous studies have shown that HSF is actively involved in plant response to heat stress, and that the HSF transcription factor AtHSF1d accumulates and enters the nucleus in Arabidopsis under high daytime temperatures, stabilizing the cytochrome-interacting factor PIF4 to adapt to the high temperature [33]. Overexpression of the wheat HSF gene TaHSFA2-10 promotes tolerance to high temperature stress in transgenic Arabidopsis thaliana [34]. With global warming, high temperature stress has become one of the major abiotic stresses limiting plant growth and development. Chloroplasts are the more sensitive organelles to high temperature in plant cells, and under high temperature stress, chloroplast homeostasis is disturbed, resulting in a decrease in chlorophyll content [35, 36]. Chlorophyll content of ZmHSF10-silenced plants was significantly reduced under high temperature stress. The stabilizing effect of the cell membrane plays an important role in protecting the stability of material exchange and homeostasis in cellular tissues and maintaining good metabolic coherence between cells. At the same time, maintaining the stability of the cell membrane can prevent or slow down the loss of cellular contents, thus maintaining cellular life activities. The strength of cell membrane stability is an important indicator of plant heat resistance [37]. Relative conductivity is a direct indicator of cell membrane stability. Under high-temperature stress, the relative conductivity of L-I and L-2 plants was significantly higher than that of WT, and the cell membrane stability was severely damaged, which reduced the heat tolerance of the plants. Zhang found that heat-tolerant plants had higher RWC under high temperature stress [38]. Under high temperature stress, the transpiration of plants is large, and RWC is one of the indicators reflecting the degree of plants subjected to high temperature stress. Tang found that StHsfA3 could improve the tolerance of transgenic potato lines to high-temperature stress, and under high-temperature stress, the transgenic potato lines still retained a high RWC [39]. In this paper, overexpression of ZmHSF10 maintained the RWC of Arabidopsis under high-temperature stress, while the RWC of VIGS plants under high temperature was significantly reduced. The above experimental results demonstrated that overexpression of the ZmHSF10 gene increased the tolerance of transgenic plants to high-temperature stress, and silencing of ZmHSF10 resulted in reduced tolerance to high-temperature stress. Interestingly, we found that overexpression of ZmHSF10 in Arabidopsis plants resulted in a 1.5-fold difference in expression levels, but exhibited consistent phenotypes under high temperature stress (Supplementary Fig. S3; Fig. 6). Ren et al. found that heat shock factor 1 (HSF1) undergoes liquid-liquid phase separation under heat stress, forming transcriptional aggregates and regulating the expression of downstream genes. This process has a concentration dependent threshold [40]. And the formation of transcription aggregates in HSF1 only has a threshold. At the same time, the molecular chaperone HSP70, which targets and activates the expression of HSF1, can negatively regulate the phase separation of HSF1, manifested as HSP70 weakening the liquid-liquid phase separation of HSF1, and even preventing the liquid-solid phase transition of HSF1 during prolonged heat shock, maintaining intracellular protein homeostasis [41]. The PrDOS website(https://prdos.hgc.jp/cgi-bin/top.cgi) prediction indicates that ZmHSF10 contains 7 disordered sequences (Supplementary Fig. S5), which are the key reasons for protein formation of phase separation. It is speculated that ZmHSF10 may form transcriptional aggregates under high temperature stress to participate in the regulation of downstream genes. There are also literature reports HSFs transcription factors actively participate in processes such as phase separation, chromatin regulation, and binding to TATA motifs to regulate downstream gene expression [42]. Although we have demonstrated that ZmHSF10 has a positive effect on maize response to high temperature stress, its specific mechanism of action still needs to be further explored.

Conclusion

The HSFs family of transcription factors is widely involved in the heat response of plants, and in this study, the maize hybrid ZD819 and its parents (ZD819-F) and (ZD819-M) were subjected to heat stress treatment and transcriptome sequencing. By analyzing RNA seq data, we identified the differentially expressed gene ZmHSF10. Under high temperature stress, ZmHSF10 showed significant upregulation in maize inbred line ZD819 and its parental materials. overexpression of ZmHSF10 in Arabidopsis thaliana and high-temperature treatment of silenced material in maize showed that ZmHSF10 plays an important function in improving the heat tolerance of maize. The article lays the foundation for further exploring the mechanism of action of ZmHSF10.

Data availability

The RNA sequencing data generated in this study were archived at the Genome Sequence Archive of the China National Center for Bioinformation (https://www.cncb.ac.cn/) under project PRJCA039198.

Abbreviations

RWC:

Relative water content

BMV:

Brome mosaic virus

VIGS:

Virus-induced gene silencing

GFP:

Green fluorescence protein

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Acknowledgements

Not applicable.

Funding

Science and Technology R & D Program of Henan Province (232301420023) Henan Province Corn Industry Technology System (HARS-02-G1) State Key Laboratory of Maize Bio-Breeding (20221130).

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Author notes

  1. Liru Cao and Guo Li Co-first author.

Authors and Affiliations

  1. The Shennong Laboratory, Henan Academy of Agricultural Sciences, Zhengzhou, Henan, 450002, China

    Liru Cao, Guo Li, Dongling Zhang, Guorui Wang, Yinghui Song, Xin Zhang & Xiaomin Lu

  2. Henan Qiule Seed Industry Technology Co., Ltd, Henan, 450002, Zhengzhou, China

    Linlin Han & Mingbo Yang

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Contributions

Conceptualization, L.C. and G.L.; methodology, D.Z., G.W. and X.L.; validation, H.L.; formal analysis, L.C. and X.L.; investigation, Y.S. and G.L.; writing—original draft preparation, L.C. and X.L.; writing—review and editing, L.C. and X.Z.; visualization, Y.S., M.Y. and G.L.; project administration, X.L. All authors have read and agreed to the published version of the manuscript.

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Cao, L., Li, G., Zhang, D. et al. Heat stress response factor ZmHSF10 positively regulates heat tolerance in maize. BMC Plant Biol 25, 1473 (2025). https://doi.org/10.1186/s12870-025-07400-1

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BMC Plant Biology

ISSN: 1471-2229

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