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. 2024 Oct 18;66(11):2346–2361. doi: 10.1111/jipb.13789

The LpHsfA2‐molecular module confers thermotolerance via fine tuning of its transcription in perennial ryegrass (Lolium perenne L.)

Guangjing Ma 1,2, Zhihao Liu 3, Shurui Song 1,2, Jing Gao 1,2, Shujie Liao 4, Shilong Cao 4, Yan Xie 1, Liwen Cao 1, Longxing Hu 4, Haichun Jing 5,6,✉, Liang Chen 1,6,✉
PMCID: PMC11583844  PMID: 39422287

ABSTRACT

Temperature sensitivity and tolerance play a key role in plant survival and production. Perennial ryegrass (Lolium perenne L.), widely cultivated in cool‐season for forage supply and turfgrass, is extremely susceptible to high temperatures, therefore serving as an excellent grass for dissecting the genomic and genetic basis of high‐temperature adaptation. In this study, expression analysis revealed that LpHsfA2, an important gene associated with high‐temperature tolerance in perennial ryegrass, is rapidly and substantially induced under heat stress. Additionally, heat‐tolerant varieties consistently display elevated expression levels of LpHsfA2 compared with heat‐sensitive ones. Comparative haplotype analysis of the LpHsfA2 promoter indicated an uneven distribution of two haplotypes (HsfA2 Hap1 and HsfA2 Hap2) across varieties with differing heat tolerance. Specifically, the HsfA2 Hap1 allele is predominantly present in heat‐tolerant varieties, while the HsfA2 Hap2 allele exhibits the opposite pattern. Overexpression of LpHsfA2 confers enhanced thermotolerance, whereas silencing of LpHsfA2 compromises heat tolerance. Furthermore, LpHsfA2 orchestrates its protective effects by directly binding to the promoters of LpHSP18.2 and LpAPX1 to activate their expression, preventing the non‐specific misfolding of intracellular protein and the accumulation of reactive oxygen species in cells. Additionally, LpHsfA4 and LpHsfA5 were shown to engage directly with the promoter of LpHsfA2, upregulating its expression as well as the expression of LpHSP18.2 and LpAPX1, thus contributing to enhanced heat tolerance. Markedly, LpHsfA2 possesses autoregulatory ability by directly binding to its own promoter to modulate the self‐transcription. Based on these findings, we propose a model for modulating the thermotolerance of perennial ryegrass by precisely regulating the expression of LpHsfA2. Collectively, these findings provide a scientific basis for the development of thermotolerant perennial ryegrass cultivars.

Keywords: haplotype; heat shock transcription factors; heat tolerance, perennial ryegrass

In perennial ryegrass (Lolium perenne), the heat shock factors LpHsfA2/4/5 influence heat tolerance by self‐regulating and regulating the expression of the heat‐shock protein gene LpHSP18.2 and the ascorbate peroxidase gene LpAPX1. Natural variation in the LpHsfA2 promoter region leads to differences in heat tolerance in perennial ryegrass germplasm.

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INTRODUCTION

High‐temperature stress significantly impacts the survival and productivity of plants. Plants exhibit a wide range of variability in their adaptive capacity to temperature. Exposure to elevated temperatures beyond a specific threshold induces heat stress adversely affecting plant physiology and development (Hasanuzzaman et al., 2013; Qu et al., 2013; Gong et al., 2020; Li et al., 2023). Heat stress leads to alterations in the biomembrane's intermolecular bonds, increased membrane fluidity and, consequently, a significant increase in electrolyte leakage (Hasanuzzaman et al., 2013; Shekhawat et al., 2022). Additionally, heat stress triggers chlorophyll degradation and disrupts photosynthetic processes, resulting in the accumulation of reactive oxygen species (ROS) (Jaspers and Kangasjarvi, 2010; Hasanuzzaman et al., 2013). Meanwhile, plants immediately initiate a series of defense mechanisms collectively termed the heat stress response to protect themselves from heat stress‐induced damage.

Heat stress responses are primarily mediated by a distinct class of proteins termed as the heat shock factors (Hsfs). Moreover, heat shock proteins (HSPs) play an important role in heat stress responses (Sun et al., 2002; Gong et al., 2020; Li et al., 2023). Hsfs are highly conserved transcription factors crucial for heat stress response (HSR) and thermotolerance across eukaryotes. Multiple Hsfs have been identified in various plant species (Nover et al., 1996; von Koskull‐Doring et al., 2007; Meng et al., 2016; Yu et al., 2022). For example, Arabidopsis harbors 24 Hsf genes grouped into three major classes: HsfA, HsfB, and HsfC (Nover et al., 2001; Ogawa et al., 2007; Liu et al., 2011). In rice, 25 Hsf genes and 74 HSP genes were identified (Li et al., 2023). Notably, HsfA1s appear to be a key master regulator upstream of the thermal regulatory network (Liu et al., 2011, 2013; Liu and Charng, 2013). In Arabidopsis, Hsc70‐1 forms a trimeric complex with HsfA1d and HsfA1e under normothermic conditions, and this association is disrupted under conditions of heat stress, leading to the activation of downstream signaling mechanisms (Sung and Guy, 2003). HsfA2 was regulated by HsfA1d and HsfA1e, serving as an integral regulator of both basal and acquired thermotolerance. This regulatory role is mediated by activating the expression of stress‐related genes and several Hsf genes, HsfA7a, HsfA7b, HsfB1, and HsfB2a (Charng et al., 2007; Hahn et al., 2011; Nishizawa‐Yokoi et al., 2011). Tomato HsfA1 and HsfA2 could create hetero‐oligomeric superactivators, which synergistically activate heat stress gene expression (Scharf et al., 1990; Chan‐Schaminet et al., 2009). Furthermore, the heat stress regulatory network encompasses additional genes that function independently of HsfA1. Hsfs interacts with the promoters of other genes to further regulate the expression of downstream genes involved in the HSR. Under heat stress, Hsfs can rapidly regulate and induce the expression of HSP to assist the degradation of unfolded proteins. Concurrently, Hsfs also regulate the expression of enzymes instrumental in ROS scavenging, such as ascorbate peroxidase (APX), inositol‐3‐phosphate synthase (IPS) and galactinol synthase 1 (GolS1) (Schramm et al., 2006; Charng et al., 2007; Vanderauwera et al., 2011; Ling et al., 2021). Additionally, it has been reported that HsfA‐type transcription factors could participate in plant responses to stress by regulating cellulose synthase (ZmCesA2) (Li et al., 2024) and genes related to flavanol biosynthesis (CSH1 and FLS1) (Song et al., 2024). Hsfs act as the central regulatory factor in response to heat stress and also participate in other abiotic stresses (Yang et al., 2023a; Bakery et al., 2024).

Perennial ryegrass (Lolium perenne L.) is distinct in its vigorous growth and extensive tillering habit, making it one of the globally favored species for turf application (Ahloowalia, 1977; Taleb et al., 2023). As a critical cool‐season grass, perennial ryegrass exhibits optimal growth within temperature ranges from 15°C to 25°C. High‐temperature conditions pose a significant impediment to perennial ryegrass yield, quality, and broad applicability, thereby demanding thorough genetic‐level investigations into its mechanisms of heat tolerance. Recently, the high‐quality genome of perennial ryegrass has been decoded, making it a grass species for investigating gene functions (Byrne et al., 2015; Frei et al., 2021). In ryegrass, HsfA5, HsfC1b, and HsfC2b have been identified as positive regulators in HSR (Sun et al., 2020, 2022; Ma et al., 2022). Additionally, abscisic acid (ABA) can upregulate the expression of FaHsfA2c to improve heat tolerance in tall Fescue (Wang et al., 2017a, 2017b). However, the roles and associated regulatory mechanisms of critical LpHsf genes implicated in the high‐temperature stress response in perennial ryegrass remain to be elucidated.

Previous studies have reported that HsfA2 serves as a key regulator in the late stage of the heat shock response, influencing the persistence of plant‐acquired heat tolerance (Charng et al., 2007; Liu et al., 2023). Recently, we identified 16 Hsf genes in the perennial ryegrass genome and analyzed their expression patterns under heat stress (Ma et al., 2022). In this study, we focused on LpHsfA2, which exhibits a high degree of amino acid sequence similarity with HsfA2 in different perennial ryegrass varieties and even different species. Interestingly, variations within the promoter regions of LpHsfA2, which modulate the expression of LpHsfA2 and high‐temperature tolerance, have been identified across perennial ryegrass varieties. Importantly, we found that, in response to heat stress, LpHsfA2 not only directly regulates the expression of LpHSP18.2 and LpAPX1, but also achieves precise regulation of its own expression levels through direct binding to its own promoter. Simultaneously, we identified that LpHsfA4 and LpHsfA5 were involved in regulating the expression of LpHsfA2 during high‐temperature response. These findings underscore the potential utility of these allele variations in perennial ryegrass breeding to improve plant heat tolerance.

RESULTS

LpHsfA2 expression responses to heat stress in perennial ryegrass

As a typical cool‐season grass, perennial ryegrass is extremely sensitive to high temperatures. Exposure to 38°C for a duration of 24 h results in leaf wilting, accompanied by a sharp decline in relative water content (RWC) and an elevation in electrolyte leakage (EL). At 42°C for a mere 12 h, there is a reduction in RWC and an increase in EL. Extended exposure, lasting 48 h to these conditions, leads to complete plant mortality (Figure 1A–C). To speculate the possible role of LpHsfA2 in heat tolerance, we examined the expression pattern of LpHsfA2 during high‐temperature response. As shown in Figure 1D, the expression of LpHsfA2 is significantly upregulated under various high temperatures conditions (32°C, 38°C, and 42°C), showing nearly an 18‐fold increase at 1 h under heat stress (38°C). Furthermore, subcellular localization analysis revealed that LpHsfA2 protein is localized in the cell nucleus, corroborated by a 35S::NLS‐mCherry nuclear marker (Figure 1E). These results suggest that LpHsfA2 potentially functions as a transcriptional modulator in the response of perennial ryegrass to heat stress.

Figure 1.

Figure 1

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Expression patterns of LpHsfA2 and subcellular localization of LpHsfA2 protein

(A–C) Phenotype (A), Relative water content (RWC) (B), and electrolyte leakage (EL) (C) of perennial ryegrass following different temperature (25°C, 32°C, 38°C and 42°C) treatments. (D) The expression patterns of LpHsfA2 following different temperature (25°C, 32°C, 38°C and 42°C) treatments during 48 h. (E) Subcellular localization of 35S::LpHsfA2‐eGFP in tobacco epidermal cells under normal growth conditions. Data are the means ± SD (n = 3).

Natural variation in LpHsfA2 confers different heat tolerance in perennial ryegrass

The pronounced impact of temperature on the expression of LpHsfA2 has aroused our interest in exploring the relationship between the expression levels of LpHsfA2 within the different varieties and their heat tolerance. We initially conducted a natural summer survival experiment involving 161 perennial ryegrass accessions in Wuhan, China (2022), and measured their withered leaves rate. The results showed that there was a substantial variation in heat tolerance among different accessions (Figure S1). Five heat‐tolerant and five heat‐sensitive varieties were selected for further analysis. Under control conditions, their growth was comparable. However, when subjected to heat stress, heat‐tolerant varieties exhibited higher RWC than heat‐sensitive varieties, with a correspondingly diminished EL (Figure 2A–C). The analysis of the expression levels of the LpHsfA2 gene across these 10 varieties revealed that heat stress induces the expression of LpHsfA2. Moreover, under both control and heat treatment conditions, heat‐tolerant varieties consistently demonstrated higher levels of LpHsfA2 expression compared with heat‐sensitive varieties (Figure 2D).

Figure 2.

Figure 2

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Natural variation in LpHsfA2 is associated with heat tolerance

(A) Phenotype of five heat‐tolerant and five heat‐sensitive perennial ryegrass varieties before and after heat treatment (42°C/38°C, with 16/8 h light/dark) for 2 d and recovery 7 d. (B, C) relative water content (RWC) (B) and electrolyte leakage (EL) (C) of five heat‐tolerant and five heat‐sensitive perennial ryegrass varieties under control and after heat treatment. (D) The expression levels of LpHsfA2 in the leaves of five heat‐tolerant and five heat‐sensitive perennial ryegrass varieties under control and 1 h of heat treatment (38°C). (E) Diagrams and haplotype analysis of the promoters of LpHsfA2 gene showing the relative positions of heat shock element (HSE) elements and difference of promoter differential location. (F) Numbers of heat‐tolerant and heat‐sensitive perennial ryegrass varieties with the HsfA2 Hap1 allele or and HsfA2 Hap2 allele. (G) Analysis of the relative expression of HsfA2 Hap1 and HsfA2 Hap2 promoters using a LUC activity assay in tobacco leaves. Data are means ± SD (n = 3). T represents heat‐tolerant perennial ryegrass varieties, and S represents heat‐sensitive perennial ryegrass varieties. Letters on the bars indicate a significant difference at the P < 0.05 level (one‐way analysis of variance (ANOVA)). Columns marked with asterisks (*) indicate statistical significance compared with the control (*P < 0.05; **P < 0.01, Student's t‐test).

Considering the differential expression of LpHsfA2 in heat‐tolerant and heat‐sensitive varieties, we sought to further explore the potential haplotypes and their distribution within the perennial ryegrass population. Subsequently, 20 heat‐sensitive and 20 heat‐tolerant varieties in 161 perennial ryegrass accessions were chosen (Table S1), from which we cloned the coding sequences (CDS) of the LpHsfA2 gene. The CDS from various varieties manifested minimal disparities, and no correlation with heat tolerance of ryegrass was evident. This prompted us to further clone the promoter of the LpHsfA2 gene from these varieties. We conducted a comparison of 55 promoter sequences of LpHsfA2 cloned from 41 perennial ryegrass varieties, using the sequence from perennial ryegrass “Lark” as the reference. In total, 184 variant sites were identified. The association analysis results indicated that variations in the promoter region from −439 to −422 bp were correlated with withered leaves rates (Figures S2, S3, and 2E). Based on the variations in this region, we classified the HsfA2 promoter into two distinct haplotypes, named HsfA2 Hap1 and HsfA2 Hap2 (Figure 2E). We conducted a statistical analysis on the distribution of these two haplotypes across perennial ryegrass varieties, and comprising 75% (15/20) of the promoters of heat‐tolerant ryegrass varieties were proHsfA2 Hap1, whereas 10% (2/20) of heat‐sensitive varieties were proHsfA2 Hap1. It is noteworthy certain varieties contain two types of haplotypes, including five heat‐tolerant and two heat‐sensitive varieties (Figure 2F).

To further analyze the transcriptional activity of these differing haplotype promoters, we amplified the promoter sequences of proHsfA2 Hap1 from perennial ryegrass germplasm resources No. 26 and proHsfA2 Hap2 from resources No. 27, positioning them upstream of the LUC reporter gene. When using an empty vector containing only the 35S protomer as the effector, we found that the expression level of LUC driven by proHsfA2 Hap1 was approximately 10 times higher than that of proHsfA2 Hap2 (Figure 2G). These data reveal that the differences in LpHsfA2 promoters have a significant impact on their activities.

LpHsfA2 positively regulates heat tolerance in perennial ryegrass

To confirm the function of LpHsfA2 in perennial ryegrass, overexpression (OE) and RNA interference (RNAi) vectors of LpHsfA2 were constructed and transferred into perennial ryegrass. Two lines each with significant variations in expression levels (OEA2‐3, OEA2‐26 for overexpression and A2‐RNAi‐7, A2‐RNAi‐28 for RNAi) were selected for further assays based on the RNA expression level and GUS staining. This GUS was driven by a 35S constitutive promoter as a reporter gene for identification of transgenic perennial ryegrass (Figures 3A, S4). Under normal conditions, no noticeable phenotypic differences were observed between the transgenic perennial ryegrass lines and wild type (WT “Lark,” heat‐tolerant perennial ryegrass variety). However, after 48 h of heat stress, the overexpression lines manifested superior growth rates, while the RNAi lines exhibited compromised growth status relative to WT (Figure 3B). Comparative physiological assays were conducted among these transgenic lines and WT. After exposure to heat stress, the overexpression lines displayed relatively higher percentages in their water content ranging between 58% and 61%. In contrast, RNAi lines displayed lower percentages in their water content, which plummeted to a mere 28% (Figure 3C). Alterations of EL inversely correlate with variations of RWC (Figure 3D). Furthermore, ROS levels, as evidenced by diaminobenzidine (DAB) nitro blue tetrazolium (NBT) histochemical staining, were substantially lower in LpHsfA2 overexpression lines compared with RNAi lines and WT under heat stress conditions (Figure 3E–H). Collectively, these data strongly suggest that LpHsfA2 significantly improves the tolerance of perennial ryegrass to heat stress.

Figure 3.

Figure 3

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LpHsfA2 positively regulates heat tolerance in perennial ryegrass

(A) The expression of LpHsfA2 was analyzed by RT‐qPCR. (B) Perennial ryegrass phenotypes of wild type (WT), overexpression LpHsfA2 lines (OEA2‐3, OEA2‐26) and LpHsfA2‐RNAi lines (A2‐RNAi‐7, A2‐RNAi‐28) under heat stress for 2 d (42°C/38°C under a 16 h/8 h light/dark cycle). (C, D) Relative water content (RWC) (C) and electrolyte leakage (EL) (D) of perennial ryegrass leaves under control and after heat treatment. (E, F) DAB staining and statistics analysis using ImageJ software of leaves treated with or without heat stress. (G, H) NBT staining and statistics analysis of leaves treated with or without heat stress. Data are the means ± SD (n = 3). Columns marked with asterisks (*) indicate statistical significance compared with the control (**P < 0.01, Student's t‐test).

LpHsfA2 directly binds to the promoter of LpHSP18.2 and LpAPX1 to activate their expression

To further explore the potential mechanisms underlying heat tolerance regulation by LpHsfA2, we generated transgenic Arabidopsis plants overexpressing HsfA2 and analyzed several heat‐related marker genes. Our findings revealed that the transcription levels of AtHSP18.2 and AtAPX2 were significantly increased in the transgenic lines compared with the Col and Vector‐control under both control and heat stress treatment conditions (Figure S5). In perennial ryegrass, LpHSP18.2 and LpAPX1 are also significantly induced under heat stress (Figure 4A, B). Therefore, the expression levels of LpHsP18.2 and LpAPX1 were closely correlated with the expression of LpHsfA2 in the genetically modified perennial ryegrass (Figures 3A, 4C, D). Furthermore, heat shock element (HSE) motifs were identified in the promoters of LpHSP18.2 and LpAPX1. Yeast‐one‐hybrid (Y1H) assay indicated that LpHsfA2 is capable of activating the expression of reporter genes downstream of these promoters (Figure 4F). EMSA further confirmed that LpHsfA2 could bind to the HSE elements of LpHSP18.2 and LpAPX2 promoters in vitro, evidenced by the successful binding of recombinant LpHsfA2‐GST to the labeled probe, which was diminished by a competitive probe (Figure 4G). Subsequently, a dual‐luciferase reporter assay was performed to further verify the binding of LpHsfA2 to LpHSP18.2 and LpAPX2 promoters. As shown in Figure 4H–J, the LUC activity, as indicated by the LUC/REN ratio in leaves co‐expressing 35S::LpHsfA2 with either LpHSP18.2 or LpAPX1, was higher than that observed in control leaves infiltrated with 35S and either LpHSP18.2 or LpAPX1 to activate their expression in vivo. Collectively, these findings revealed that LpHsfA2 is an important regulator involved in the direct regulation of the expression of LpHSP18.2 and LpAPX1 in perennial ryegrass.

Figure 4.

Figure 4

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LpHsfA2 directly binds to the promoter of LpHSP18.2 and LpAPX1 and activates their expression

(A, B) LpHSP18.2 (A) and LpAPX1 (B) expression in response to different temperature (25°C, 32°C, 38°C and 42°C) treatments over 48 h. (C, D) Expression analysis of LpHSP18.2 and LpAPX1 genes in wild type (WT), overexpression LpHsfA2 lines and LpHsfA2‐RNAi lines perennial ryegrass under control (25°C) and heat treatment (38°C) by RT‐qPCR. (E) Diagrams of the promoters of LpHSP18.2 and LpAPX1 genes showing the relative positions of heat shock element (HSE) (GAAnnTTC) elements. (F) Analysis of LpHsfA2 binding to the LpHSP18.2 and LpAPX1 promoters via yeast‐one‐hybrid assay. Yeast cells EGY48 harboring the prey (pJG4‐5‐LpHsfA2) plasmid were transformed with the baits (pLacZi‐ProLpHSP18.2 or pLacZi‐ProLpAPX1). (G) Electrophoretic mobility shift assays (EMSA) of the interaction between GST‐LpHsfA2 and the LpHSP18.2 and LpAPX1 promoter. The GST‐LpHsfA2 fusion protein was incubated with the biotin‐labeled probes. The unlabeled probe was used as a competitor (10× and 100×). The bound DNA–protein complex is indicated by the arrows. +, presence; −, absence. Biotin‐labeled probes incubated with GST protein alone served as the negative control. (H) Diagrams of the constructed vectors for LUC activity assay. (I, J) Analysis of LpHsfA2 binding to promoters of ProLpHSP18.2 (I) and ProLpAPX1 (J) in vivo using LUC activity assay in tobacco leaves. The ratio of LUC:REN for the co‐transformant with the empty vector and promoter was set as 1 for normalization. The error bars indicate the SD values from at least three repetitions of each treatment. Columns marked with asterisks (*) indicate statistical significance compared with the control (*P < 0.05; **P < 0.01, Student's t‐test).

LpHsfA2 expression is upregulated directly by itself, LpHsfA4 and LpHsfA5

To identify upstream regulators of LpHsfA2, we cloned a 1,500‐bp segment of the LpHsfA2 promoter (ProLpHsfA2) from perennial ryegrass “Lark.” One HSE binding site was identified at position −482 to −475 in ProLpHsfA2 (Figure 2E). This led us to consider whether self‐regulation and cross‐regulation existed within the HsfA gene family such as that found in other transcription factors (Wang et al., 2022). We conducted a Y1H assay and discovered that LpHsfA2, as well as LpHsfA4 and LpHsfA5 (genes previously reported by Ma et al., 2022), could bind to ProLpHsfA2 (Figures 5A, S6A). EMSA was used to ascertain the direct binding of LpHsfA2, LpHsfA4, and LpHsfA5 (the CDS of LpHsfA5 is the same as that of LmHsfA5) to the HSE site at ProLpHsfA2. As shown in Figures 5B and S6B, LpHsfA2, LpHsfA4 and LpHsfA5 displayed specific binding affinity toward the probe containing the HSE motif (GAAGATTC) within the ProLpHsfA2. A competitive binding assay was further used to confirm our results. Subsequently, a dual‐luciferase reporter assay was performed to further demonstrate the binding of LpHsfA2, LpHsfA4 and LpHsfA5 to ProLpHsfA2 in vivo. The luciferase activity of ProLpHsfA2::LUC driven by the LpHsfA2 promoter was compared with that of REN driven by the 35S promoter transiently in tobacco leaves. Relative to the control group co‐transformed with the blank 35S vector, 35S::LpHsfA2, 35S::LpHsfA4 and 35S::LpHsfA5 all notably displayed elevated levels in the LUC/REN ratio (Figures 5C, S6C, D). These results suggested that LpHsfA2, LpHsfA4 and LpHsfA5 could directly upregulate the expression of LpHsfA2 for modulating its downstream genes. In addition, with the introduction of the 35S::LpHsfA2 as the effector, there was a noticeable augmentation in LUC expression for both haplotypes. The expression level of LUC driven by proHsfA2 Hap1 was approximately 10 times higher than that of proHsfA2 Hap2 (Figure 5D). These data revealed that the differences in the promoters had a significant impact on their activities.

Figure 5.

Figure 5

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LpHsfA2 binds directly to the LpHsfA2 promoter and activates its expression

(A) Analysis of LpHsfA2 binding to the LpHsfA2 promoter via yeast‐one‐hybrid assay. Yeast cells Y1H Gold harboring the prey AD‐LpHsfA2 plasmid were transformed with baits (pAbAi‐ProLpHsfA2). (B) Analysis of LpHsfA2 binding to LpHsfA2 promoter using electrophoretic mobility shift assays (EMSA). The GST‐LpHsfA2 fusion proteins were incubated with the biotin‐labeled probes. The unlabeled probe was used as a competitor (10× and 100×). The bound DNA–protein complex is indicated by the arrows. +, presence; −, absence. Biotin‐labeled probes incubated with GST protein alone served as the negative control. (C) Analysis of LpHsfA2 binding to LpHsfA2 promoter using LUC activity assay. (D) Analysis of the relative expression of HsfA2 Hap1 and HsfA2 Hap2 promoters with 35S or 35S::HsfA2 using LUC activity assay in tobacco leaves. The ratio of the LUC:REN of co‐transformant with the empty vector and promoter was set as 1 for normalization. The error bars indicate the SD values from at least three repetitions of each treatment. Columns marked with asterisks (*) indicate statistical significance compared with the control (*P < 0.05; **P < 0.01, Student's t‐test).

Overexpression of LpHsfA4 enhances heat tolerance in transgenic perennial ryegrass

We have ascertained that LpHsfA2 enhances plant thermotolerance, and LpHsfA4 and LpHsfA5 positively regulate the expression of LpHsfA2. In light of our previous research elucidated that LmHsfA5 augments plant thermotolerance (Ma et al., 2022), we are now inclined to investigate the specific role of LpHsfA4 in enhancing plant resistance to elevated temperatures. Under heat stress, LpHsfA4 exhibits an expression pattern similar to that of LpHsfA2, with its peak value within 1 h under heat treatment (Figure S7). Furthermore, the LpHsfA4 protein is situated within the cell cytoplasm and nucleus (Figure 6A). To further elucidate the role of LpHsfA4 in the high‐temperature response of perennial ryegrass, three LpHsfA4‐overexpressing lines (OEA4‐8, OEA4‐13, OEA4‐19) of transgenic perennial ryegrass were selected and subjected to heat stress for 48 h

Figure 6.

Figure 6

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LpHsfA4 positively regulates heat tolerance in perennial ryegrass

(A) Subcellular localization of 35S::LpHsfA4‐eGFP in tobacco protoplasts cell under normal growth conditions. (B) The expression of LpHsfA4 was analyzed by RT‐qPCR. (C) Perennial ryegrass phenotypes of wild type (WT) and overexpression LpHsfA4 lines (OEA4‐6, OEA42‐136, OEA4‐19) under heat stress for 2 d (42°C/38°C under 16 h/8 h light/dark condtions). (D, E) Relative water content (RWC) (D) and electrolyte leakage (EL) (E) of perennial ryegrass leaves in response to heat stress. (F, G) DAB staining and statistics analysis using ImageJ software of leaves treated with or without heat stress. (H, I) NBT staining and statistical analysis of leaves treated with or without heat stress. Data are the means ± SD (n = 3). Columns marked with asterisks (*) indicate statistical significance compared with the control (**P < 0.01, Student's t‐test).

(Figures 6B, S8). These transgenic lines exhibited enhanced resistance to heat stress (Figure 6C). Under normal conditions, there was no difference in both RWC and EL between the transgenic lines and WT. Under heat stress, however, the RWC in the LpHsfA4 overexpressing lines was significantly higher than that of the WT (Figure 6D), whereas their EL exhibited a converse trend (Figure 6E). These results indicated that overexpression of LpHsfA4 significantly improved the heat tolerance of perennial ryegrass. Subsequent DAB and NBT staining experiments revealed that, similar to LpHsfA2, the overexpression of LpHsfA4 in perennial ryegrass also led to a reduction in the levels of hydrogen peroxide (H2O2) and O2− (Figure 6F–I). Additionally, silencing of LpHsfA4 (A4‐RNAi‐6, A4‐RNAi‐7 for RNAi) increased the sensitivity in perennial ryegrass compared with WT under heat stress (Figure S9A–D).

LpHsfA4 exhibits identical target genes, LpAPX1 and LpHSP18.2, as LpHsfA2

Given that both LpHsfA4 and LpHsfA2 belong to the HsfA family and may share regulatory targets, the expression of LpHSP18.2 and LpAPX1 was significantly increased in the LpHsfA4 overexpression lines compared with the WT under both control and heat stress treatment conditions (Figure 7A, B). However, silencing LpHsfA4 resulted in significant downregulation of the expression of LpHSP18.2 and LpAPX1 (Figure 9SE, F). Therefore, we conducted further analysis to investigate the interactions between LpHsfA4 and the promoters of LpHSP18.2 and LpAPX1. Y1H assay validated that LpHsfA4 can bind to these promoters (Figure 7C). Subsequently, EMSA further indicated that LpHsfA4 specifically recognized the HSE sequences on these promoters (Figure 7D). Dual‐luciferase assays confirmed that LpHsfA4 could activate the expression of downstream genes LpHSP18.2 and LpAPX1 (Figure 7E, F). Collectively, these findings suggested that LpHsfA4 not only regulated the expression of LpHsfA2 but also modulated the same target genes as LpHsfA2 to enhance the heat tolerance of perennial ryegrass.

Figure 7.

Figure 7

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LpHsfA4 directly binds to the promoter of LpHSP18.2 and LpAPX1 and activates their expression

(A, B) Expression analysis of LpHSP18.2 and LpAPX1 genes in wild type (WT) and overexpression LpHsfA4 lines perennial ryegrass under control (25°C) and heat treatment (38°C) by RT‐qPCR. (C) Analysis of LpHsfA4 binding to the LpHSP18.2 and LpAPX1 promoters via yeast‐one‐hybrid assay. Yeast cells EGY48 harboring the prey (pJG4‐5‐LpHsfA4) plasmid were transformed with the baits (pLacZi‐ProLpHSP18.2 or pLacZi‐ProLpAPX1). (D) Electrophoretic mobility shift assays (EMSA) of the interaction between GST‐LpHsfA4 and the LpHSP18.2 and LpAPX1 promoter. The GST‐LpHsfA4 fusion protein was incubated with the biotin‐labeled probes. The unlabeled probe was used as a competitor (10× and 100×). The bound DNA–protein complex is indicated by the arrows. +, presence; −, absence. Biotin‐labeled probes incubated with GST protein alone served as the negative control. (E, F) Analysis of LpHsfA4 binding to promoters of LpHSP18.2 (E) and LpAPX1 (F) using LUC activity assay in tobacco leaves. The ratio of LUC:REN of co‐transformant with the empty vector and promoter was set as 1 for normalization. The error bars indicate the SD values from at least three repetitions of each treatment. Columns marked with asterisks (*) indicate statistical significance compared with the control (*P < 0.05; **P < 0.01, Student's t‐test).

DISCUSSION

The Hsf family is pivotal in mediating plant response to assorted abiotic stressors, especially imparting heat tolerance. The discovery of plant Hsf transcription factors was first chronicled in tomato and, since then, members of the Hsf family have been identified across diverse plant taxa (Scharf et al., 1990). In tomato, HsfA2 is an important co‐activator of HsfA1a, which enhances antioxidant capacity, protein repair, and protein degradation‐related genes, thus improving the plant's ability to withstand heat stress (Scharf et al., 1998; Chan‐Schaminet et al., 2009). Similarly, Arabidopsis HsfA1 is considered to be the principal regulator and HsfA2 protein is prominently induced by HsfA1 in response to heat stress (Nishizawa‐Yokoi et al., 2011; Ohama et al., 2017). A recent study unveiled that Arabidopsis HY5‐HDA9 can dynamically regulate HsfA2 in response to salt and heat stress (Yang et al., 2023a). In perennial ryegrass, LpHsfC1b and LpHsfC2b have been identified to enhance heat tolerance (Sun et al., 2020, 2022). In this study, we substantiated that both LpHsfA2 and LpHsfA4 enhanced plant heat tolerance, further indicating that HsfA family genes have conservative functions in plant response to high temperatures.

Upon heat stress exposure, plants typically accumulate misfolded proteins and exhibit escalated ROS levels (Yang et al., 2023b). In the HSR cascade, Hsfs play a cardinal role, with their target genes encoding HSPs and enzymes that scavenge ROS (Driedonks et al., 2015; Chen et al., 2022). APX is an important reactive ROS‐scavenging enzyme, which catalyzes the conversion of H2O2 to H2O (Apel and Hirt, 2004). Small heat shock proteins (sHSP) have been strongly implicated as molecular chaperones in heat tolerance in plants, among which HSP18.2 is an important heat stress memory sHSP (Shekhawat et al., 2022). In this study, we revealed that LpHSP18.2 and LpAPX1 are direct downstream target genes regulated by LpHsfA2 and LpHsfA4. Overexpression of both LpHsfA2 and LpHsfA4 significantly increased the expression of LpHSP18.2 and LpAPX1 in perennial ryegrass under both normal conditions and heat stress treatment relative to WT. The outcomes from ROS staining in transgenic perennial ryegrass aligned with the variations observed in the expression of LpAPX1. These results provided evidence that LpHsfA2 and LpHsfA4 modulate plant thermotolerance by orchestrating ROS regulation. Building upon our prior research (Ma et al., 2022), we postulated that LpHsfA2, LpHsfA4 and LpHsfA5 together coordinated a molecular cascade, amplifying the expression of pivotal proteins such as LpHSP18.2 and LpAPX1. Consequently, this attenuated ROS accumulation and misfolding of proteins and therefore enhanced plant thermotolerance.

Studies in other species have shown that HsfA2's role in modulating heat tolerance is intricately linked to HsfA1 (Nishizawa‐Yokoi et al., 2011; Ohama et al., 2017). In perennial ryegrass, the LpHsfA2 promoter region harbors HSE motifs, and we confirmed that LpHsfA2 is not only regulated by the HsfA1 homologue, LpHsfA4, but also by an HsfA9 homologue, LpHsfA5. Interestingly, we also observed that LpHsfA2 is capable of modulating its own expression. This finding implies the existence of potential cascaded amplification or autoregulatory mechanisms among these HsfA genes in response to elevated temperature.

The natural variation of heat stress‐related genes in germplasm holds profound significance for the development and breeding of heat‐tolerant varieties. In multiple species, natural variation loci associated with heat stress‐related genes have been continuously discovered. In rice, the heat tolerance‐associated gene SRL10, encoding a double‐stranded RNA binding protein, has been identified with excellent heat tolerance alleles (Wang et al., 2023). The E3 ligase TT3.1, localized in the plasma membrane, might function as a heat sensor by safeguarding chloroplasts in conjunction with the chloroplast precursor protein TT3.2. African rice TT3 CG14 (TT3.1 CG14/TT3.2 CG14) uncovered one amino acid substitution, respectively, contributing to be more tolerant than its isogenic control, the Asian rice variety (WYJ) TT3 WYJ (TT3.1 WYJ/TT3.2 WYJ), upon heat stress (Zhang et al., 2022a). The G protein TT2 facilitates the influx of calcium ions upon exposure to heat stress, and a single nucleotide polymorphism (SNP) causes a premature stop codon in the open reading frame of TT2 conferring thermotolerance in African rice (Kan et al., 2022). A heat tolerance‐related gene, TaHST2, has been identified in wheat. In common wheat, this gene is present as a heat‐tolerant haplotype. However, it displays considerable diversity in the Aegilops tauschii D‐genome donor species, a variation that is likely to have been due to strong artificial selection during domestication (Zhang et al., 2022b). In grapevine, the variation of a single amino acid (Thr315Ile) in HSFA2 leads to higher transcriptional activation activities of VdHSFA2 than VvHSFA2 (Liu et al., 2023). In this study, we identified the variant regions in the promoter of LpHsfA2 associated with heat tolerance in perennial ryegrass. Based on these findings, we categorized the promoters into two distinct haplotypes HsfA2Hap1 and HsfA2Hap2. The varieties carrying the HsfA2Hap1 allele generally exhibited higher heat tolerance compared with those carrying the HsfHap2 allele. This association was further substantiated through comprehensive analyses of the promoter activity coupled with gene expression. Notably, certain perennial ryegrass germplasm resources harbor both haplotypes. This duality can be ascribed to the inherent nature of perennial ryegrass as an outbreeding species exhibiting self‐incompatibility, leading to a genomic landscape characterized by pronounced heterozygosity. Further, our findings also revealed a marked conservation of the HsfA2 promoter across both perennial and annual ryegrass, and the natural variation allele analyses are adapted in the two species of ryegrass. These results indicated that the variation in the LpHsfA2 promoter could be a contributing factor to the observed disparities in thermal tolerance among perennial ryegrass varieties.

In summary, our research elucidated pivotal heat stress‐responsive genes belonging to the Hsf family, namely LpHsfA2, LpHsfA4, and LpHsfA5. The interplay of mutual regulation and self‐regulation among these HsfA genes, along with their regulation of LpAPX1 and LpHSP18.2, collectively constitutes a synergistic mechanism influencing the heat tolerance of perennial ryegrass (Figure 8). Markedly, we have pioneered the identification of a crucial haplotype tied to the HSR in perennial ryegrass. This seminal finding carries profound implications for advancing genetic augmentation and breeding of heat‐tolerant perennial ryegrass cultivars.

Figure 8.

Figure 8

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Proposed model of LpHsfA2 ‐mediated heat tolerance

Due to the allelic variation of LpHsfA2 promoter, the regulation of downstream genes LpHSP18.2 and LpAPX1 is different. Therefore, perennial ryegrass has different phenotypes under heat stress. Solid lines represent the direct regulation of downstream genes or characters. Arrows indicate upregulation. The thicknesses of the lines represent the strength of the regulation. The number of the circles represents the level of protein expression.

MATERIALS AND METHODS

Plant materials and growth conditions

For heat treatment of perennial ryegrass “Lark” materials, transgenic perennial ryegrass plants were grown for approximately 2 months under normal conditions in the field. After this period, perennial ryegrass “Lark” materials were transitioned to growth chambers (25°C/22°C) under a 16 h/8 h light/dark regime for 1 week, followed by control (25°C) and heat treatment (32°C, 38°C or 42°C) under 16 h/8 h light/dark conditions for 2 d. The leaves from perennial ryegrass were collected as samples at different times (0, 1, 12, 24, 48 h) for measuring RWC, and EL. Samples taken at different times (0, 1, 3, 6, 12, 24, 48 h) were confirmed for RNA extraction and gene expression analysis. Heat‐resistant, heat‐sensitive, and transgenic materials were transitioned to growth chambers (25°C/22°C) under 16 h/8 h light/dark conditions for 1 week, followed by a 2‐d heat treatment. Following this, physiological indicators RWC and EL were measured, and DAB and NBT staining were performed. Samples were also collected 1 h after heat treatment for RNA extraction and gene expression analysis.

For natural summer survival experiments, perennial ryegrass varieties were sown as a single seed, and reproduced asexually with new tillers' production. Two‐month‐old perennial ryegrass plants were treated for natural summer survival in an open greenhouse in Wuhan, China (2022). The growth status and withered leaves rate of perennial ryegrass varieties were observed and measured periodically.

RNA extraction and gene expression analysis

The extraction of total RNA from plant materials was carried out using the RNAiso Plus kit (TaKaRa, Japan), following the manufacturer's instructions. The extracted RNA was reverse transcribed (RT) into cDNA using MonScript™ RTIII allin‐One Mix with dsDNase (Monad, Wuhan, China). Quantitative PCR (qPCR) was performed using SYBR Green (Monad, Wuhan, China). The reaction was performed on an ABI StepOne Plus Real‐Time PCR System from Applied Biosystems, USA, following a previously described protocol (Ma et al., 2020). AtACTIN2 was used as the reference genes for Arabidopsis and eEF1A was used as the reference genes for perennial ryegrass. All the RT‐qPCR primers used are listed in Table S2.

Construction of vectors, generation of transgenic perennial ryegrass

The CDS of LpHsfA2 and LpHsfA4 were cloned from perennial ryegrass “Lark” (heat‐tolerant perennial ryegrass variety). The transgenic background was the perennial ryegrass “Lark.” Transgenic materials from perennial ryegrass were transformed using the gene gun method (PDS‐1000/He Hepta System from Bio‐Rad), and the specific steps were carried out according to Instrument‐Based Transfection Methods. In brief, the CDS of LpHsfA2 and LpHsfA4 were inserted into the pXU1305.2‐UBI vector, and HsfA2‐RNAi and HsfA4‐RNAi were controlled by the UBI promoter. The vectors used for the genetic transformation of perennial ryegrass contain the GUS gene driven by a CaMV 35S promoter. Therefore, transgenic plants can be identified through GUS histochemical staining. The embryogenic calli of the perennial ryegrass “Lark” were used for gene gun transformation, the gene construct is typically coated onto microscopic gold, and the transformed calli were selected on a medium containing 25 mg/L hygromycin. Obtaining and screening perennial ryegrass calli followed the previously described protocol (Zhang et al., 2013). Plants generated from different calli were considered independent transgenic events. Putative transgenic lines were confirmed by GUS staining and RT‐qPCR analysis.

Subcellular localization analysis

The LpHsfA2 and LpHsfA4 without the stop codon were cloned into the pBI121‐eGFP vector. The recombinant plasmids of 35S::LpHsfA2‐eGFP and 35S::LpHsfA4‐eGFP were transformed respectively into GV3101, which was injected into the lower epidermis of tobacco (Nicotiana tabacum). The NLS‐mCherry nucleus was used as the nucleus‐located marker gene (Li et al., 2022). Tobacco protoplasts preparation and transformation were operated according to the method described previously (Rolland, 2018). After 48 h of incubation, green and red fluorescence was observed in leaves of tobacco under a confocal microscope (Leica, Germany; excitation wavelength with GFP: 488 nm and RFP: 552 nm) (Li et al., 2022).

Analysis of variations in the promoters of LpHsfA2

The promoters of LpHsfA2 were cloned from different perennial ryegrass cultivars and sequenced by Sanger sequencing. Multiple sequence alignments were performed on the sequenced data, leading to the development of molecular markers in the LpHsfA2 promoter region. These molecular markers were used to conduct an association analysis with withered leaves rates from the natural summer survival experiment in perennial ryegrass cultivars, utilizing the GLM model in Tassel 5.0. The significance threshold was determined by 0.05/(number of markers). LDBlockShow software was used to visualize the results of the association analysis and linkage disequilibrium.

Measurement of relative water content and electrolyte leakage

The leaves of perennial ryegrass were collected as samples for measuring RWC, and EL with three replicates per group. For RWC measurement, fresh leaves were weighed and recorded as fresh weight, and then the leaves were submerged in water. After 12 h, the soaked leaves were weighed and recorded as turgid weight. Finally, all leaves were dried at 80°C for 72 h and were weighed and recorded as dry weight. The RWC was calculated according to the formula: RWC (%) = (fresh weight − dry weight)/(turgid weight − dry weight) × 100%. The EL was measured as described previously (Ma et al., 2020) and was calculated using the following formula: EL (%) = (initial conductivity/total conductivity) × 100%.

Histochemical staining

For GUS staining, the leaves of WT and transgenic perennial ryegrass were stained in the GUS solution (Biorun, Wuhan, China) for 24 h at 37°C in an incubator. Leaf samples were immersed in 50 mM DAB solution (Solarbio, China) for 24 h. For NBT staining, leaf samples were immersed in 50 mM NBT solution (Solarbio, China) for 12 h. These were subsequently decolorized in 95% [v/v] ethanol until the color turned white, and then images were taken. Statistical analysis was conducted using ImageJ software.

Yeast‐one‐hybrid assay

For the EGY48 yeast‐one‐hybrid system, the promoter regions of LpHSP18.2, LpAPX1, AtHSP18.2, and AtAPX2 were fused to a pLacZi vector as bait. The CDS of LpHsfA2 and LpHsfA4 were ligated into the pJG4‐5 vector to construct the prey, and both the bait and the prey were co‐transformed into the yeast strain EGY48. The yeast strain was tested for β‐galactosidase activity on selective medium (SD/−Trp/−Ura/BU salt/X‐gal) to confirm whether the prey and bait interacted.

For the Matchmaker Gold yeast‐one‐hybrid system, the promoter of LpHsfA2 (proLpHsfA2) was inserted into the pAbAi vector to construct the bait, and the CDS of LpHsfA2, LpHsfA4 and LpHsfA5 were fused to the GAL4 AD in the pGADT7 vector to generate the prey vector. A BstBI‐cut of the pAbAi‐proLpHsfA2 bait was transferred into the Y1H Gold yeast strain, and efficiently integrated into the genome of the Y1HGold yeast strain by homologous recombination. After selecting the transformants on SD/−Ura plates, the 500 ng/mL AbA antibiotic was determined. The prey was introduced into the Y1H Gold yeast strain containing the pAbAi‐proLpHsfA2. Positively co‐transformed cells were screened on SD/−Leu/AbA medium.

Dual‐luciferase assay

For the dual‐luciferase assay, the promoter regions of LpHSP18.2, LpAPX1 and LpHsfA2 were ligated to a pGreenII 0800‐LUC vector, while 35S::LpHsfA2, 35S::LpHsfA4 and 35S::LpHsfA5 constructs were made. These vectors were then co‐transferred into Nicotiana benthamiana leaves and subjected to a dual‐luciferase assay to measure the activity of firefly and Renilla luciferase according to the manufacturer's instructions in the Dual‐Luciferase Reporter Gene Assay Kit (Yeasen, Shanghai, China). Five repetitions were conducted for each sample.

Electrophoretic mobility shift assay

The CDS of LpHsfA2/LpHsfA4/LpHsfA5 were cloned into pGEX‐6p‐1. The GST‐LpHsfA2 fusion protein was induced by 1 mM isopropyl‐β‐d‐thiogalactopyranoside (IPTG) at 30°C for 8 h, the GST‐LpHsfA4 fusion protein was induced by 1 mM IPTG at 18°C for 16 h, and the GST‐LpHsfA5 protein induction was carried out following a previously described protocol (Ma et al., 2022). The fusion proteins were purified using the GSTSep Glutathione Agarose Resin (Yeasen, Shanghai, China) according to the manufacturer's instructions. The pairs of oligonucleotides, each containing an HSE element, were synthesized and labeled with biotin. Each pair of oligonucleotides was annealed to double‐chain DNA probes. The purified GST‐LpHsfA2/LpHsfA4/LpHsfA5 fusion protein and the biotin‐labeled DNA probes containing either WT or mutated HSE (Table S2) were used for EMSA. The EMSA (Light Shift Chemiluminescent EMSA kit. Beyotime, Nanjing, China) was performed according to the manufacturer's instructions. Photographs were obtained using a multifunctional imaging system (FluorChem R, ProteinSimple, USA).

Statistical analysis

Statistical analysis was performed using the software SPSS 16.0. Experimental data were analyzed using one‐way analysis of variance (ANOVA) or Student's t‐test and mean separations were performed using the least significant difference multiple range tests, with P < 0.05, as the significance criteria used to show differences. GraphPad Prism 8 (GraphPad Software, San Diego, California, USA) was used to construct graphs.

CONFLICTS OF INTEREST

The authors declare no conflict of interest.

AUTHOR CONTRIBUTIONS

G.M. performed the experiments, analyzed the data and wrote the manuscript; Z.L. and S.S. contributed to manuscript writing; Y.X., L.C, J.G., S.L., and S.C. participated in experiments; L.H. provided perennial ryegrass germplasm; L.C. and H.J. designed the research, revised the manuscript and supervised the project. All authors read and approved of its content.

Supporting information

Additional Supporting Information may be found online in the supporting information tab for this article: http://onlinelibrary.wiley.com/doi/10.1111/jipb.13789/suppinfo

Figure S1. Withered leaves rate of 161 ryegrass germplasm accessions of perennial ryegrass in the summer season

Figure S2. Variations in the LpHsfA2 promoter region affect heat tolerance in perennial ryegrass

Figure S3. Multiple sequence alignment of the LpHsfA2 sequence of heat‐sensitive and 20 heat‐tolerant ryegrass varieties

Figure S4. LpHsfA2 transgenic ryegrass was identified by GUS staining

Figure S5. Expression analysis of heat stress‐responsive and antioxidant‐associated genes in Arabidopsis

Figure S6. LpHsfA4 and LpHsfA5 directly bind to the LpHsfA2 promoter and activate its expression

Figure S7. Expression analysis of LpHsfA4 in response to heat stress treatment (38°C) over 24 h

Figure S8. LpHsfA4 transgenic ryegrass was identified by GUS staining

Figure S9. LpHsfA4 positively regulates heat tolerance in perennial ryegrass

JIPB-66-2346-s003.docx (11.2MB, docx)

Table S1. Information and withered leaves rate of 20 heat‐sensitive and 20 heat‐tolerant perennial ryegrass materials

JIPB-66-2346-s001.xlsx (12.6KB, xlsx)

Table S2. List of all primers used in this study

JIPB-66-2346-s002.xlsx (12.5KB, xlsx)

ACKNOWLEDGEMENTS

This work was supported by the National Key R&D Program of China (2022YFF1003200), the National Natural Science Foundation of China (NSFC) (Grant Nos. 32001394, 32102431 and 32101430), the Science & Technology Specific Projects in Agricultural High‐tech Industrial Demonstration Area of the Yellow River Delta (Grant No. 2022SZX13), the Strategic Priority Research Program of Chinese Academy of Sciences (Grant No. XDA26050201), and the Major Science and Technology Innovation Project of Shandong Province (2022LZGC018). We thank Prof. Xuebao Li (Central China Normal University) and Miss Gichovi Bancy Wandiri for language polishing. This paper is dedicated to the memory of Mrs. Y.P. Zhang.

Biographies

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graphic file with name JIPB-66-2346-g011.gif

Ma, G. , Liu, Z. , Song, S. , Gao, J. , Liao, S. , Cao, S. , Xie, Y. , Cao, L. , Hu, L. , Jing, H. , et al. (2024). The LpHsfA2‐molecular module confers thermotolerance via fine tuning of its transcription in perennial ryegrass (Lolium perenne L.). J. Integr. Plant Biol. 66: 2346–2361.

Edited by: Honghui Lin, Sichuan University, China

Contributor Information

Haichun Jing, Email: hcjing@ibcas.ac.cn.

Liang Chen, Email: chenliang888@wbgcas.cn.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Additional Supporting Information may be found online in the supporting information tab for this article: http://onlinelibrary.wiley.com/doi/10.1111/jipb.13789/suppinfo

Figure S1. Withered leaves rate of 161 ryegrass germplasm accessions of perennial ryegrass in the summer season

Figure S2. Variations in the LpHsfA2 promoter region affect heat tolerance in perennial ryegrass

Figure S3. Multiple sequence alignment of the LpHsfA2 sequence of heat‐sensitive and 20 heat‐tolerant ryegrass varieties

Figure S4. LpHsfA2 transgenic ryegrass was identified by GUS staining

Figure S5. Expression analysis of heat stress‐responsive and antioxidant‐associated genes in Arabidopsis

Figure S6. LpHsfA4 and LpHsfA5 directly bind to the LpHsfA2 promoter and activate its expression

Figure S7. Expression analysis of LpHsfA4 in response to heat stress treatment (38°C) over 24 h

Figure S8. LpHsfA4 transgenic ryegrass was identified by GUS staining

Figure S9. LpHsfA4 positively regulates heat tolerance in perennial ryegrass

JIPB-66-2346-s003.docx (11.2MB, docx)

Table S1. Information and withered leaves rate of 20 heat‐sensitive and 20 heat‐tolerant perennial ryegrass materials

JIPB-66-2346-s001.xlsx (12.6KB, xlsx)

Table S2. List of all primers used in this study

JIPB-66-2346-s002.xlsx (12.5KB, xlsx)

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