Next Article in Journal
Effect of A PLC-Based Drinkers for Fattening Pigs on Reducing Drinking Water Consumption, Wastage and Pollution
Previous Article in Journal
Effect of Moringa oleifera Leaf Powder Supplementation on Growth Performance, Digestive Enzyme Activity, Meat Quality, and Cecum Microbiota of Ningdu Yellow Chickens
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Agriculture
Volume 14
Issue 9
10.3390/agriculture14091524
agriculture-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Maize Class C Heat Shock Factor ZmHSF21 Improves the High Temperature Tolerance of Transgenic Arabidopsis

by
Yurong Xie
1,*,† and
Yuhan Ye
2,†
1
Research Institute of Biology and Agriculture, University of Science and Technology Beijing, Beijing 100096, China
2
College of Medical, Veterinary & Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Agriculture 2024, 14(9), 1524; https://doi.org/10.3390/agriculture14091524
Submission received: 9 July 2024 / Revised: 23 August 2024 / Accepted: 3 September 2024 / Published: 4 September 2024
(This article belongs to the Special Issue Breeding and Genetics of Maize)
Browse Figures
Versions Notes

Abstract

:
High temperatures seriously threaten the global yield of maize. The objectives of the present study were to explore the key candidate gene involved in heat shock responses in maize and its potential biological function to heat stress. Here, we identified a Class C heat shock factor, ZmHSF21, from maize leaves and used molecular biological and plant physiological assays to investigate its roles in transgenic Arabidopsis. ZmHSF21 encodes a putative protein of 388 amino acids. We showed that ZmHSF21 was expressed in most tissues of maize with relatively high expression in leaves and silks but rather low in roots and stalks, and its expression level in leaves was significantly up-regulated by heat treatment. We also showed that overexpression of ZmHSF21 in Arabidopsis significantly improved the seed germination frequency and plant survival rate when exposed to heat stress. We demonstrated that, compared with wild-type plants, the activities of peroxidase, superoxide dismutase, and catalase increased while the reactive oxygen species accumulation decreased in ZmHSF21 overexpressors under heat stress conditions. We further demonstrated that ZmHSF21 promoted the transcriptional level of AtAPX2, AtGolS1, and several AtHSPs. Collectively, the first-class C HSF in maize (ZmHSF21) is cloned in this study, and the combined results suggest that ZmHSF21 is a positive regulator of heat shock response and can be applied to develop maize high-temperature-tolerant varieties for more yield.

1. Introduction

According to the World Food Security Summit Declaration, it is estimated that to meet the food demand of 9 billion people in 2050, food production must increase by 70%, which means that annual crop production growth in the coming decades will need to be 38% higher than historical levels. As one of the most important environmental factors, temperature plays a decisive role in the growth and development of crops and the ultimate yield. Climate warming has become an increasingly significant global problem, especially the frequent occurrence of high (or extremely high) temperatures in recent years. Projections from the Fourth Assessment of the Intergovernmental Panel on Climate Change (IPCC) state that the average global temperature rise ranges from 1.8 °C to 4.0 °C by 2100, which may cause plants to suffer from heat stress and hinder their growth and development, as well as the process of flowering and setting, and eventually seriously reduce the yield [1,2].
Heat sensing is the first step for plants to respond to increased temperature. It translates extracellular temperature signals into intracellular physiological responses or directly senses warming inside the cell and changes its ribonucleic acid (RNA) and protein levels. There are at least four kinds of sensing in objects to initiate heat shock response (HSR), including plasma membrane sensing, light receptor sensing, unfolded protein sensing in endoplasmic reticulum and cytoplasm sensing, and reactive oxygen species (ROS) sensing [3]. Plants have evolved a variety of strategies to adapt to high temperature or heat stress. Four major mechanisms have been found to cope with high temperature stress in plants; these mechanisms combine several typical pathways, including the heat shock factor–heat shock protein (HSF-HSP) pathway, the calcium ion–calmodulin (Ca2+-CaM) pathway, the reactive oxygen species pathway, and the hormone pathway [4]. Among them, the HSF-HSP pathway is the main pathway for plants. HSF is firstly discovered in yeast that regulates the expression of heat response genes [5] and is found to be prevalent in most species, such as 1 HSF in drosophila, 4 HSFs in humans, 21 HSFs in Arabidopsis, 24 HSFs in tomato, 52 HSFs in soybean, and 61 HSFs in wheat [6]. HSP is a type of protein specifically induced to express under heat shock conditions, which mainly protects against heat damage by maintaining the stability of plant proteins. HSF is the central regulator of HSP expression and the main regulator of heat shock response [7].
Plant HSFs harbor conserved functional domains, including the N-terminal deoxyribonucleic acid-binding structure for locating and binding to the heat shock element and the oligomerization domain for the formation of homologous trimers. According to the number of amino acids inserted into the two hydrophobic seven-peptide HR-A-HR-B of the oligomerization domain, HSF family members are divided into three classes: A, B, and C [8]. HSF members in Class A have the C-terminal AHA domain, which is essential for transcriptional activation, while members in Class B and C lack this AHA domain and do not have the ability to activate autonomously. HSF members in Class A are the primary regulators of heat shock response, and Class A HSF1 is the master regulator of this response and alters the transcription of a large number of genes encoding the chaperones and proteases [9,10,11,12]. Class B HSF members mainly act as repressors to negatively regulate the expression of many heat-induced HSF and HSP and negatively regulate plant heat tolerance. In Arabidopsis, the survival frequency of hsfb1/hsfb2b double mutants was significantly higher than that of the wild-type (WT) plants when exposed to heat stress conditions [13]. SlHSFB4a overexpressors showed a larger scorched area and significantly lower survival rate than control plants, and the ROS accumulation and cell death of plants silencing SlHSFB4a were significantly reduced [14,15]. However, the function of Class C HSF remains largely unknown.
Maize (Zeal mays L.) is one of the world’s most important crops, which originates in Mexico, Guatemala, and Honduras in Central America. Maize has different optimum temperatures at different growth stages, such as 24–31 °C for seed germination, 22–28 °C for growth, 25–26 °C for flowering, and 20–24 °C for grain filling. High temperatures play a detrimental effect on seed germination, grain yield, and grain quality [16,17,18]. The maize yields are projected to decline by about 10.3% for every 1 °C increase in temperature [6]. Accordingly, high temperature stress has become the major abiotic stress in maize, and it is urgent to develop new maize varieties with heat resistance [19]. However, the key candidate genes involved in the maize heat shock response and their molecular regulatory mechanisms are still largely unknown. Thus, it is urgent to explore the key genes and dissect their regulation to provide strategies for developing new varieties and offer genetic resources for directed manipulations to improve the heat-tolerance maize. Here, we report the identification of the first-class C HSF and its expression profiles, including tissue-specificity and responses to heat stress, germination frequency, survival rate, and regulation of potential target genes.

2. Materials and Methods

2.1. Plant Materials and Growth Conditions

Arabidopsis seeds were sown on half-strength Murashige and Skoog (MS) (Sigma-Aldrich, St. Louis, MO, USA) solid media containing 0.8% agar and 1% sucrose after surface sterilization. After vernalizing at 4 °C in the dark for 3 days, seedlings were transferred to a growth chamber under 22 °C, 50% humidity, and a 16-h light/8-h dark photoperiod with 100 µmol m−2 s−1 light intensity for 5 days before further heat treatment or transferring into soil (Pindstrup Substrate No. 2, Pindstrup Mosebrug A/S, Ryomgaard, Denmark) in a culture room (Beijing, China) under the same conditions.
To clone ZmHSF21 and detect its expression pattern in different tissues, the seeds of the maize B73 inbred line were sown in the field (Langfang, Heibei province, China; 39.53°_N, 116.72°_E) in May 2023.
To investigate the expression change of ZmHSF21 when exposed to heat stress, the maize seeds were sown in 7 cm × 7 cm pots filled with soil as above and grown in a growth chamber (28 °C 12-h light/22 °C 12-h dark photoperiod, 50% humidity). For heat treatment, the v3-stage seedlings were transferred into a pre-heated growth chamber (45 °C 12-h light/45 °C 12-h dark photoperiod, 50% humidity) and harvested leaves at the given time. The seedlings without heat treatment were used as a parallel control.

2.2. RNA Extraction and Quantitative Real-Time PCR Assay

To detect the expression levels in Arabidopsis transgenic plants, 10-day-old seedlings were harvested, and the RNA was extracted with RNeay Plant Mini Kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions. The first strand of cDNA was synthesized starting with 1 µg of total RNA with the PrimeScript II 1st Strand cDNA Synthesis Kit (TaKaRa, Tokyo, Japan). Then, the cDNA was used as a template, and qRT-PCR was carried out with Fast SYBR® Green master mix (Applied Biosystems, Ottawa, ON, Canada) on an ABI 7500 Fast Real-Time PCR System with the following program: 10 min at 95 °C, 40 cycles of 15 s at 95 °C, and 1 min at 60 °C. The Arabidopsis AtACTIN2 was adopted as an internal control. For tissue-specific expression detection in maize, different tissues from seedlings or adult plants were harvested, and ZmTubulin5 was used as an internal control. The relative abundance of the target gene was calculated using the 2ΔCt method [18], in which ΔCt = Ct(ZmTUBULIN5/AtACTIN2) − Ct(target) [20]. All the primers used for qRT-PCR above are enlisted in Supplementary Table S1.

2.3. Plasmid Construction and Transformation

To generate ZmHSF21 overexpressors, the coding sequence of ZmHSF21 (GRMZM2G086880, Zm00001d011406, and Zm00001eb358700 for versions V3, V4, and V5, respectively) available in the maize genome database (https://maizegdb.org/) was amplified with high-fidelity KOD-FX neo DNA polymerase (TOYOBO, Osaka, Japan) and ligated into the pCPB vector to generate the 35S::ZmHSF21 construct (ZmHSF21-OE). After confirmation by sequencing, the construct was transformed into Agrobacterium tumefaciens strain LBA4404 and further introduced into WT Arabidopsis plants using the floral-dip method [21]. The primer pair used for the construct above was given in Supplementary Table S1.

2.4. Screening of Transgenic Plants

The seeds (in T0 generation) were directly sown into soil as above and grown at 22 °C (light 16 h/darkness 8 h) for 10 days, then 10% Basta solution (1:2000 dilution) was sprayed onto the seedlings once every two days. The genomic DNA from the Basta-resistant plants was extracted for genotyping with the primer pair ZmHSF21-F/R. The T3 homozygous transgenic lines of the positive plants were used for further study.

2.5. Heat Tolerance Analysis

The seeds from WT plants and ZmHSF21 overexpressors were sterilized and sown onto half-strength MS solid media and then grown at 22 °C (light 16 h/dark 8 h) for 5 days. The seedlings were treated at 45 °C for the given time and then returned to 22 °C to resume growth for 7 days. The seedlings that could re-grow and become green during recovery were considered to have survived. The survival rate was determined by the percentage of the number of survived seedlings to the number of total seedlings before heat treatment.

2.6. Nitro Blue Tetrazolium Staining

Four-week-old WT plants and ZmHSF21 overexpressors were treated in a chamber at 45 °C (50% humidity). The leaves were then quickly immersed in 1 mg mL−1 nitro blue tetrazolium solution for 12 h; subsequently, the color was decolorized with 75% ethanol for three times.

2.7. Measurements of Reactive Oxygen Species Content and Enzyme Activities

The sixth leaves of WT and ZmHSF21 transgenic plants with or without heat treatment for 1 h were taken for physiologic investigation. The content of reactive oxygen species (ROS) and the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) were measured with a commercial kit (Jiancheng, Nanjing, China) following the manufacturer’s instructions. The kit numbers for ROS, SOD, POD, and CAT measurements are J38834, A001-1-1, A084-3-1, and A007-1-1, respectively.

2.8. Statistical Analysis

All the measurements or investigations were performed for at least three biological replicates. The least significance difference test and Student’s t test were conducted to analyze the significant differences among multiple groups and between two groups, respectively. And the Bonferroni calibration of SPSS software (version IBM SPSS Statistics 22.0) further carried out the determination of the significant differences. The graphs with dots and bars were produced using GraphPad Prism software (version 7.04).

3. Results

3.1. ZmHSF21 Belongs to the Class C HSF Group

To clone ZmHSF21, specific primers were designed according to its sequence published in the maize genome database (https://maizegdb.org/), and v3-stage leaf cDNA from maize seedlings grown in the field was used as a template. The sequencing results confirmed the cloned sequence was consistent with the sequence in the maize genome database, indicating that ZmHSF21 had been successfully cloned. Sequence analysis showed that the open reading frame of ZmHSF21 was 1164 bp in length and was predicted to encode 388 amino acids. In Arabidopsis, there are 21 HSFs, and they are divided into three classes, including 15 Class A HSFs, 5 Class B HSFs, and 1 Class C HSF. Further comparison of the amino acid sequence of ZmHSF21 with Arabidopsis 21 HSFs and several Class C HSFs from rice, wheat, and Festuca arundinacea revealed that ZmHSF21 clusters with all Class C HSFs in the same group, and the homology of ZmHSF21 with AtHSFC1 of Class C was higher than that of any Class A or B HSF (Figure 1), indicating that ZmHSF21 is a typical Class C HSF.

3.2. ZmHSF21 Is Expressed in Diverse Tissues and Is Up-Regulated by Heat Stress

To detect the tissue expression specificity of ZmHSF21 in maize, total RNA was extracted from different tissues of seedlings or mature plants grown in the field, including roots, leaves, sheaths, and stalks from v3-stage seedlings (~20 days old), silks, husks, and pollens from R1-stage plants (3 days after silking and tasseling), and kernels from R2-stage ears (15 days after pollination). Quantitative PCR analysis showed that ZmHSF21 was detectible in all the tissues tested, with a high expression level in leaves and silks and rather low expression in roots and stalks (Figure 2a).
To detect the role of ZmHSF21 in maize heat stress, the expression pattern of ZmHSF21 in response to heat stress was investigated. The results showed that, compared with control, the ZmHSF21 expression was rapidly induced by high temperature treatment (45 °C) and reached the peak within 1 h after treatment. The expression level declined with the extension of treatment time but was still significantly higher than that of control (0 h) (Figure 2b). These results suggest that the expression of ZmHSF21 is up-regulated by high temperature stress and may play an important role in response to heat stress.

3.3. Screening and Identification of Plants Overexpressing ZmHSF21

To study the biological function of ZmHSF21, the coding sequence of ZmHSF21 was ligated to the pCPB vector to produce a 35S::ZmHSF21 construct and further transformed into WT Arabidopsis. The T0 transgenic seeds were sown into soil, and the 5-day-old seedlings were sprayed with Basta solution for screening the positive (Basta-resistant) seedlings. A total of 14 T1-positive plants were obtained in this study. The genomic DNA of these T1-positive plants was further extracted and used as a template for PCR detection with a special primer pair. The results showed that the fragments with the same size as the coding region of ZmHSF21 could be amplified from the genomic DNA of all positive plants but not from WT plants (Figure 3b). Further sequencing of these fragments verified the consistency with the sequence of ZmHSF21, indicating that the ZmHSF21 had been integrated into the Arabidopsis genome. The further detection of ZmHSF21 expression showed that the T3 homozygous transgenic Line 1 (OE#1), OE#4, and OE#11 had high expression levels (Figure 3c).

3.4. Response of Wild-Type Plants to High Temperature Stress

To study the response of ZmHSF21 to high temperature stress, the wild-type Arabidopsis seedlings that germinated and grown at normal temperature (22 °C) for 5 days were moved into a chamber at 45 °C and treated for 0.5, 1.0, 1.5, or 2.0 h, respectively, and then returned to 22 °C for 7-day resumed growth. Comparison of their survival rate between the treatments showed that there was no significant difference in survival rate between the 0.5 h high temperature treatment and the control (0 h), indicating that Arabidopsis seedlings could tolerate high temperature stress for a short time. However, the survival rate of Arabidopsis seedlings significantly declined with the continuation of high temperatures. The seedling survival rates were only 64.76%, 25.24%, and 0% of that of control (0 h) for 1.0, 1.5, and 2.0 h high temperature treatment, respectively (Figure 4).
The accumulation of ROS in plants reflects the severity of stress. In this study, NBT staining demonstrated that the SOD accumulation in leaves increased significantly with the increase in heat stress duration (Figure 5a,b). The physiological measurement further showed that, with the extension of high temperature stress, the activities of superoxide dismutase SOD, POD, and CAT in leaves increased significantly (Figure 5b), indicating that high temperature stress causes the ROS outbreak and the enzyme activities increase within a short time.

3.5. Overexpression of ZmHSF21 Improved Seed Germination and Heat Resistance of Plants under High Temperature Stress

To test the role of ZmHSF21 in seed germination, the seeds of WT or T3 homozygous ZmHSF21 overexpressors were directly heat-stressed at 50 °C for 60 min and then were plated onto 1/2MS and returned to 22 °C for germination. The results showed that the germination frequency of WT and ZmHSF21 overexpressors all decreased significantly compared with their counterparts under normal conditions without heat treatment (Figure 6). However, after heat treatment, the germination frequency of ZmHSF21 overexpressors was significantly higher than that of WT (65.8%, 82.3%, 85.7%, and 88.3% for WT ZmHSF21 overexpressors OE#1, OE#4, and OE#11, respectively), indicating that ZmHSF21 overexpression could significantly improve the seed germination frequency under high temperature stress (Figure 6).
To further test the effect of ZmHSF21 overexpression on plant growth under high temperature stress, the 5-day-old seedlings were treated at 45 °C for 90 min and then resumed growth at 22 °C for 7 days. The results found that the survival rate of ZmHSF21 overexpressors and WT seedlings was significantly decreased after high temperature treatment (Figure 7). However, the survival rate of ZmHSF21 overexpressors was significantly improved compared with WT when exposed to high temperature treatment (1.25%, 19.75%, 25.75%, and 28.75% for WT, ZmHSF21 overexpressor #OE1, #OE4, #OE11, respectively) (Figure 7), indicating that ZmHSF21 overexpress also improves the plant’s high temperature tolerance.

3.6. ZmHSF21 Significantly Promoted the Expression Levels of HSPs

HSF is reported to be a class of transcription factors that regulate the expression of heat-response genes, mainly through regulating the expression of target genes HSPs, which is specifically induced to express proteins under heat shock conditions and resists heat damage by maintaining the stability of plant proteins. In this study, the results showed that the overexpression of ZmHSF21 significantly promoted the expression of a series of HSP in Arabidopsis, including AtHSP17.7, AtHSP18.5, AtHSP22.0, AtHSP60.2, AtHSP26.5, AtHSP70.13, and AtHSP90.5 (Figure 8). In addition, ZmHSF21 also promoted the expression levels of oxidative stress-related genes AtAPX2 and AtGOLS1 (5–30 folders higher than that of the WT).

3.7. Overexpression of ZmHSF21 Significantly Decreased ROS Content When Plants Exposed to Heat Stress

To reveal the physiological basis for the high temperature tolerance of ZmHSF21 overexpressors, the ROS content was further investigated in this study. The nitro blue tetrazolium staining showed that, under normal conditions, the SOD content in the leaves from both WT plants and ZmHSF21 overexpressors was rather low and there was no significant difference between them (Figure 9a,b). However, when plants are exposed to heat stress for 2 h, the SOD content in the leaves from WT plants and ZmHSF21 overexpressors is obviously higher than their counterparts before heat stress. Comparison of the ROS content between WT plants and ZmHSF21 overexpressors after heat stress showed that the ROS content in ZmHSF21 overexpressors was significantly lower than that in WT plants, only around 54.07%, 53.10%, and 68.22% of WT plants for ZmHSF21OE#1, ZmHSF21OE#4, and ZmHSF21OE #11, respectively (Figure 9a,b).
SOD, POD, and CAT are the main components of the ROS clearance system. They play an extremely important role in preventing membrane lipid peroxidation, delaying plant senescence, and mitigating membrane damage caused by adversity. Thus, we further compared the activities of CAT, SOD, and POD between WT and ZmHSF21 overexpressors before and after heat stress. The results showed that the activities of CAT, SOD, and POD of WT plants and ZmHSF21 overexpressors after heat stress are significantly higher than their counterparts before heat stress (Figure 9b). The results also showed that, after heat stress, the activities of CAT, SOD, and POD of ZmHSF21 overexpressors are significantly higher than the counterparts of WT plants, around 170%, 149%, and 195% of WT plants for the activities of CAT, SOD, and POD, respectively (Figure 9b).

4. Discussion

Heat stress is one of the most common abiotic stresses for crops. Maize is one of the most important crops that originated in the tropical region and is currently widely cultured in temperate regions. However, frequent occurrences of high and extremely high temperatures worldwide have led to a serious decline in its yield and threaten global food security. Thus, it is urgent to explore key candidate genes and elucidate the molecular mechanisms of heat stress in order to understand how maize responds and adapts to heat stress and to cultivate maize with improved heat tolerance to cope with a warming environment. HSFs are the most important regulatory factors involved in heat shock response. HSF, together with the target gene HSPs (HSF-HSP module), control most physiological and biochemical processes of heat shock response. Previous studies have shown that there are 31 HSFs in the maize genome, including 16 Class A HSFs, 10 Class B HSFs, and 5 Class C HSFs [22]. Overexpression of maize Class A ZmHSF1 or ZmHSF05 in Arabidopsis or rice can significantly enhance the heat resistance of plants [23,24]. A very recent study reported that Class A ZmHSF20 could reduce the heat tolerance of seedlings by inhibiting another maize, Class A ZmHSF4 and ZmCesA2, and thus balancing growth and defense of seedlings [25]. ZmHSF4 directly activates ZmCesA2 transcription and thus increases maize heat tolerance [25]. In maize Class B HSFs, overexpression of ZmHSF11 in Arabidopsis and rice significantly increased cell death and decreased plant survival rate under heat shock treatment [26]. ZmHSF11 transgenic rice accumulated more H2O2 and less proline content, and the expression levels of oxidative stress-related genes APX2 and DREB2A were significantly decreased [26]. However, no maize Class C HSF has been reported, and their function is still largely unknown.
This research mainly focused on the identification of maize Class C HSF and its biological function under heat stress. In this study, we identified a Class C HSF ZmHSF21 from maize leaves, which encodes a putative protein of 388 amino acids. We showed that ZmHSF21 was expressed in most tissues with relatively high expression in leaves and silks, which indicates the biological function of ZmHSF21 in leaves and silks. The phylogenetic tree constructed with ZmHSF21 and other HSFs, including 21 AtHSF (15 Class A HSFs, 5 Class B HSFs, 1 Class C HSF) from Arabidopsis, 2 Class C OsHSFs from rice, 3 Class C TaHSFs from wheat, and 1 FaHSFC from Festuca arundinacea, shows ZmHSF21, AtHSFC1, 2 OsHSFCs, 3 TaHSFCs, and FaHSFC all cluster in the same group, suggesting that there are similar structures and motif composition between ZmHSF21 and Class C HSFs from other species tested and ZmHSF21 is a typical Class C HSF. ZmHSF21 expression was significantly up-regulated during 6-h heat treatment. Further study showed that overexpression of ZmHSF21 in Arabidopsis significantly improved the seed germination frequency and seedling survival rate under heat stress conditions, which is similar with maize other class HSFs, such as ZmHSF1, ZmHSF4, and ZmHSF5 of Class A, but not ZmHSF20, which reduced the heat tolerance of seedlings. The improving heat tolerance of Class C HSFs has also previously been reported in other species. For instance, overexpression of TaHSFC2 in wheat improved heat tolerance of transgenic plants, and the expression of HSP70s was up-regulated [27]. Overexpression of Festuca arundinacea FaHSFC1b in Arabidopsis also significantly up-regulated the expression of several HSPs, chlorophyll content, and photochemical efficiency, which resulted in an increase in plant survival rate under heat stress [28]. Compared with FaHSFC1b and TaHSFC2, our results demonstrated that overexpression of ZmHSF21 in Arabidopsis not only up-regulated the expression level of HSP60, HSP70, and HSP90 but also up-regulated the expression level of small HSPs, including AtHSP17.7, AtHSP18.5, AtHSP22.0, and AtHSP26.5. Especially, our results showed that ZmHSF21 overexpression obviously promoted the expression of oxidative stress-related genes such as AtAPX2 and AtGolS1, which directly respond to oxidative damage and increase tolerance to stress. Considering the up-regulation of ZmHSF21 when exposed to heat stress, it is reasonable to suppose that the expression of HSPs above and AtAPX2 and AtGolS1 up-regulated, resulting in the decline of ROS content and the increase in the activities of ROS, POD, and CAT, and subsequently improving the heat tolerance and survival rate. Additionally, the up-regulation of both chaperone HSPs and oxidative stress-related genes by ZmHSF21 overexpression indicates that ZmHSF21 may regulate heat shock response through two independent pathways: one is to promote HSPs and the other is to up-regulate oxidative stress-related genes. Collectively, our combined results suggested that ZmHSF21 is a positive regulator of heat shock response and can be applied as genetic resources to develop high-temperature-tolerant varieties of maize and other crops, especially in tropical regions, which would provide a potential way to dramatically improve food security in the 21st century.
Although our results demonstrated that overexpression of ZmHSF21 in Arabidopsis obviously improved the germination frequency and survival rate of transgenic materials under heat stress, the detailed molecular mechanism underlying it is still largely unknown and is worth further analysis. In addition, it remains to be verified and tested whether overexpression of ZmHSF21 in maize can achieve similar effects and improve the germination frequency and survival rate in practice. Considering the complex genome of maize and the diversity of inbred lines, it is necessary to systematically analyze the haplotypes of ZmHSF21 in different inbred lines from different regions, such as tropical regions and temperate regions, and to investigate its transcriptional level differences between inbred lines under heat stress, so as to explore its excellent haplotypes and then directly apply to breeding in practice for novel heat-resistant maize germplasm and varieties, as well as provide effective genetic resources for maize and other crops to cope with global warming.

5. Conclusions

In conclusion, ZmHSF21, the first-class C HSF, was cloned and identified in this study. The expression of ZmHSF21 is up-regulated by heat stress. Overexpression of maize ZmHSF21 in Arabidopsis significantly down-regulates the expression level of several HSPs and the oxidative stress-related genes. We demonstrated that overexpression of ZmHSF21 promotes the activities of SOD, POD, and CAT, reduces the content of ROS, and results in the improvement of seed germination frequency and survival rate of transgenic plants under heat stress. Taken together, our data demonstrated that ZmHSF21 is a positive regulator of heat shock response and may serve as a potential useful genetic resource for maize.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agriculture14091524/s1. Table S1: Primers used in this study.

Author Contributions

Y.X. conceived and designed the research. Y.Y. and Y.X. performed experiments. Y.Y. collected and analyzed the data. Y.Y. and Y.X. wrote the manuscript. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by grants from National Natural Science Foundation of China (32270264) and Beijing Natural Science Foundation (5222030).

Institutional Review Board Statement

Not applicable.

Data Availability Statement

All data supporting the findings of this study are included within the article or available upon request from the authors.

Conflicts of Interest

The authors declare no conflicts of interest.

References

  1. Arshad, M.S.; Farooq, M.; Asch, F.; Krishna, J.S.V.; Prasad, P.V.V.; Siddique, K.H.M. Thermal stress impacts reproductive development and grain yield in rice. Plant Physiol. Biochem. 2017, 115, 57–72. [Google Scholar] [CrossRef] [PubMed]
  2. Janni, M.; Maestri, E.; Gullì, M.; Marmiroli, M.; Marmiroli, N. Plant responses to climate change, how global warming may impact on food security: A critical review. Front. Plant Sci. 2024, 14, 1297569. [Google Scholar] [CrossRef]
  3. Mittler, R.; Finka, A.; Goloubinoff, P. How do plants feel the heat? Trends Biochem. Sci. 2012, 37, 118–125. [Google Scholar] [CrossRef]
  4. Zhao, J.; Lu, Z.; Wang, L.; Jin, B. Plant responses to heat stress: Physiology, transcription, noncoding RNAs, and epigenetics. Int. J. Mol. Sci. 2020, 22, 117. [Google Scholar] [CrossRef] [PubMed]
  5. Wlederrecht, G.; Seto, D.; Parker, C.S. Isolation of the gene encoding the & cerevisiae heat shock transcription factor. Cell 1988, 54, 841–853. [Google Scholar]
  6. Tolosa, L.N.; Zhang, Z. The role of major transcription sactors in Solanaceous food crops under different stress conditions: Current and Future Perspectives. Plants 2020, 9, 56. [Google Scholar] [CrossRef] [PubMed]
  7. Kan, Y.; Mu, X.R.; Gao, J.; Lin, H.X.; Lin, Y. The molecular basis of heat stress responses in plants. Mol. Plant. 2023, 16, 1612–1634. [Google Scholar] [CrossRef]
  8. Haider, S.; Raza, A.; Iqbal, J.; Shaukat, M.; Mahmood, T. Analyzing the regulatory role of heat shock transcription factors in plant heat stress tolerance: A brief appraisal. Mol. Biol. Rep. 2022, 49, 5771–5785. [Google Scholar] [CrossRef]
  9. Anckar, J.; Sistonen, L. Regulation of HSF1 function in the heat stress response: Implications in aging and disease. Annu. Rev. Biochem. 2011, 80, 1089–1115. [Google Scholar] [CrossRef]
  10. Mishra, S.K.; Tripp, J.; Winkelhaus, S.; Tschiersch, B.; Theres, K.; Nover, L.; Scharf, K.D. In the complex family of heat stress transcription factors, HsfA1 has a unique role as master regulator of thermotolerance in tomato. Genes Dev. 2002, 16, 1555–1567. [Google Scholar] [CrossRef]
  11. Li, P.S.; Yu, T.F.; He, G.H.; Chen, M.; Zhou, Y.B.; Chai, S.C.; Xu, Z.S.; Ma, Y.Z. Genome-wide analysis of the Hsf family in soybean and functional identification of GmHsf-34 involvement in drought and heat stresses. BMC Genom. 2014, 15, 1009. [Google Scholar] [CrossRef]
  12. Gai, W.X.; Ma, X.; Li, Y.; Xiao, J.J.; Khan, A.; Li, Q.H.; Gong, Z.H. CaHsfA1d improves plant thermotolerance via regulating the expression of stress- and antioxidant-related genes. Int. J. Mol. Sci. 2020, 21, 8374. [Google Scholar] [CrossRef]
  13. Ikeda, M.; Mitsuda, N.; Ohme-Takagi, M. Arabidopsis HsfB1 and HsfB2b act as repressors of the expression of heat-inducible Hsfs but positively regulate the acquired thermotolerance. Plant Physiol. 2011, 157, 1243–1254. [Google Scholar] [CrossRef] [PubMed]
  14. Fragkostefanakis, S.; Simm, S.; El-Shershaby, A.; Hu, Y.; Bublak, D.; Mesihovic, A.; Darm, K.; Mishra, S.K.; Tschiersch, B.; Theres, K.; et al. The repressor and co-activator HsfB1 regulates the major heat stress transcription factors in tomato. Plant Cell Environ. 2019, 42, 874–890. [Google Scholar] [CrossRef] [PubMed]
  15. Rao, S.; Gupta, A.; Bansal, C.; Sorin, C.; Crespi, M.; Mathur, S. A conserved HSF:miR169:NF-YA loop involved in tomato and Arabidopsis heat stress tolerance. Plant J. 2022, 112, 7–26. [Google Scholar] [CrossRef]
  16. Graham, G.G.; Lembcke, J.; Morales, E. Post-prandial plasma aminograms in the assessment of protein quality for young children: Maize and grain amaranth, alone and combined. Eur. J. Clin. Nutr. 1990, 44, 35–43. [Google Scholar]
  17. Zhu, P.; Jin, Z.; Zhuang, Q.; Ciais, P.; Bernacchi, C.; Wang, X.; Makowski, D.; Lobell, D. The important but weakening maize yield benefit of grain filling prolongation in the US Midwest. Glob. Chang. Biol. 2018, 24, 4718–4730. [Google Scholar] [CrossRef] [PubMed]
  18. Qiao, S.; Liu, Z.; Zhang, Z.; Su, Z.; Yang, X. The heat stress during anthesis and the grain-filling period of spring maize in Northeast China is projected to increase toward the mid-21st century. J. Sci. Food Agric. 2023, 103, 7612–7620. [Google Scholar] [CrossRef]
  19. Frey, F.P.; Urbany, C.; Hüttel, B.; Reinhardt, R.; Stich, B. Genome-wide expression profiling and phenotypic evaluation of European maize inbreds at seedling stage in response to heat stress. BMC Genom. 2015, 16, 123. [Google Scholar] [CrossRef]
  20. Livak, K.J.; Schmittgen, T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001, 25, 402–408. [Google Scholar] [CrossRef]
  21. Clough, S.J.; Bent, A.F. Floral dip: A simplified method for Agrobacterium- mediated transformation of Arabidopsis thaliana. Plant J. 1998, 16, 735–743. [Google Scholar] [CrossRef] [PubMed]
  22. Zhang, H.; Li, G.; Fu, C.; Duan, S.; Hu, D.; Guo, X. Genome-wide identification, transcriptome analysis and alternative splicing events of Hsf family genes in maize. Sci. Rep. 2020, 10, 8073. [Google Scholar] [CrossRef] [PubMed]
  23. Li, G.L.; Zhang, H.N.; Shao, H.; Wang, G.Y.; Zhang, Y.Y.; Zhang, Y.J.; Zhao, L.N.; Guo, X.L.; Sheteiwy, M.S. ZmHsf05, a new heat shock transcription factor from Zea mays L. improves thermotolerance in Arabidopsis thaliana and rescues thermotolerance defects of the athsfa2 mutant. Plant Sci. 2019, 283, 375–384. [Google Scholar] [CrossRef] [PubMed]
  24. Zhang, H.; Li, G.; Hu, D.; Zhang, Y.; Zhang, Y.; Shao, H.; Zhao, L.; Yang, R.; Guo, X. Functional characterization of maize heat shock transcription factor gene ZmHsf01 in thermotolerance. Peer J. 2020, 8, e8926. [Google Scholar] [CrossRef]
  25. Li, Z.; Ji, Y.; Wang, C.; Wang, S.; Shi, Y.; Le, J.; Zhang, M. The heat shock factor 20-HSF4-cellulose synthase A2 module regulates heat stress tolerance in maize. Plant Cell 2024, 36, 2652–2667. [Google Scholar] [CrossRef]
  26. Qin, Q.; Zhao, Y.; Zhang, J.; Chen, L.; Si, W.; Jiang, H. A maize heat shock factor ZmHsf11 negatively regulates heat stress tolerance in transgenic plants. BMC Plant Biol. 2022, 22, 406. [Google Scholar] [CrossRef]
  27. Hu, X.J.; Chen, D.; Lynne, M.C.; Fernanda, D.M.; Zhang, Z.B.; Drenth, J.; Kalaipandian, S.; Chang, H.; Xue, G.P. Heat shock factor C2a serves as a proactive mechanism for heat protection in developing grains in wheat via an ABA-mediated regulatory pathway. Plant Cell Environ. 2018, 41, 79–98. [Google Scholar] [CrossRef]
  28. Zhuang, L.; Cao, W.; Wang, J.; Yu, J.; Yang, Z.; Huang, B. Characterization and functional analysis of FaHsfC1b from Festuca arundinacea conferring heat tolerance in Arabidopsis. Int. J. Mol. Sci. 2018, 19, 2702. [Google Scholar] [CrossRef]
Agriculture 14 01524 g001
Figure 1. Phylogenetic relationship between maize ZmHSF21 and Class C HSFs from other species. The software Clustal X (version 3.0 46) was used to align the protein sequence of all HSFs. The software Treeview (version 1.16r4 47) was adopted to produce the phylogenetic tree by the neighbor jointing method. The ZmHSF21 cloned in this study is in red. The red open box indicated the Class C HSFs from Arabidopsis, maize, rice, wheat, and Festuca arundinacea. The amino acid sequences of all proteins aligned are given in the supplementary datasheet.
Figure 1. Phylogenetic relationship between maize ZmHSF21 and Class C HSFs from other species. The software Clustal X (version 3.0 46) was used to align the protein sequence of all HSFs. The software Treeview (version 1.16r4 47) was adopted to produce the phylogenetic tree by the neighbor jointing method. The ZmHSF21 cloned in this study is in red. The red open box indicated the Class C HSFs from Arabidopsis, maize, rice, wheat, and Festuca arundinacea. The amino acid sequences of all proteins aligned are given in the supplementary datasheet.
Agriculture 14 01524 g001
Agriculture 14 01524 g002
Figure 2. Expression patterns of ZmHSF21 in different tissues and under heat stress. (a) Comparison of expression levels of ZmHSF21 between different tissues. Bar represents standard deviation (SD). Values are means ± SD (n = 3 biological replicates). The different letters indicate significant differences at p < 0.05. (b) The up-regulation of ZmHSF21 expression after high temperature treatment. V3-stage maize seedlings were treated with high temperature (45 °C) for a time course (0, 15, 30, 60, 120, and 240 min), and the second leaves were harvested for RNA extraction. All data represent the mean ± SD of three biological replicates. The different lowercase letters indicate significant differences at p < 0.05.
Figure 2. Expression patterns of ZmHSF21 in different tissues and under heat stress. (a) Comparison of expression levels of ZmHSF21 between different tissues. Bar represents standard deviation (SD). Values are means ± SD (n = 3 biological replicates). The different letters indicate significant differences at p < 0.05. (b) The up-regulation of ZmHSF21 expression after high temperature treatment. V3-stage maize seedlings were treated with high temperature (45 °C) for a time course (0, 15, 30, 60, 120, and 240 min), and the second leaves were harvested for RNA extraction. All data represent the mean ± SD of three biological replicates. The different lowercase letters indicate significant differences at p < 0.05.
Agriculture 14 01524 g002
Agriculture 14 01524 g003
Figure 3. Identification of ZmHSF21 transgenic plants. (a) Screening of T1 transgenic seedlings. Five-day-old T1 seedlings were sprayed with 10% Basta (1:2000 dilution) for three times. The white triangles indicated the Basta-resistant (positive) seedlings. (b) Genotyping of ZmHSF21 transgenic plants. The genomic DNA from WT plants was used as a negative control. (c) Detection of the expression level of ZmHSF21 in different homozygous transgenic lines. All data represent the mean ± SD of three independent biological replicates. The different lowercase letters indicate significant differences at p < 0.05. The T3 homozygous seeds of OE#1, OE#4, and OE#11 were used for further study.
Figure 3. Identification of ZmHSF21 transgenic plants. (a) Screening of T1 transgenic seedlings. Five-day-old T1 seedlings were sprayed with 10% Basta (1:2000 dilution) for three times. The white triangles indicated the Basta-resistant (positive) seedlings. (b) Genotyping of ZmHSF21 transgenic plants. The genomic DNA from WT plants was used as a negative control. (c) Detection of the expression level of ZmHSF21 in different homozygous transgenic lines. All data represent the mean ± SD of three independent biological replicates. The different lowercase letters indicate significant differences at p < 0.05. The T3 homozygous seeds of OE#1, OE#4, and OE#11 were used for further study.
Agriculture 14 01524 g003
Agriculture 14 01524 g004
Figure 4. Comparison of survival rates of WT seedlings after different durations of high temperature treatment. Five-day-old seedlings were placed at 45 °C for given time and then resumed growth at 22 °C. (a) 0 h; (b) 0.5 h; (c) 1.0 h; (d) 1.5 h; and (e) 2.0 h. (f) Statistical analysis of survival rates of seedlings with or without heat treatment. The data represent the mean ± SD (n = 3 biological replicates) of three independent. The different letters indicate significant differences at p < 0.05.
Figure 4. Comparison of survival rates of WT seedlings after different durations of high temperature treatment. Five-day-old seedlings were placed at 45 °C for given time and then resumed growth at 22 °C. (a) 0 h; (b) 0.5 h; (c) 1.0 h; (d) 1.5 h; and (e) 2.0 h. (f) Statistical analysis of survival rates of seedlings with or without heat treatment. The data represent the mean ± SD (n = 3 biological replicates) of three independent. The different letters indicate significant differences at p < 0.05.
Agriculture 14 01524 g004
Agriculture 14 01524 g005
Figure 5. The reactive oxygen species is significantly accumulated with heat stress. (a) NBT staining showing the SOD accumulation. (b) Quantification of the content of ROS and the enzyme activities. Values are means ± SDs of three replicates. The different letters indicate significant differences at p < 0.05.
Figure 5. The reactive oxygen species is significantly accumulated with heat stress. (a) NBT staining showing the SOD accumulation. (b) Quantification of the content of ROS and the enzyme activities. Values are means ± SDs of three replicates. The different letters indicate significant differences at p < 0.05.
Agriculture 14 01524 g005
Agriculture 14 01524 g006
Figure 6. Comparison of seed germination frequency of WT and ZmHSF21 transgenic seeds under heat stress. (a) Control; (b) After heat shock; (c) Diagram of the seeds in (a); (d) Statistical analysis of germination frequency of seeds with or without heat treatment. Seeds were placed at 50 °C for 60 min and then transferred to 22 °C for germination and growth. The data represent the means ± SDs (n = 3). The different letters indicate significant differences at p < 0.05 (Student’s t-test).
Figure 6. Comparison of seed germination frequency of WT and ZmHSF21 transgenic seeds under heat stress. (a) Control; (b) After heat shock; (c) Diagram of the seeds in (a); (d) Statistical analysis of germination frequency of seeds with or without heat treatment. Seeds were placed at 50 °C for 60 min and then transferred to 22 °C for germination and growth. The data represent the means ± SDs (n = 3). The different letters indicate significant differences at p < 0.05 (Student’s t-test).
Agriculture 14 01524 g006
Agriculture 14 01524 g007
Figure 7. Survival rates of WT and ZmHSF21 transgenic seedlings under heat stress. (a) Control; (b) After heat shock; (c) Diagram of the seedlings in (a); (d) Statistical analysis of survival rate of seedlings with or without heat treatment. Five-day-old seedlings were placed at 45 °C for 90 min and then resumed growth at 22 °C for 7 days. The data represent the means ± SDs (n = 3). The different letters indicate significant differences at p < 0.05 (Student’s t-test).
Figure 7. Survival rates of WT and ZmHSF21 transgenic seedlings under heat stress. (a) Control; (b) After heat shock; (c) Diagram of the seedlings in (a); (d) Statistical analysis of survival rate of seedlings with or without heat treatment. Five-day-old seedlings were placed at 45 °C for 90 min and then resumed growth at 22 °C for 7 days. The data represent the means ± SDs (n = 3). The different letters indicate significant differences at p < 0.05 (Student’s t-test).
Agriculture 14 01524 g007
Agriculture 14 01524 g008
Figure 8. ZmHSF21 significantly up-regulated the expression of HSPs. Ten-day-old seedlings grown under normal conditions were harvested for RNA extraction. All data represent the mean ± SD of three independent biological replicates. * p < 0.05; ** p < 0.01. AtACTIN2 was used as an internal control.
Figure 8. ZmHSF21 significantly up-regulated the expression of HSPs. Ten-day-old seedlings grown under normal conditions were harvested for RNA extraction. All data represent the mean ± SD of three independent biological replicates. * p < 0.05; ** p < 0.01. AtACTIN2 was used as an internal control.
Agriculture 14 01524 g008
Agriculture 14 01524 g009
Figure 9. Comparison of the ROS content in the leaves between WT plants and ZmHSF21 overexpressors with or without high temperature treatment. (a) NBT staining showing the SOD accumulation between WT plants and ZmHSF21 overexpressors before heat stress (HS) or after HS. (b) Quantification of the content of ROS and the activities of CAT, SOD, and POD between WT leaves and ZmHSF21 overexpressors. Values are means ± SDs of three replicates. The different letters indicate significant differences at p < 0.05.
Figure 9. Comparison of the ROS content in the leaves between WT plants and ZmHSF21 overexpressors with or without high temperature treatment. (a) NBT staining showing the SOD accumulation between WT plants and ZmHSF21 overexpressors before heat stress (HS) or after HS. (b) Quantification of the content of ROS and the activities of CAT, SOD, and POD between WT leaves and ZmHSF21 overexpressors. Values are means ± SDs of three replicates. The different letters indicate significant differences at p < 0.05.
Agriculture 14 01524 g009
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Xie, Y.; Ye, Y. Maize Class C Heat Shock Factor ZmHSF21 Improves the High Temperature Tolerance of Transgenic Arabidopsis. Agriculture 2024, 14, 1524. https://doi.org/10.3390/agriculture14091524

AMA Style

Xie Y, Ye Y. Maize Class C Heat Shock Factor ZmHSF21 Improves the High Temperature Tolerance of Transgenic Arabidopsis. Agriculture. 2024; 14(9):1524. https://doi.org/10.3390/agriculture14091524

Chicago/Turabian Style

Xie, Yurong, and Yuhan Ye. 2024. "Maize Class C Heat Shock Factor ZmHSF21 Improves the High Temperature Tolerance of Transgenic Arabidopsis" Agriculture 14, no. 9: 1524. https://doi.org/10.3390/agriculture14091524

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop