Introduction

Wheat (Triticum aestivum L.) is a major cereal for human consumption and its production needs to be considerably increased to meet global food demand (Sun et al. 2022). Wheat is very sensitive to heat and its production is estimated to decrease by 6% for each degree rise in global temperature (Zhao et al. 2017). Global temperature is predicted to rise by 1.6 °C in 2021–2040, 2.4 °C in 2041–2060, and 4.4 °C in 2081–2100, unless adequate mitigation measures of human interventions are taken (Masson-Delmotte et al. 2021). Heat stress (HS) during grain filling stage of wheat is detrimental to grain yield and quality (Rangan et al. 2020). A simulation experiment shows yield reduction due to HS during grain filling stage alone can have a significant effect on global food security (Asseng et al. 2011). Significant grain yield reduction (57.3%) is reported in wheat due to short-term severe HS at grain filling stage (Schittenhelm et al. 2020). Heat tolerance breeding targeting grain filling stage can mitigate the effect of heat on wheat production.

Heat stress can cause a range of morphological changes in plant ultimately affecting the final yield. Morphological traits such as TKW, chlorophyll fluorescence, rate of senescence has been used widely as parameter to measure heat tolerance in crops. Higher TKW is associated with heat tolerance and higher grain yield in heat tolerant genotypes under HS (Fleitas et al. 2020; Sharma et al. 2008). Chlorophyll fluorescence has been used to detect genetic difference in heat tolerance in wheat (Sharma et al. 2012). Stay green is one of the major morphological characters for heat tolerance studies (Rehman et al. 2021). Similarly, strong association of senescence with grain yield under post-anthesis heat stress has been elucidated in different studies (Chapman et al. 2021; Prasad et al. 2008; Ullah et al. 2021).

Grain yield is determined by grain number and grain weight at harvest. HS at grain development shortens the grain filling duration, decreases starch and protein accumulation through restricted activity of grain biosynthesis enzymes and impaired flag leaf assimilatory efficiency and stem reserves mobilization in wheat reducing grain weight (Ullah et al. 2021; Abdelrahman et al. 2020). Expression of HS tolerance involves activation of a set of heat responsive genes by transcription factors (TF) and production of metabolites (Singh et al. 2019). AP2/ERF is a large TF family in plant which forms cross-regulatory network with hormone signaling to regulates abiotic stress response (Ma et al. 2024). It plays important role in response to multiple stresses through regulating downstream gene expression (Nie and Wang 2023). AP2/ERF genes bind to DRE cis-elements of heat responsive genes (HSFA3 and HSPs) under HS and enhance their expressions to regulate heat tolerance (Cheng et al. 2013). The AP2/ERF superfamily TF genes respond to various phytohormones, biotic and abiotic stimuli by regulating the expression of stress responsive genes through phytohormone-mediated signaling pathways in plants (Feng et al. 2020). The regulatory role of AP2/ERF TF genes for heat tolerance has been reported in various transgenic studies. For example, overexpression of AtERF53 in rg1g1rg1g2 double mutant induced heat tolerance by regulating expression of heat responsive downstream genes, accumulation of endogenous ABA and proline, and stomatal movement in Arabidopsis (Hsieh et al. 2013). Similarly, AtDREB2C activates transcription of HsfA3 in mesophyll protoplast of Arabidopsis and induce expression of HSP genes under HS regulating heat tolerance (Chen et al. 2010). Despite, the importance of this gene family in heat tolerance, there have been limited studies in wheat for heat tolerance. Heat responsive expressions of TaAP2/ERF genes in wheat seedlings have also been reported in our previous study (Magar et al. 2022), whereas their roles at grain filling stage are yet to be explored.

The expression and expression regulation of target genes by AP2/ERF TF genes involves complex network of hormones and metabolic pathways. The abscisic acid (ABA) and ethylene (ET) are major stress hormones regulating abiotic stress response by AP2/ERF genes, and ET and jasmonic acid (JA) cross-talk with each other and with signaling pathways of ABA, gibberellic acid (GA) and auxin related to abiotic stress tolerance (Sah et al. 2016; Kazan 2015). ET signaling pathway is important transcriptional regulator for different biological functions in rice and Arabidopsis, and there exist cross-talks between ET and other hormone affecting plant morphology and stress response (Zhao et al. 2021). Also, ERF1 induce heat tolerance by regulation of heat responsive genes via. JA, ET, and ABA signaling in Arabidopsis (Cheng et al. 2013). Similarly, HSDA1b activates accumulation of JA and JA signaling regulates expression of DREB2A gene in Arabidopsis mutant under HS and exhibits enhanced heat tolerance with higher survival rate as compared to wild type (Tian et al. 2020). The genes are expressed in sets under stress condition and co-expressed genes with similar biological function can be identified using co-expression network analysis. AtAP2/ERF co-expression network was analyzed to understand the role of AP2/ERF genes in hormone and abiotic stress-responsive pathways in Arabidopsis thaliana (Chockalingam et al. 2017). In wheat, the gene co-expression network analysis was used to identify hub genes associated with drought resistance (Lv et al. 2020) and to select putative candidate TF for stress tolerance (Saidi et al. 2022). However, detailed heat response mechanism, signaling pathways, and co-expressed TaAP2/ERF TF genes in wheat is yet to be explored. In this study, we compared the HS response of contrasting wheat genotypes at grain filling stage using morphological traits, gene expression and co-expression network analysis of TaAP2/ERF TF genes to identify candidate genes and major signaling pathways regulating heat tolerance in wheat.

Materials and Methods

Plant Materials

Wheat genotypes contrasting in heat tolerance, W156 (tolerant) and Brazil 32 (susceptible), were selected for a HS experiment targeting at the grain filling stage. They were identified as contrasting genotypes for heat tolerance at seedling stage (Magar et al. 2022), and showed consistent heat tolerance/susceptibility at both seedling and reproductive stages (Lu et al. 2022). The seeds of the genotypes were obtained from the Australian Winter Cereal Collection, and two seeds were sown in each pot (8 × 8 × 16 cm) containing potting mixture (5:2:3 compost: peat: sand at pH 6.0). Twelve pots per genotype (three replicates for each control and 2 time points of heat treatment) were grown in two sets (set A for sampling and set B for harvesting to measure yield related traits) in a completely randomized block design in a naturally illuminated glasshouse at The University of Western Australia, Crawley, Western Australia (31° 59′ S, 115° 49′ E) from May to November. The controlled environment conditions (relative humidity, temperature, and ambient light level) are shown in the Supplementary Fig. 1. The plants were thinned at three leaf stage to maintain a single plant per pot and were regularly watered and fertilized fortnightly with ‘Dimond red’ (Campbells Fertilisers Australasia Pty Ltd, Laverton North, Australia) from 4 weeks after sowing until the end of the grain-filling period.

Heat Stress Treatment

Each plant was observed individually and tagged to record the anthesis date. On 10th day post-anthesis (10 DPA), six pots each from Set A and B (12 pots) were shifted to a control environment room (CER) for heat treatment set at 37/27 °C (day/night), 16/8-h photoperiod, and 420 mmol m−2 s−1 light intensity while the remaining 12 pots (six pots each from Set A and B) were left in glasshouse to grow as control. Each pot was watered regularly to ensure there is no water stress. Immediately after 1 day (24 h) heat treatment, on 11th day post-anthesis (11 DPA) three plants from control and three plants from CER (both set A and B) were measured for morphological parameters including chlorophyll content with SPAD meter, leaf temperature, and chlorophyll fluorescence with Pocket PEA. The flag leaf of three plants from set A in CER room were sampled and snap frozen in liquid nitrogen, while the other three plants from set B were returned to glasshouse to grow naturally till physiological maturity. Similarly, after 3-day (72 h) heat treatment, on 13th day post-anthesis (13 DPA) three plants from control and three plants from CER (both set A and B) were measured for morphological parameters the same as on 11 DPA. The flag leaf of three plants from set A in CER room were sampled and snap frozen in liquid nitrogen, while other three plants from set B were returned to glasshouse to grow naturally till physiological maturity. The samples taken from both 11 DPA and 13 DPA from 4 to 6 pm and were stored at − 80 °C for RNA extraction. The days to flag leaf senescence and exposed peduncle senescence were recorded for both control and heat-treated plants. At maturity, morphological traits including number of fertile tiller per plant, plant height of main tiller (cm), and panicle length of main tiller (cm) were measured. The main tiller was harvested separately for each pot and the remaining tillers were bulked. They were dried at 60 °C oven for 3 days and then measured for dry biomass of main tiller (g), total biomass (g), grain number of main tiller, total grain number, grain weight of main tiller (g), and total grain weight/yield (g).

Sample Preparation, RNA Extraction, and cDNA Synthesis

RNA was extracted from 24 samples (2 genotypes × 3 replications × 2 treatments × 2 time points) using RNeasy Plant Mini Kit (Qiagen) with an on-column DNase digestion with RNase-free DNase I (Qiagen). The extracted total RNA was quantified using Nanodrop spectrophotometer ND-1000, and the concentration was also verified by using Qubit RNA BR Assay kit in Qubit 3.0 Fluorometer (Invitrogen by life technologies Ref: Q33216). The integrity of RNA was tested using gel electrophoresis with 5 μl RNA in 1.5% agarose gel. The quantified RNA was used for the cDNA synthesis using SensiFAST cDNA Synthesis Kit from Meridian Bioscience (BIO-65054) following the kit protocol with 1 μg RNA in 20 μl reaction volume.

Gene Selection and Primer Design

Wheat AP2/ERF superfamily TF genes highly expressed under abiotic stress were selected from 630 TaAP2/ERF TF genes identified in wheat (Magar et al. 2022), based on the insilico gene expression data from wheat expression database (http://www.wheat-expression.com/). Gene specific primers were designed by Geneious Prime for the 22 representative genes and validated by PCR amplification. The primers that amplified a single specific band within a defined range (100–200 bp) were selected for further RT-qPCR analysis. The primers specificity was also validated for the single peak in the melting curve to avoid primer-dimer and genomic DNA contamination influencing the assay. The RT-qPCR analysis were conducted using a 10 μl mixture containing 25 ng cDNA, 8 μM gene-specific primer mix, 5 μl 2 × SensiFAST SYBR Lo-ROX mix and water, which was amplified on Applied Biosystem 7500/7500 Fast Real-Time PCR System. The protocol was carried out by initial denaturation at 95 °C for 30 s, 40 cycles of denaturation at 95 °C for 3 s, and primer annealing at specific annealing temperature for 30 s, followed by default melt curve analysis. Each sample was analyzed in three biological replications and two technical repeats. The housekeeping gene β-actin (accession number AB181991.1) was used as a reference gene for qPCR reaction and gene expression was calculated using the 2−ΔΔCT method (Livak and Schmittgen 2001), which showed a stable expression throughout various treatment and across genotypes in wheat (Magar et al. 2022).

Co-expression Network Analysis

In this study, the 20 AP2/ERF genes significantly differently expressed under HS compared to control condition were used to build the global co-expression network using RNA-seq data in the WheatCENet database (http://bioinformatics.cau.edu.cn/WheatCENet/index.php) and visualized using Cytoscape version 3.9.1 (Shannon et al. 2003). The PCC and MR values were used to measure the co-expression relationship between genes. The thresholds for negative and positive correlation were determined by the values of the lowest 5% PCC (− 0.25) and highest 5% PCC (0.5) as in the T. aestivum global network, respectively. The co-expression network also contains genes whose protein products are reported to co-exist in the tissues along with respective TaAP2/ERF genes in different analysis (Li et al. 2022), connected by brown lines showing other-gene interactions (OGI).

Further, the Gene Set Enrichment Analysis of 20 TF genes was done using The Plant Gene Set Enrichment Analysis (GSEA) Toolkit (http://bioinformatics.cau.edu.cn/WheatCENet/GSEA.php) and the Gene Ontology (GO) analysis was done using GO Analysis Toolkit and Database for Agricultural Community (AGRiGO version 2) http://systemsbiology.cpolar.cn/agriGOv2/SEAresult2.php (Tian et al. 2017). Both the GSEA and GO analysis results with p-value < 0.05 were considered significant.

Expression Validation of Distinctly Expressed TaAP2/ERF Genes in Other Genotypes

Two genotypes Perenjori (heat tolerant) and Yitpi (heat susceptible), identified in previous study (Lu et al. 2022), were selected for further validation of significantly differentially expressed TaAP2/ERF TF genes (DEGs) which are linked to ethylene-mediated signaling pathway in this study. The two genotypes were grown in similar condition as mentioned above (Sect. 2.2), flag leaf was sampled and used for RNA extraction and cDNA synthesis. The markers used in the above experiment were amplified with the cDNAs from these two genotypes and gene expression was analyzed as per Materials and Methods, Sect. 2.4.

Statistical Analysis

Morphological data were analyzed using ANOVA and Tukey test to estimate the significant differences in the response of genotypes and treatment. The relative expression values were calculated by using the 2−ΔΔCT method (Livak and Schmittgen 2001). The expression values obtained were used to calculate the fold change (FC) for each gene by comparing the expression under treatment over control for both tolerant and susceptible genotypes, and Log2FC values were used to analyze the pattern of gene regulation. The genes were considered significantly up- or down-regulated when their Log2FC was ≥ 1 and ≤ − 1, respectively (Li et al. 2020a). The topological parameter of the network was analyzed using NetworkAnalyzer (Assenov et al. 2008).

Results

Effect of HS on Morphological Traits of Wheat Genotypes

Heat stress significantly reduced thousand kernel weight (TKW), days to flag leaf senescence (FLS100) and days to exposed peduncle senescence (PS) in the susceptible genotype (Brazil 32) after 13 DPA, while there was non-significant difference in the tolerant genotype (W156) both at 11 DPA and 13 DPA between heat treatment and the control (Fig. 1). The chlorophyll fluorescence (Fv/Fm) was significantly affected at both 11 DPA and 13 DPA in Brazil 32, while the effect was non-significant in W156. Heat damage was also significant in Brazil 32 for single grain weight of the main tiller (SgwtMT) and single grain weight of the whole plant (Sgwt), while it was non-significant in Brazil 32. However, the changes in chlorophyll content (SPAD), canopy temperature (CT), and grain weight of the main tiller (GwtMT) and total grain weight (yield) (Gwt) were significantly affected by HS in both W156 and Brazil 32 as shown in Supplementary Table 1 (Table S1). The result indicates that yield-attributing traits in W156 were less affected by HS as compared to Brazil 32.

Fig. 1
figure 1

Effect of heat stress (HS) on yield attributing factors of two contrasting wheat genotypes at grain filling stage. a change in thousand kernel weight (TKW); b change in chlorophyll fluorescence (Fv/Fm); c change in days to flag leaf senescence (FLS100); and d change in days to exposed peduncle senescence (PS), compared between controls at 11 DPA (C1) and 13 DPA (C2) and HS at 11 DPA (T1) and 13 DPA (T2). The susceptible genotype (Brazil 32) is shown in purple boxes and tolerant genotype (W156) in green boxes. Boxplots with the same letters indicate no significant difference according to the Tukey test at \(\alpha\) = 0.05 (Color figure online)

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Differential Expression of TaAP2/ERF Genes in Contrasting Genotypes Under HS

The genes showed significant expression difference between HS and control condition in RT-qPCR expression analyses were considered DEGs. Out of 22 TaAP2/ERF genes tested using gene-based markers (Table 1) in both W156 and Brazil 32 at 11 DPA and 13 DPA under heat treatment, 20 DEGs were identified. Some DEGs were expressed only in W156, some were expressed only in Brazil 32 and some were expressed in both W156 and Brazil 32, while some TF genes were not differentially expressed in either of the genotypes (Fig. 2). At the same time, DEGs were either upregulated or downregulated. Based on the expression variation in the contrasting genotypes, TF genes were categorized into four groups: Group (1) DEGs expressed only in W156 (tolerant genotype) (five genes: TraesCS1A02G221900, TraesCS7D02G158500, TraesCS6B02G331000, TraesCS4D02G298600, and TraesCS6D02G324200); Group (2) DEGs expressed only in Brazil 32 (susceptible genotype) (three genes: TraesCS2A02G542700, TraesCS5B02G193200, and TraesCS2A02G427700); and Group (3) DEGs switching their expression pattern either in one or in both W156 and Brazil 32 (12 genes:, TraesCS4B02G299600, TraesCS6B02G375400, TraesCS5B02G214400, TraesCS4B02G268100, TraesCS2D02G425700, TraesCS6B02G334700, TraesCS7B02G062200, TraesCS1A02G058400, TraesCS5D02G317100, TraesCS3A02G099200, TraesCS1A02G314300, and TraesCS2D02G515800); Group (4) TF genes not expressed differentially in either genotype (two genes: TraesCS5A02G468800 and TraesCS7A02G158000) (Table 2 and Fig. 2).

Table 1 List of 22 AP2/ERF super-family TF genes with corresponding primer sequences used in the gene expression analysis
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Fig. 2
figure 2

Expression patterns of 22 TaAP2/ERF family genes under HS in two contrasting wheat genotypes. The expression of genes after a 11 days post-anthesis (11 DPA) HS in tolerant genotype (T1) and susceptible genotype (S1); b 13 days post-anthesis (13 DPA) HS in tolerant genotype (T2) and susceptible genotype (S2). The tolerant genotypes are shown in blue color and susceptible genotypes are in orange color. The bars above 1 or below − 1 indicate significant expression of genes. The genes are classified into four groups: Group (1) genes expressed significantly only in the tolerant genotype highlighted in green; Group (2) genes expressed significantly only in the susceptible genotype highlighted in pink; Group (3) eight genes expressed significantly in both tolerant and susceptible genotypes highlighted in blue; Group (4) gene with no significant expression in either genotype with no highlight. The number of significantly expressed genes are more at 13 DPA as compared to 11 DPA under HS condition (Color figure online)

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Table 2 Expression values of 22 TaAP2/ERF family TF genes under HS in W156 (heat tolerant) and Brazil 32 (heat susceptible)
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Expression of TaAP2/ERF Genes Vary at Different Time Points Under HS

Heat treatment duration (time points) also affected the pattern of gene expression. The differential expression of some genes was induced early at 11 DPA, while some were induced later at 13 DPA. They were differentially expressed either at both treatment time points (11 DPA and 13 DPA), in only one treatment time point or not expressed differentially at both time points. These genes were classified into four groups: Group 1) DEGs at both time points; Group 2) DEGs only at 11 DPA; Group 3) DEGs only at 13 DPA; Group 4) genes with no differential expression at both time points in either genotypes. In the W156 (tolerant genotype), nine genes (TraesCS1A02G221900, TraesCS7D02G158500, TraesCS6B02G331000, TraesCS4D02G298600, TraesCS6D02G324200, TraesCS2D02G425700, TraesCS6B02G334700, TraesCS4B02G299600, and TraesCS5B02G214400) were differentially expressed at both time points (Group 1), one gene (TraesCS6B02G375400) was differentially expressed only at 11 DPA (Group 2), four genes (TraesCS1A02G314300, TraesCS4B02G268100, TraesCS3A02G099200, and TraesCS2D02G515800) were differentially expressed only at 13 DPA (Group 3) and the remaining eight genes were not differentially expressed at either time point (Group 4) (Fig. 3A). The results suggest gene expressed at 11 DPA were also expressed and remained consistent with increasing duration of HS.

Fig. 3
figure 3

Expression patterns of 22 TaAP2/ERF family genes under HS between two heat treatment time points in contrasting wheat genotypes. a The expression of genes in tolerant genotype after 11 days post-anthesis HS (11 DPA) in blue color and after 13 days post-anthesis HS (13 DPA) in orange color. b The expression of genes in susceptible genotypes after 11 DPA HS (11 DPA) in pink color and after 13 DPA HS (13 DPA) in green color. The bars above 1 or below − 1 indicate significant expression of genes. The genes are classified into four groups: Group (1) genes expressed in both time points highlighted in orange; Group (2) genes expressed only at 11 DPA highlighted in yellow; Group (3) genes expressed only at 13 DPA highlighted in purple; Group (4) gene with no significant expression in any time points with no highlight. The number of significantly expressed genes are higher in tolerant genotype as compared to susceptible genotype and more at 13 DPA as compared to 11 DPA under HS condition (Color figure online)

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Similarly, in Brazil 32 (susceptible genotype), four genes (TraesCS2A02G542700, TraesCS5B02G193200, TraesCS2A02G427700, and TraesCS4B02G268100) were differentially expressed at both time points (Group 1); two genes (TraesCS2D02G425700 and TraesCS6B02G334700) were differentially expressed only at 11 DPA (Group 2); eight genes (TraesCS7B02G062200, TraesCS1A02G058400, TraesCS5D02G317100, TraesCS4B02G299600, TraesCS3A02G099200, TraesCS6B02G375400, TraesCS5B02G214400, and TraesCS2D02G515800) were differentially expressed only at 13 DPA (Group 3); and the remaining eight genes were not differentially expressed at either time point (Group 4) (Fig. 3B). The results suggest that longer duration of HS induces the expression of more heat responsive genes.

Some genes were consistent in their expression pattern at both treatment time points (11 DPA and 13 DPA) including five genes (TraesCS1A02G221900, TraesCS7D02G158500, TraesCS6B02G331000, TraesCS4D02G298600, and TraesCS6D02G324200) differentially expressed only in W156, three genes (TraesCS2A02G542700, TraesCS5B02G193200, and TraesCS2A02G427700) differentially expressed only in Brazil 32 and two genes (TraesCS5A02G468800 and TraesCS7A02G158000) not expressed differentially in both genotypes. However, a number of genes showed differential expression pattern at 11 DPA and 13 DPA (Fig. 3 and Table 2).

Co-expression Network Analysis of TaAP2/ERF Genes in Wheat

Gene co-expression network analysis of 20 DEGs, based on RT-qPCR expression analysis showed that TaAP2/ERF genes identified and validated in our study were involved in three different types of co-expression relationship with their target genes. Some genes showed positive co-expression relationship with target genes (351), negative co-expression relationship with target genes (269) and co-expression relationship with genes whose protein products were reported to co-exist in the tissues along with respective TaAP2/ERF genes in different analysis, indicated here as Other-Gene Interaction (OGI) (149) (Fig. 4 and Supplementary Table 2). In this study, eight DEGs were identified with relatively higher degree of nodes (number in bracket): TraesCS6B02G375400 (131), TraesCS1A02G221900 (107), TraesCS6D02G324200 (105), TraesCS6B02G331000 (90), TraesCS4B02G299600 (80), TraesCS5B02G193200 (64), TraesCS4D02G298600 (61), and TraesCS1A02G058400 (59) which showed that these DEGs were relatively important hub TF genes in the wheat gene regulatory network with major functional importance. The details of all the genes involved in the co-expression network are given in Supplementary Table 2 (Table S2).

Fig. 4
figure 4

Co-expression network of 20 TaAP2/ERF TF genes responsive to HS at grain filling stage and their target genes showing positive co-expression relationship (351) in pink rectangles, negative co-expression relationship (269) in light blue rectangles, and other-gene interaction with co-existence of protein products (149) in brown rectangles. The query TaAP2/ERF TF genes are highlighted with three different shapes and colors; green diamond for genes expressed only in tolerant genotype, red circle for gene expressed only in susceptible genotype and blue triangles for genes which are expressed in both tolerant and susceptible genotypes. The hub TF genes are highlighted with lime ellipse are TF hub genes related to Ethylene-activated signaling pathway and yellow ellipse are TF hub genes not related to ethylene signaling (Color figure online)

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The Gene Ontology (GO) enrichment analysis showed DEGs were significantly enriched with GO terms related to heat acclimation, positive regulation of gene expression, negative regulation of gene expression, ethylene-activated signaling pathway, and regulation of different biosynthetic and metabolic processes (RNA, nitrogen compound, cellular macromolecules, wax, nucleobase-containing compound, etc.) as given in Supplementary Table 3 (Table S3). Most of the DEGs related to heat acclimation, positive and negative regulation of gene expression were involved in ethylene-activated signaling pathway (TraesCS2D02G425700, TraesCS6B02G375400, TraesCS5B02G193200, TraesCS6D02G324200, TraesCS7D02G158500, TraesCS2D02G515800, TraesCS4B02G268100, TraesCS4D02G298600, TraesCS7B02G062200, TraesCS1A02G058400, TraesCS3A02G099200, TraesCS5B02G214400, TraesCS2A02G427700, TraesCS2A02G542700, TraesCS6B02G331000, and TraesCS1A02G314300) except TraesCS1A02G221900, which suggested that heat responsive differential expression of TaAP2/ERF TF gene was dominantly regulated by ethylene-activated signaling pathway to induce heat acclimation (Table S3). The significance level of GO terms was determined by adjusted p-value (p ≤ 0.05) and represented with degree of color and named from level 1 (lowest at p-value ≤ 0.05) to level 9 (highest at p-value ≤ 5e-10). The Fig. clearly showed that the heat acclimation and positive regulation of differential gene expression was highly significant, reaching significance level 6 (p-value ≤ 5e−7) and negative regulation of differential gene expression is also significant with slightly lower level of significance to level 3 (p-value ≤ 5e−04) (Fig. 5). In addition, the six TF hub genes (TraesCS6B02G375400, TraesCS6D02G324200, TraesCS6B02G331000, TraesCS5B02G193200, TraesCS4D02G298600, and TraesCS1A02G058400) identified from co-expression network analysis were also related to ethylene-activated signaling pathway.

Fig. 5
figure 5

Direct acyclic graph (DAG) of GO terms illustrating how biological processes are involved in heat responsive gene expression and heat acclimation. The GO terms labeled by their GO ID, term definition, and statistical information, mentioned in the above boxes are related to significantly expressed candidate TaAP2/ERF genes under HS condition in contrasting wheat genotypes. The highlighted color pattern in the chart shows the level of significance positively correlated to the enrichment level of the terms and the color and pattern of arrows show the relationship between two GO terms as shown in the legend given on top-right corner of the graph. The significance level 1 is the lowest at p-value ≤ 0.05 and the significant level 9 is the highest at p-value ≤ 5e−10. The rank direction of the graph is set from top to bottom (Color figure online)

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Further, Gene Set Enrichment Analysis (GSEA) revealed that 20 DEGs were significantly enriched with 68 gene sets related to different biological processes, cellular components, and molecular functions as shown in Supplementary Table 4 (Table S4). The gene sets related to response to heat, heat acclimation, seed development, tissue development, along with hormone signaling pathways (ethylene, cytokinin, auxin, Jasmonic acid, abscisic acid) and red or far-red light signaling pathways were significantly enriched (p-value ≤ 0.05). Among the hormone signaling pathways, the ethylene-activated signaling pathway consists of 16 (80%) DEGs (TraesCS2D02G425700, TraesCS6B02G375400, TraesCS5B02G193200, TraesCS3A02G099200, TraesCS7D02G158500, TraesCS2D02G515800, TraesCS7B02G062200, TraesCS4B02G268100, TraesCS4D02G298600, TraesCS2A02G542700, TraesCS1A02G058400, and TraesCS1A02G314300). These DEGs were also related to heat response (TraesCS2D02G515800 and TraesCS1A02G058400); heat acclimation (TraesCS6B02G375400, TraesCS7D02G158500, TraesCS7B02G062200, and TraesCS4D02G298600); seed development (TraesCS2D02G515800 and TraesCS1A02G058400); tissue development (TraesCS6B02G375400); and red or far-red light signaling pathway (TraesCS4B02G268100) (Table S4). This result revealed that ethylene-activated signaling pathway is major transcriptional regulatory pathway in heat tolerance by TaAP2/ERF TF genes in wheat.

Validation of Selected DEGs Expressed at Both Time Points in Other Genotypes

The DEGs associated with ethylene-activated signaling pathway and consistently expressed at both time points were further validated for their expression in an additional pair of wheat genotypes (Perenjori and Yitpi), including four genes in the tolerant genotype W156 (TraesCS7D02G158500, TraesCS6B02G331000, TraesCS4D02G298600, and TraesCS6D02G324200), and three genes in the susceptible genotypes Brazil 32 (TraesCS2A02G542700, TraesCS5B02G193200, and TraesCS2A02G427700). Three genes (TraesCS6B02G331000, TraesCS6D02G324200, and TraesCS4D02G298600) showed differential expression in both tolerant genotypes (Perenjori and W156), and two genes (TraesCS2A02G542700 and TraesCS5B02G193200) were expressed similarly in both susceptible genotypes (Brazil32 and Yitpi), showing consistency across wheat genotypes (Table 3).

Table 3 Comparison of expression patterns of ethylene-activated signaling pathway related DEGs consistently expressed at both time points (11 DPA and 13 DPA) in heat tolerant (W156) and heat susceptible (Brazil 32) genotypes and in additional one pair of wheat genotypes (heat tolerant: Perenjori; heat susceptible: Yitpi)
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Discussion

Wheat production is limited by HS on a large scale and the grain filling stage is highly vulnerable to HS (Rangan et al. 2020). The physiological properties of a plant under stress conditions are largely influenced by plant hormones. Hormonal regulation of ERFs is a complex process. The major phytohormones regulating ERF under environmental stresses involve ethylene and it interacts with other phytohormones such as auxin, gibberellin, brassinosteroid, and abscisic acid. However, the regulation of ERF by phytohormone is highly temperature dependent. For example, the tomato ERF which were regulated by ethylene and brassinosteroid at room temperature (20 °C) were inhibited by auxin, while these ERF were inhibited by gibberellin when the fruits were transferred from cold storage (4 °C) to retailer condition (20 °C) (Park et al. 2023). The major hormones produced in plants regulating HS response are ABA, JA, ethylene, and SA, and ethylene response factors (ERFs) has been reported to play critical role in ethylene signaling and HS response (Huang et al. 2022). Ethylene plays important role in different aspects of plant growth and development including seed germination, shoot and root growth, flowering, fruit ripening, senescence, abscission, response to biotic and abiotic stresses (Piya et al. 2018; Lü et al. 2018; Iqbal et al. 2017; Huang et al. 2021). The major components of ethylene-signaling pathway include ethylene receptor (ER) in the membrane of endoplasmic reticulum; a protein kinase, constitutive triple response 1 (CTR1); an ER-localized transmembrane protein called ethylene-insensitive 2 (EIN2); and transcription factors such as EIN3, EIN3-like (EIL) (Binder 2020). ERF acts as downstream receptors of EIN3 (Huang et al. 2021). Ethylene is produced in wheat grain under heat stress (Hays et al. 2007). Therefore, heat induced ethylene production activates EIN3, which induced expression of ERF as downstream genes, thus regulating the expression of ERFs under HS (Binder 2020).

Under HS condition, plant naturally activates antioxidant defense. However, ethylene treatment of heat stressed plant boosts the level of antioxidant enzymes (SOD, APX, and GR) contributing to enhanced heat tolerance (Gautam et al. 2022). Thus, ethylene regulates heat tolerance through signaling and activation of oxidative defense. Ethylene regulated basal heat tolerance by signaling active oxidative defenses during HS has been reported in Arabidopsis (Larkindale et al. 2005). Also, ethylene-mediated signaling is reported to enhance heat tolerance in rice by reducing oxidative damage and maintaining chlorophyll content under HS (Wu and Yang 2019). The AP2/ERF superfamily TF genes play important regulatory roles in biotic and abiotic stress tolerance in plants. Various studies have reported that ethylene production and signaling are highly influenced by heat stress and ethylene-mediated response to heat stress is regulated by AP2/ERF TF gene (Huang et al. 2023). In this study, we analyzed the expression pattern of AP2/ERF TF genes in contrasting wheat genotypes at the grain filling stage under HS. The yield attributing morphological traits were suppressed in Brazil 32 as compared to W156 and TF genes were differentially expressed in W156 and Brazil 32 under HS. These DEGs were associated with heat response, heat acclimation, seed development, regulation of gene expression, and ethylene-activated signaling pathway. The ethylene signaling related DEGs were also differentially expressed in one additional pair contrasting wheat genotypes under HS.

Post-Anthesis HS Affected Seed Development

In this study, HS imposed at grain filling stage significantly reduced the TKW in susceptible Brazil 32 at 3-day stress treatment (13 DPA), while the effect was non-significant in tolerant W156 (Fig. 1). This result is in line with the results of heat tolerant genotypes of CIMMYT spring wheat which outperformed the susceptible genotypes showing higher TKW under HS (Fleitas et al. 2020). The TKW is a measure of grain weight, and it is determined by the duration and rate of grain filling. HS at grain development shortens the grain filling duration, decrease starch and protein accumulation through restricted activity of grain biosynthesis enzymes and impaired flag leaf assimilatory efficiency, and stem reserves mobilization in wheat (Ullah et al. 2021; Abdelrahman et al. 2020). Reduced kernel weight due to grain filling stage HS induced ethylene production was also reported in wheat, where 12-fold increase in ethylene production in flag leaf of heat susceptible genotype “Karl 92,” and no change in ethylene content of tolerant genotype “Halberd” was observed, which accelerated senescence and arrested grain development (Hays et al. 2007).

Flag leaf plays a pivotal role in heat tolerance at reproductive stage of wheat, which is measured by canopy temperature, leaf senescence, chlorophyll fluorescence, SPAD (Soil Plant Analysis Development) meter, along with grain number, grain weight (Narayanan 2018; Driedonks et al. 2016). In this study, HS significantly accelerated senescence of flag leaf and exposed peduncle in the Brazil 32 at 13 DPA HS, while W156 maintained greenness and showed minimal damage. Similarly, significantly enhanced leaf senescence and reduced grain size and weight in heat susceptible genotype, while non-significant effect in tolerant genotype was reported by Ullah et al. (2019). Transcription factors integrates ethylene signals with environmental and developmental stresses to regulates leaf senescence (Koyama 2014). Heat induced endogenous ethylene biosynthesis activates ethylene insensitive 2 (EIN2) receptor, which enhance expression of ERF, WRKY, and NAC genes contributing to upregulation of senescence-associated genes (SAG) leading to premature flag leaf senescence (Abdelrahman et al. 2017). Ethylene induced premature leaf senescence and early increase in mRNA level of SAGs was also reported in Arabidopsis, where etr1-1 mutant with suppressed ethylene production exhibited delayed senescence with delayed induction of SAGs and enhanced expression of photosynthesis-associated genes (PAGs) (Grbic and Bleecker 1995). Similarly, ZmEIN3 induce expression of ZmNAC126 enhanced chlorophyll degradation and ethylene-triggered leaf senescence in maize and Arabidopsis (Yang et al. 2020). The delayed senescence allows longer grain filling duration with extended photosynthesis in plant contributing to better grain development (Chapman et al. 2021) and resulting in higher yield (Yadav et al. 2022) and heat tolerance (Pinto and Reynolds 2015).

Calvin cycle in stroma and thylakoid lamellae in chloroplast are the primary organelles for heat response and signaling. Chlorophyll fluorescence is considered as selection tool for thermostability and better functioning of photochemical reactions and photo-assimilation in wheat cultivar under HS (Jain et al. 2017). In this study, both 11 DPA and 13 DPA HS was sufficient to significantly reduce chlorophyll fluorescence (Fv/Fm) in flag leaf in Brazil 32. The lower Fv/Fm values indicate higher thylakoid membrane damage, limiting the seed development in susceptible wheat genotypes under post-anthesis HS (Haque et al. 2014). Similar effect of post-anthesis HS is reported by Pradhan and Prasad (2015), where the heat induced reduction in Fv/Fm and chlorophyll content was able to decrease individual grain weight by 44% resulting in lower yield. HS induced generation of reactive oxygen species disrupts the thylakoid membrane, chloroplast, and plasma membrane, which deactivate photosystem, reduce photosynthetic efficiency, and inactivate rubisco affecting photosynthate production and allocation during grain filling (Lal et al. 2022). Exogenous application of ethylene is reported to enhance the proline accumulation production of antioxidants and expression of psbA and psbB, while reduces the production of glutathione, which provides defense against abiotic stresses (Gautam et al. 2022). The psbA and psbB are photosynthetic genes, which encodes D1 protein and CP47, respectively, and their expressions improve photosynthetic performance under HS (Sehar et al. 2022). Similarly, exogenous ethylene application in rice regulated photosynthesis via carbohydrate metabolism and antioxidant system, thus inducing heat tolerance (Gautam et al. 2022). The morphological traits in Brazil 32 were significantly compromised under HS possibly due to heat induced endogenous ethylene production, accelerating senescence and limiting photosynthetic efficiency of wheat.

Heat Induced Expression of TaAP2/ERF Genes at Grain-Filling Stage in Wheat

The AP2/ERF TF genes play important role in abiotic stress response through transcriptional regulation of heat responsive genes, hormonal pathways, and metabolite production. Ethylene, a gaseous hormone which influence ethylene insensitive 3 (EIN3) protein, a master regulator of ethylene signaling pathway, and AP2/ERF TF genes, activates expression of AP2/ERF genes under stress condition (Chang et al. 2013). Ethylene signaling is complicated processes and regulation of heat tolerance by ethylene signaling vary in different plant species (Li et al. 2020b). In this study, the RT-qPCR expression analysis of 22 TaAP2/ERF TF genes in two contrasting wheat genotypes under HS at 11 DPA and 13 DPA show differential expression of genes (Fig. 2). The number of differentially expressed TaPA2/ERF genes (DEGs) in W156 (five) was almost double the number of DEGs in Brazil 32 (three), while remaining 12 DEGs were inconsistent in their expression between W156 and Brazil 32, showing intervarietal difference in heat induced gene expression. Similarly, significant intervarietal differences in the expression level of HMW-GS, GBSSI, and Pinb gene was observed after post-anthesis heat treatment (10 DPA to 17 DPA) in seven European wheat varieties (Tomás et al. 2020). In addition, higher upregulation of Squamosa promoter binding protein (SPL) TF gene by 10.8 folds in heat tolerant rice genotype (N22) and only twofold downregulation in heat susceptible genotype (Vandana) was reported after long duration HS at reproductive stage (Bhogireddy et al. 2021). Also, HS induce expression of OsAP2 TF genes in indica rice genotypes showed differential expression pattern under stress in different genotypes (Ahmed et al. 2021). Singh et al. (2021) has explained heat induced ethylene production activates expression of ERF TF genes via ethylene signaling pathway, which regulate expression of HsfA2 to induce heat tolerance in plants. Several transgenic studies have also reported role of AP2/ERF genes in heat tolerance. Overexpression of DREB2C in transgenic Arabidopsis enhanced heat tolerance by regulating the expression of heat-inducible genes containing DRE/CRT elements in their promoter region (Lim et al. 2007). AtDREB2C activates transcription of HsfA3 in mesophyll protoplast of Arabidopsis and induce expression of HSP genes under HS regulating heat tolerance (Chen et al. 2010). The AP2/ERF are integral part of signaling cascade which regulate expression of stress responsive genes thus producing developmental effects in phenotypes and ethylene is important component of AP2/ERF mediated plant responsive pathways (Phukan et al. 2017). The rice ERF gene (OsERF115) regulated the ethylene-mediated grain development, and its overexpression positively affected the grain size and weight by enhancing grain filling (Liu et al. 2022).

Further, the number of DEGs analyzed in 2 time points (11 DPA and 13 DPA), showed higher number of DEGs (nine) in tolerant W156 as compared to the number of DEGs (six) in susceptible Brazil 32 at 11 DPA, whereas the number of DEGs increased to 13 in W156 and 12 in Brazil 32 at 13 DPA. This coincides with the results of Su et al. (2019) where, increase in the number of DEGs in flag leaf of wheat was found when duration of HS changed from five minutes (276 DEGs) to 4 h (2382 DEGs). In addition, transcript analysis showed highest expression level (5.29) AP2 transcription factor (TaDREB2) at 12 DAF as compared to 10 DAF heat treatment in wheat (Ko et al. 2018). The number DEGs in both time points was nine in W156 and four in Brazil 32, which suggested the early induction and consistent expression of higher number of TaAP2/ERF TF genes in W156 with increase in duration of HS was responsible for heat tolerance through initial heat acclimation. Similar results of consistent high expression of HSP101 genes in leaves of heat tolerant C306 as compared to heat susceptible PBW343, was responsible for heat tolerance of C306 under long-term (15 DPA) HS (Almeselmani et al. 2011). Rapid HS induction and sustained expression pattern of HSPs (HSP 17, HSP 40, HSP 70, and HSP 90) in heat tolerant peanut genotypes (ICGS 44 and GG 7) as compared to heat susceptible genotypes (DRG 1 and Ak 159) maintained protein aggregation and regulated downstream gene expression for heat tolerance (Chakraborty et al. 2018). The ethylene biosynthesis and signaling regulated by ERF1, is required for CO2-induced HS response and induction of heat shock factor A2, HSP70, and HSP90 in Tomato (Pan et al. 2019). AP2/ERF TF regulate expression of downstream genes through ethylene-signaling pathways under abiotic stress conditions such as drought stress in cassava (Ren et al. 2017), salt stress in Arabidopsis (Zhang et al. 2011), freezing tolerance in tobacco and tomato (Zhang and Huang 2010).

Ethylene-Signaling Regulated Heat Tolerance at Grain-Filling Stage in Wheat

Heat response of TF genes involves interaction of different biological pathways regulated by coordinated interaction of cluster of genes or co-expressed genes, metabolic and signaling pathways. A single TF can regulate multiple target genes and manipulate all or many steps in metabolic pathways (Grotewold 2008). The WheatCENet, generates co-expression networks using the Pearson’s correlation coefficient (PCC) and mutual rank (MR) algorithm to measure the co-expression relationship between wheat genes which helps to identify co-expression modules containing key genes associated with stress tolerance (Lv et al. 2020). In this study, the global co-expression network analysis of 20 DEGs identified eight hub TF genes (Table S2 and Fig. 4) which were connected with large number of target genes (Deng et al. 2019) co-expressing either positively, negatively or interacting with their protein products to perform biological function suggests their biological importance (Fig. 4). Similarly, co-expression network was used to identify hub genes associated with drought resistance in wheat (Lv et al. 2020) and to select hub TF as putative candidates for stress tolerance in wheat (Saidi et al. 2022).

The Gene Ontology (GO) analysis and pathway enrichment analysis are useful tools to understand the heat sensing, signaling, functions, and metabolic pathways involved in heat tolerance in wheat (Su et al. 2019; Wang et al. 2019). By these methods, four DEGs (TraesCS1A02G221900, TraesCS7D02G158500, TraesCS4D02G298600, and TraesCS6D02G324200) were found involving in positive regulation of gene expression, response to heat, heat acclimation, and four DEGs (TraesCS7D02G158500, TraesCS6B02G331000, TraesCS4D02G298600, and TraesCS6D02G324200) were related to ethylene-activated signaling pathway (Fig. 5 and Table S3). Two of these DEGs have homologs in Arabidopsis TraesCS1A02G221900 (AT5G05410 and AT3G11020) and TraesCS6B02G331000 (AT1G22190, AT1G78080, AT1G36060). These results suggested TaAP2/ERF TF genes regulate heat tolerance in grain filling stage of wheat through ethylene-mediated positive regulation of gene expression, influencing heat response and heat acclimation. Different studies reported AP2/ERF involvement in regulating expression of downstream genes through ethylene-signaling pathways under abiotic stress conditions such as drought stress in cassava (Ren et al. 2017), salt stress in Arabidopsis (Zhang et al. 2011), freezing tolerance in tobacco and tomato (Zhang and Huang 2010). The major components of ethylene-signaling pathway are ethylene receptor (ER) in the membrane of endoplasmic reticulum; a protein kinase, constitutive triple response 1 (CTR1); an ER-localized transmembrane protein called ethylene-insensitive 2 (EIN2); transcription factors such as EIN3, EIN3-like (EIL), ethylene response factors (ERF) (Binder 2020). ERF1 is downstream gene activated by EIN3 in response to ethylene gas in ethylene signaling pathway (Solano et al. 1998). Ethylene-mediated signaling maintains chlorophyll content, reduce oxidative damage, enhance heat tolerance in rice. It also regulates the mRNA transcripts of Hsfs and ethylene signaling related genes under HS and their interaction confer heat tolerance (Wu and Yang 2019).

Similarly, in Brazil 32, two DEGs TraesCS2A02G542700 and TraesCS2A02G427700 were involved in negative regulation of gene expression, and all three DEGs (TraesCS2A02G542700, TraesCS5B02G193200, and TraesCS2A02G427700) were related to ethylene-activated signaling pathway. This suggests TaAP2/ERF TF genes regulate heat susceptibility by negatively regulating the gene expression in Brazil 32 through ethylene signaling. Similarly, overexpression of ERF49 in transgenic Arabidopsis suppressed the expression of HS inducible DREB2A gene and decreased heat tolerance (Chen et al. 2022). It also coincides with results of (Clarke et al. 2009), where ethylene acts as negative regulator of HS response in Arabidopsis, and ethylene-insensitive 2–1 (ein2-1) mutant lacking ethylene signaling pathway was heat tolerant.

Also, the nine DEGs expressed in both genotypes were associated with ethylene-activated signaling pathway. The two DEGs (TraesCS1A02G314300 and TraesCS2D02G425700) were involved in ethylene-mediated negative regulation of gene expression, while three DEGs (TraesCS7B02G062200, TraesCS6B02G375400, and TraesCS5B02G214400) were involved in ethylene-mediated positive regulation of gene expression, response to HS and heat acclimation for heat tolerance. This reveals that DEGs involved in positive regulation of gene expression are only involved in heat acclimation conferring heat tolerance in heat tolerant genotype. Interestingly, one DEG (TraesCS3A02G099200) was involved in both positive and negative regulation of gene expression through ethylene signaling, which suggest it can act both as activator and suppressor of downstream target gene expression. In addition, two DEGs (TraesCS1A02G058400 and TraesCS2D02G515800) were involved in heat response and seed development under HS through ethylene signaling. Similarly, the rice ERF gene (OsERF115) regulates the ethylene-mediated grain development, and its overexpression positively affected the grain size and weight by enhancing grain filling (Liu et al. 2022). These results suggested that HS at grain filling stage of wheat induce ethylene production, which activate EIN3 to influence expression of TaAP2/ERF TF genes through ethylene activated signaling pathway and maintain heat tolerance via heat response, heat acclimation, regulating expression of downstream heat responsive genes while maintain seed development.

The three major DEGs (TraesCS6B02G331000, TraesCS6D02G324200, and TraesCS4D02G298600) validated in both tolerant genotypes (Perenjori and W156), are potential heat tolerant genes for grain filling stage heat tolerance. Similarly, two DEGs (TraesCS2A02G542700 and TraesCS5B02G193200) validated in both susceptible genotypes (Brazil32 and Yitpi) suggested their potential role as heat susceptible genes at grain filling stage (Table 3). In addition, three DEGs (TraesCS7B02G062200, TraesCS3A02G099200, and TraesCS2D02G425700) were expressed consistently in both tolerant and susceptible genotypes, where TraesCS7B02G062200 is involved in ethylene-mediated heat response, heat acclimation through positive regulation of gene expression and TraesCS2D02G425700 is involved in ethylene-mediated negative regulation of gene expression. However, TraesCS3A02G099200 is involved in both positive and negative regulation of gene expression through ethylene signaling (Table 3). These genes consistently expressed across time points and genotypes might be useful resource for heat tolerance breeding in wheat for grain filling stage HS. A good understanding of transcriptional regulation of TF can be used to produce novel variation by altering the gene expression through genetic engineering in crop improvement for complex traits like yield and heat tolerance (Springer et al. 2019). Therefore, our result suggest that selective TF genes can be used as promising tools for heat tolerance breeding in wheat for grain filling stage heat stress.

Conclusion

Heat stress response of contrasting wheat genotypes at grain filling stage showed pronounced effect on chlorophyll fluorescence, senescence, and thousand kernel weight in heat susceptible genotype. The heat induced expression of TaAP2/ERF TF genes was affected by genotype, growth stage, and duration of HS. However three TF genes (TraesCS6B02G331000, TraesCS6D02G324200, and TraesCS4D02G298600) consistently expressed in both tolerant genotypes (W156 and Perenjori), were associated with ethylene-mediated heat response, heat acclimation and positive regulation of gene expression, while two TF genes (TraesCS2A02G542700 and TraesCS5B02G193200) expressed in both susceptible genotypes (Brazil 32 and Yitpi) were associated with ethylene-mediated gene expression regulation and does not show any role in heat acclimation. The TF genes regulated expression of a cluster of target genes under HS which are associated with different metabolic and biosynthetic pathways required for seed development and heat acclimation. We suggest that HS induced ethylene production in wheat leaf activates EIN3 to induce expression of TaAP2/ERF TF genes, which positively regulate expression of heat responsive target genes in tolerant genotype via. ethylene-activated signaling pathway and generate heat tolerant phenotype such as response to HS, heat acclimation to maintain grain development at grain filling stage. Therefore, regulatory role of TaAP2/ERF TF genes might be associated with ethylene-activated signaling pathway in wheat under HS condition.