Somatic embryogenetic receptor kinase TaSERL2 regulates heat stress tolerance in wheat by influencing TaBZR2 protein stability and transcriptional activity

Abstract

Heat stress is a major factor limiting crop yield, a challenge intensified by climate change. Initial findings indicate that BES1/BZR1 may use heat shock to regulate plant thermal adaptability independently of BIN2‐mediated brassinosteroid signalling, although the exact molecular mechanism remains unclear. In this study, we identified TaBZR2, a wheat gene whose expression showed a strong positive correlation with heat stress tolerance, based on transcriptome analysis of heat‐tolerant wheat cultivars. Overexpression of TaBZR2 enhanced heat stress tolerance, while RNA interference of TaBZR2 reduced it. Further analysis revealed that TaBZR2 interacts with and is phosphorylated by wheat somatic embryogenesis receptor‐like kinase 2 (TaSERL2). Overexpression of TaSERL2 reduced heat stress tolerance by promoting TaBZR2 degradation and inhibiting its regulation of wheat heat stress response genes. However, heat stress reduced the phosphorylation levels of both TaSERL2 and TaBZR2, lessening TaSERL2's inhibitory effect on TaBZR2 and enhancing the stability of TaBZR2. These results reveal that the TaSERL2‐TaBZR2 module negatively regulates plant heat stress tolerance. This study expands the current model of heat stress responses and provides evidence for the role of BES1/BZR1 in heat stress regulation independent of brassinosteroid signalling.

Introduction

Heat stress is a major constraint on plant growth and development, a problem exacerbated by climate change, which presents serious challenges for plant survival. High temperatures can lead to extensive cellular damage, often resulting in plant death (Kan et al., 2023). The plant heat stress response, an ancient signalling pathway, enables mitigation of cell damage caused by high temperatures and adaptation to hot environments (Ding et al., 2020). This adaptive response has evolved through long-term interactions with extreme environments. Central to this response are heat shock transcription factors (HSFs), which protect plant cells from heat damage and restore normal cellular and physiological functions by regulating the expression of heat shock proteins (HSPs; Larkindale et al., 2005; Kim and An, 2013; Ohama et al., 2017; Gu et al., 2019; Kan et al., 2023; Li et al., 2024). The role of specific phytohormones in the heat stress response has also been explored. For instance, ethylene response factors ERF95 and ERF97 regulate basal thermotolerance by modulating HsfA2 expression through EIN3 activity in response to ethylene (Huang et al., 2021). Similarly, abscisic acid (ABA) plays a role in acquiring thermotolerance by activating HSFA6b expression, which is essential for initiating thermotolerance mechanisms (Huang et al., 2016). Furthermore, recent studies have identified the brassinosteroid (BR) signalling pathway as a key regulator of plant heat stress responses, establishing a link between BRs and thermotolerance (Albertos et al., 2022).

BRs, a group of phytosterol hormones, are crucial for plant growth and development, influencing processes such as cell division, elongation, and abiotic stress responses (Dhaubhadel et al., 2002; Divi et al., 2016; Nolan et al., 2020; Quint et al., 2016). In plants, BRs are perceived at the plasma membrane by receptor-like kinases brassinosteroid insensitive 1 (BRI1) and somatic embryogenesis receptor kinase3 (SERK3)/BRI1-associated kinase1 (BAK1). These receptors transmit the BR signal to BRI1-emssuppressor1 (BES1) and brassinazole-resistant1 (BZR1) transcription factors by dephosphorylating the glycogen synthase kinase 3 (GSK3)-like kinase brassinosteroid insensitive2 (BIN2), which in turn regulates gene expression and other vital processes (He et al., 2002; Wang et al., 2002; Yin et al., 2002; Li et al., 2002; Yu et al., 2011; Fridman and Savaldi-Goldstein, 2013). BES1 and BZR1, which are critical in the BR signalling pathway, have been shown to play significant roles in modulating plant stress tolerance. For example, the wheat BES/BZR transcription factor TaBZR2 is positively regulated during plant drought stress (Cui et al., 2019), while overexpression of PpBZR1 in peach enhances cold stress tolerance by preventing sucrose degradation (Zhang et al., 2023). The role of BZR/BES in regulating heat stress tolerance is beginning to be clarified. In Arabidopsis, BES1 interacts with HSFA1 to enhance heat stress tolerance by promoting HSP expression (Albertos et al., 2022). Notably, heat stress has been shown to accelerate BES1 dephosphorylation, a process that appears to function independently of BIN2, which typically acts as a negative regulator in the BR signalling pathway (Albertos et al., 2022). Despite these insights, the key proteins regulating BES/BZR activity under heat stress and the specific mechanism by which BES/BZR modulates plant heat tolerance remain unclear.

Somatic Embryogenesis Receptor-like Kinase (SERK) is a transmembrane kinase primarily responsible for regulating intercellular communication and transmitting signals into the cell interior through co-receptors and phosphorylated substrates (Ma et al., 2016). SERK plays an essential role in plant morphological development and responses to environmental cues, including light, hormones, and other external factors (Fan et al., 2016; Ma et al., 2016). The Arabidopsis genome includes five members of the SERK family, including SERK3 (also known as BAK1), a kinase that associates with BRI1 and is integral to BR signalling (Chinchilla et al., 2009; Li et al., 2002). Studies demonstrated the role of SERK proteins in plant disease resistance. For instance, BAK1 serves as a central component of pattern recognition receptor (PRR)-mediated immunity (PTI), sensing pathogen invasion and transmitting extracellular signals to intracellular effectors, thereby triggering a hypersensitive response and programmed cell death (Postma et al., 2016; Sun et al., 2013; Wu et al., 2020). In wheat, TaSERK1 interacts with TaRLK-6A to enhance resistance to Fusarium crown rot (Qi et al., 2024). In rice, Xanthomonas outer protein K (XopK) targets and ubiquitinates OsSERK2, degrading it and compromising the plant's disease resistance response (Qin et al., 2018). SERK proteins also play roles in abiotic stress-related responses. For instance, the SERK protein OsSERK-like 2 (OsERL2) in rice interacts with COG1, which activates OsERL2 to mediate cold signal transduction, aiding in chilling defence (Xia et al., 2023). However, the link between SERK proteins and heat stress remains underexplored.

Wheat, a globally essential food crop, is particularly vulnerable to elevated temperatures (Ni et al., 2018; Ullah et al., 2021). Persistent high temperatures now threaten wheat production, causing considerable yield losses. Studies estimate that each 1 °C rise in global temperature results in an average 6.0% decrease in worldwide wheat yields (Asseng et al., 2015; Zhao et al., 2017). The IPCC report projects a 1.5 °C increase in global mean temperature over the next two decades, further intensifying this risk (Kim et al., 2022; Pörtner et al., 2022). Consequently, understanding the genetic networks involved in heat stress perception and adaptation is essential for improving heat tolerance in wheat through molecular breeding. In this study, we demonstrated a significant positive association between TaBZR2 and heat stress tolerance in wheat. Our findings showed that TaBZR2 interacted with TaSERL2 in wheat and was phosphorylated by TaSERL2, leading to accelerated TaBZR2 degradation and reduced heat tolerance. Moreover, heat stress could inhibit TaSERL2's phosphorylation activity, allowing the accumulation of non-phosphorylated TaBZR2, enhancing TaBZR2's regulatory capacity and promoting heat stress tolerance in wheat. Our findings demonstrate the critical role of the TaSERL2-TaBZR2 module in the regulation of wheat heat stress tolerance, advancing the understanding of heat stress responses and BR signalling in plants.

Results

TaBZR2 induction by heat stress and promotes its nuclear localization in wheat

Recent studies have demonstrated BES/BZR's role in regulating plant heat stress tolerance (Albertos et al., 2022; Chen et al., 2022; Yin et al., 2018). However, the regulatory mechanisms that enhance plant heat tolerance by controlling BES activity remain unclear. Therefore, we examined the link between TaBZR molecular features and heat tolerance in wheat, identifying 15 wheat BES/BZR gene family members via multiple sequence alignment (Figure S1 and Table S1). Analysis of transcriptome data from prior studies on heat-tolerant wheat cultivars (Liu et al., 2015) revealed that TaBZR2 had a stronger heat response than other family members (Figure S2a). qPCR analysis confirmed a significant induction of TaBZR2 under heat treatment compared to other TaBZR genes (Figure S2b), indicating TaBZR2's critical role in wheat's heat response.

In wheat protoplasts, TaBZR2 was localized to the nucleus and cytoplasm under normal conditions (Figure S3a,b). However, under heat stress (40 °C for 30 min), TaBZR2 translocated significantly to the nucleus (Figure S3c). Correspondingly, western blot analysis also showed that the control protein (NLS-RFP) exhibited no significant change in the nucleus between normal and heat stress conditions (Figure S3d), suggesting that heat stress did not affect NLS-RFP degradation. Heat stress increased TaBZR2-GFP levels in the nucleus while reducing them in the cytoplasm (Figure S3e). Fluorescence intensity analysis showed a substantial increase in nuclear TaBZR2-GFP and a decrease in cytoplasmic levels under heat stress (Figure S3f,g), suggesting that heat stress triggers TaBZR2 translocation from the cytoplasm to the nucleus.

Wheat heat stress tolerance is enhanced by overexpression of TaBZR2 and weakened by its RNA interference

To further assess TaBZR2's role in heat tolerance, we produced two TaBZR2 overexpression lines (TaBZR2-OE6 and -OE8) and two RNAi lines targeting three homeologs of TaBZR2 (TaBZR2-RNAi2 and -RNAi4; Figure S4). Under normal conditions, all lines showed similar phenotypes at the seedling stage (Figure S5a). However, after heat stress, TaBZR2-OE lines exhibited enhanced tolerance with higher survival rates, fresh weight, chlorophyll content, proline content, and glutathione reductase (GR) levels, compared to wild type (WT) and TaBZR2-RNAi plants (Figure S5b–h). In contrast, TaBZR2-RNAi plants were more sensitive to heat (Figure S5b–h), indicating that TaBZR2 positively regulates heat tolerance in wheat.

During the grain-filling stage, under normal conditions, TaBZR2-OE plants showed increased tillering, spike length, grain numbers per spike, grain length, grain width, and thousand-grain weight compared to WT and TaBZR2-RNAi plants (Figure 1; Figure S6). After 1 day of heat exposure (36 °C/30 °C day/night), TaBZR2-RNAi plants displayed lower proline levels than WT and TaBZR2-OE plants (Figure 1a,b). After 20 days of heat exposure, TaBZR2-RNAi plants wilted rapidly, showing yellowing and lower chlorophyll content in their panicles compared to WT and TaBZR2-OE plants (Figure 1a,c). Additionally, while TaBZR2-RNAi plants showed a decline in wheat grain traits, their susceptibility to heat was more pronounced than WT and TaBZR2-OE plants (Figure 1d).

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Agronomic analysis under heat stress revealed that TaBZR2-OE plants exhibited smaller reductions in shoot fresh weight (31.83%), grain length (5.91%), grain width (20.68%), and thousand-grain weight (52.92%) than WT plants, which showed reductions of 35.69%, 6.43%, 23.01%, and 56.70%, respectively. The largest reductions were observed in TaBZR2-RNAi plants, which had decreases of 36.89% in shoot fresh weight, 8.78% in grain length, 24.90% in grain width, and 58.85% in thousand-grain weight (Figure 1e–h). Field experiments further confirmed that TaBZR2-RNAi lines showed poorer agronomic traits under heat stress compared to WT and TaBZR2-OE plants (Figure 2), supporting that TaBZR2 positively regulates heat stress tolerance in wheat.

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TaSERL2 interacts with and phosphorylates TaBZR2 in wheat

To investigate TaBZR2's role in heat stress tolerance, we employed a yeast two-hybrid (Y2H) screening using a wheat cDNA library derived from RNA of the heat-tolerant cultivar ‘ZM1860’ to identify potential interacting protein partners. We assessed the influence of TaBZR2 protein self-activation on Y2H and used the N segment of TaBZR2 (TaBZR2-N) as the bait for screening candidate proteins (Figure S7). Among the interacting proteins identified (Table S2) somatic embryogenesis receptor kinase-like 2 (SERL2), a typical member of the membrane-localized SERK family capable of transducing cellular signals (Figure 3a; Figure S8, Table S1). It contains a signal peptide, two LRR domains, a transmembrane domain, and a protein kinase domain (Figure S8).

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Y2H assays demonstrated that TaBZR2 interacts with TaSERL2 proteins across the three different wheat genomes (A, B, and D; Figure 3b). Luciferase complementation imaging (LCI) assays in Nicotiana benthamiana leaves confirmed with luciferase activity within the co-injected area of TaBZR2-nLUC and TaSERL2A/B/D-cLUC, supporting the interaction between TaBZR2 and TaSERL2 (Figure 3c). Bimolecular fluorescence complementation (BiFC) assays further demonstrated that TaBZR2 interacts with TaSERL2 in the cell membrane and cytoplasmic compartments (Figure 3d; Figure S9a,b). Additionally, GST pulldown assays demonstrated that TaSERL2s-His could pull down TaBZR2-GST protein in vitro (Figure 3e), indicating in vivo and in vitro interactions.

Subcellular localization assays in wheat protoplasts revealed that TaSERL2 was specifically localized to the plasma membrane (Figure S10a). Notably, the N-terminal of the TaSERL2 protein contains a transmembrane domain (Figure S8b), which has been confirmed to be localized to the plasma membrane (Figure S10b). Conversely, the C-terminal of the TaSERL2 protein has been verified to be localized to the cytoplasm (Figure S10b). The subsequent analysis revealed that TaBZR2 exclusively interacted with the C-terminal region of the TaSERL2 protein in the cytoplasm (Figure S10c). Furthermore, we discovered that four additional members (TaSERL3, TaSERL4, TaSERL5, and TaSERL6) of the TaSERK family exhibited the capability to interact with TaBZR2, however, their interaction strength was comparatively weaker than that of TaSERL2 (Figure S9c–e).

To confirm TaSERL2's ability to phosphorylate TaBZR2, we performed an in vitro phosphorylation assay, incubating purified TaSERL2s-His and TaBZR2-GST proteins in a buffer containing ATP. Using Phos-tag™ acrylamide AAL-107 assays with anti-GST monoclonal antibodies, phosphorylation was detected by the slower migration in the gel (Figure 3f), indicating that TaBZR2 can phosphorylate TaSERL2. Co-Immunoprecipitation (Co-IP) and phosphorylation assays further demonstrated the interaction and phosphorylation of TaBZR2 by TaSERL2A/B/D (Figure 4a–f). Mass spectrometry identified two potential phosphorylation sites on TaBZR2 (Figure 4g). Mutation analysis revealed that only the 47th amino acid serine (S) residue was essential for TaBZR2 phosphorylation by TaSERL2 (Figure 4i). Further mass spectrometry identified two potential S autophosphorylation sites on TaSERL2 (Figure 4h). Notably, mutating S at position 602 significantly reduced both TaSERL2's autophosphorylation and its ability to phosphorylate TaBZR2 (Figure 4j,k).

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Plant heat stress tolerance is reduced by TaSERL2 overexpression and enhanced by its silencing

To investigate the impact of TaSERL2 on plant heat stress tolerance, two independent TaSERL2-overexpressing rice lines (TaSERL2-OE2 and -OE8) were generated and subjected to 45 °C for 48 h. After 5 days of recovery, these transgenic lines exhibited lower tolerance to heat stress than WT, as evidenced by lower survival rates, fresh weight, and chlorophyll content (Figure 5a–c). In addition, three independent TaSERL2-overexpressing wheat lines (TaSERL2-OE1, -OE3, and -OE7) were established (Figure S11a–c). The TaSERL2-overexpressing wheat lines, exposed to 42 °C and 45 °C for 48 h, exhibited severe wilting, lodging, significantly elevated malondialdehyde (MDA) levels, and decreased proline content compared to WT plants (Figure 5d–f). After a 3-day recovery period, the fresh weight of TaSERL2-overexpressing lines remained significantly lower than that of WT plants (Figure 5d–f). The findings indicate that TaSERL2 overexpression diminishes plant tolerance to heat stress.

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The expression of TaSERL2 in wheat was interfered with using BSMV-mediated VIGS (Figure S12a–c), suggesting that the expression level of TaSERL2 in wheat was significantly lowered by the BSMV-mediated VIGS interference system (Figure S12b,c). Following 30 h of 42 °C, control wheat plants (Mock and BSMV-γ groups) exhibited more severe wilting than the two BSMV-TaSBRL2 lines (Figure 5g). After a 3-day recovery, most BSMV-TaSBRL2 plants (with survival rates of 84.7% and 79.2%) returned to normal, while only 34.7% and 30.6% of control plants recovered fully recovered, with most control plants exhibiting signs of mortality (Figure 5g,h). Fresh weight and chlorophyll levels were significantly higher in BSMV-TaSBRL2 than the controls after recovery (Figure 5i,j). These results indicate that TaSBRL2 downregulation enhances wheat heat stress tolerance, suggesting a negative regulatory role of TaSBRL2 in heat stress tolerance.

TaBZR2 modulates heat stress-related gene expression

To further examine the mechanism underlying TaBZR2 and TaSERL2's roles in heat tolerance, DAP-seq analysis was conducted on TaBZR2 using the heat-tolerant cultivar ‘ZM1860’ (Figure S13), and RNA-seq analysis was performed to assess its regulatory activity (Figure S14a–c). Correlation analysis between DAP-seq and RNA-seq identified a significant overlap of 22 up-regulated genes and a substantial overlap of 38 down-regulated genes (Figure S14d,e). Sequencing analysis uncovered two potential heat-related gene targets, TaHSFb2a and TaHSP21, both containing E-box (CANNTG) cis-acting elements in their promoters (Figure 6a–b and Figure S14f). RNA-seq and qPCR results revealed TaHSFb2a expression was significantly upregulated in TaBZR2-RNAi plants under heat stress but downregulated in TaBZR2-overexpressing plants (Figure S14g). Conversely, TaHSP21 expression increased in TaBZR2-overexpressing plants under heat stress and decreased in TaBZR2-RNAi plants (Figure S14g). These results identify TaHSFb2a and TaHSP21 as potential downstream targets of TaBZR2.

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The promoters (1 kb upstream of ATG) of TaHSFb2a and TaHSP21 were cloned from the ZM1860 genome, and electrophoretic mobility shift assay (EMSA) confirmed TaBZR2 binding to the E-box cis-acting elements within these promoters. Competition assays demonstrated that binding was specific, as unlabelled WT probes but not mutant probes could compete for TaBZR2 binding (Figure 6c,d). In a yeast one-hybrid assay, co-transformation of TaBZR2-AD with proTaHSFb2a-AbAi or proTaHSP21-AbAi enabled transgenic yeast growth on solid screening medium supplemented with 300 mg/mL AbA (Figure 6e), further supporting the interaction between TaBZR2 and the promoters of TaHSFb2a and TaHSP21. Additionally, dual-luciferase reporter assay showed that TaBZR2 inhibited Luciferase (LUC) activity from the TaHSFb2a promoter, while it enhanced LUC activity induced by the TaHSP21 promoter (Figure 6f–j), suggesting that TaBZR2 suppresses TaHSFb2a expression while promoting TaHSP21 expression.

Silencing of TaHSFb2a and TaHSP21 can affect the wheat heat stress tolerance

To assess the roles of TaHSFb2a and TaHSP21 in wheat's response to heat stress, we used BSMV-mediated VIGS to inhibit their expression (Figure S15). The silencing of these genes was confirmed by a significant reduction in their expression levels (Figure S16a–d). Notably, the expression levels of TaHSFb2a and TaHSP21 exhibited no significant differences between mock and BSMV-γ groups (Figure S16b,d). After exposing the plants to 42 °C for 30 h, the BSMV-TaHSFb2a lines exhibited less wilting, greater fresh weight, and higher recovery rates than control plants, with survival rates of 33.33% and 41.67% after 3 days of recovery (Figure S16f–g). However, after exposing the plants to 42 °C for 24 h, wheat lines with silenced TaHSP21 displayed reduced growth and recovery following heat stress, with a lower fresh weight and survival rate than control plants (62.22%; Figure S16i,j). These findings suggest that TaHSFb2a negatively regulates heat stress tolerance, while TaHSP21 enhances it.

TaSERL2 modulates TaBZR2's regulatory activity by affecting its protein stability

To investigate howTaSERL2 influences TaBZR2 protein levels, we measured their phosphorylation changes under different heat stress. TaBZR2 exhibited high phosphorylation levels under normal conditions, which reduced significantly under heat stress (Figure 7a,b). Similar reductions in TaSERL2 phosphorylation levels occurred with increasing temperatures (Figure 7c,d). Interestingly, we observed no significant variation in the autophosphorylation levels of TaSERL2 with increasing temperature in vitro (Figure S17a). Degradation assays revealed that TaSERL2 overexpression accelerates TaBZR2 degradation, although heat stress reduces this effect, allowing TaBZR2 accumulation (Figure 7e,f). Mutations at specific TaBZR2 (47th residue) and TaSERL2 (602nd residue) sites stabilized TaBZR2, indicating TaSERL2's role in regulating TaBZR2 stability through phosphorylation (Figure S17b,c).

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To test TaSERL2's effect on TaBZR2's regulatory function, we conducted a LUC assay usingTaHSP21 as a downstream target gene regulated by TaBZR2 to examine their interaction. We inoculated Agrobacterium strain GV3101 carrying four different combinations (proTaHSP21-LUC, proTaHSP21-LUC/GFP, proTaHSP21-LUC/TaBZR2-GFP, and proTaHSP21-LUC/TaBZR2-GFP/TaSERL2-GFP) into different regions of tobacco leaves to measure LUC signals. The presence of TaBZR2 protein significantly enhanced LUC activity relative to the control (proTaHSP21-LUC/TaBZR2-GFP; Figure 7g). However, a lowered luminescence signal was observed in the co-injection region of proTaHSP21-LUC/TaBZR2-GFP/TaSERL2-GFP compared to proTaHSP21-LUC/TaBZR2-GFP (Figure 7g), indicating that TaSERL2 exerts a negative regulatory effect on TaBZR2 activity. This result was validated in wheat protoplasts, where LUC activity in cells containing proTaHSP21-LUC/TaBZR2-GFP was significantly higher than that in the control (proTaHSP21-LUC/GFP), but reduced in the presence of TaSERL2 (Figure 7h,i). This finding demonstrated TaSERL2's inhibitory effect on TaBZR2's regulatory activity.

TaBZR2 transcript levels are positively correlated with heat stress tolerance in wheat

Our results indicated that TaBZR2 activity could exert a positive regulatory effect on heat stress tolerance (Figure 7a–i). To further investigate this, we introduced six wheat varieties with varying levels of heat stress tolerance: Zhengmai 1860 (ZM1860), Zhengmai 7698 (ZM7698), Xinmai 26 (XM26), Zhengmai 1835 (ZM1835), Bainong 207 (BN207), and Chinese Spring (CS). The heat stress tolerance of these varieties was assessed according to the Technical Specification for Heat Tolerance Identification of Wheat Varieties (TSHTIWV; Paliwal et al., 2012). The results demonstrated that ZM1860 and ZM7698 were heat-tolerant wheat varieties, while XM26 and ZM1835 exhibited moderate susceptibility to heat stress. In contrast, BN207 and CS displayed high susceptibility to heat stress (Table S3). We subsequently analysed the expression patterns of TaBZR2 in these varieties under different heat stress conditions. ZM1860 and ZM7698 displayed higher heat stress tolerance than the other cultivars, with ZM1860 showing the greatest chlorophyll content and shoot fresh weight under stress (Figure 7j–l). Notably, TaBZR2 expression levels were significantly upregulated in ZM1860 and ZM7698 in response to heat stress, particularly in ZM1860 (Figure 7m). Moderate heat- sensitive cultivars XM26 and ZM1835 also showed increased TaBZR2 expression, whereas the heat-sensitive cultivars BN207 and CS cultivars had comparatively low induction levels (Figure 7m). We observed a significant decrease in TaBZR2 degradation in ZM1860 and ZM7698 cultivars compared to XM26, ZM1835, BN207, and CS after a 90-min heat stress treatment (Figure 7n). This further supported the positive correlation between TaBZR2 expression and wheat heat tolerance.

Discussion

High temperatures pose a major threat to wheat growth and yield (Asseng et al., 2015; Trnka et al., 2014; Yang et al., 2017; Zhao et al., 2017). Although key thermotolerance factors in wheat, such as TaHSFA1, TaHAG1, TaMBF1c, TaHSFA6e, and TaSC-D1, have been identified, the underlying regulatory networks and mechanisms governing heat responses remains poorly understood (Cao et al., 2024; Lin et al., 2022; Tian et al., 2022; Wang et al., 2023; Wen et al., 2023). Our findings demonstrate a strong association between TaBZR2 expression levels and heat stress tolerance in wheat (Figure S2 and Figure 7j–m), with elevated TaBZR2 induction in heat-tolerant wheat cultivars ZM1860 and ZM7698 (Figure 7j–m). These results support that TaBZR2 enhances heat stress tolerance in wheat, as shown by its overexpression, which markedly improves heat stress tolerance in transgenic wheat lines (Figures 1 and 2). Interestingly, overexpressing TaBZR2 also increased tillering number and grain size under normal conditions (Figures 1 and 2; Figure S6), paralleling observations in TaBZR1-overexpressing plants, likely due to changes in BR signalling (Lyu et al., 2024). TaBZR2 has previously been shown to positively regulate drought tolerance and stripe rust resistance by modulating glutathione S-transferase-1 (TaGST1) and chitinase Cht20.2 activities (Bai et al., 2021; Cui et al., 2019). However, we found that TaBZR2 positively regulated wheat heat stress tolerance by upregulating TaHSP21 expression, playing a critical role in enhancing wheat heat stress tolerance, while simultaneously inhibiting the transcription of TaHSFb2a, a negative regulator of wheat heat stress tolerance (Figures 5 and 6). We also observed that TaGST1 enhances wheat heat tolerance, while TaCht20.2 silencing had no effect on heat stress resilience (Figure S18), indicating that TaCht20.2's role may be specific to biotic stress responses. Together, these results highlight TaBZR2 as a key integrator in multiple stress signalling pathways in wheat.

BES/BZR1 proteins, targets of BIN2 kinase in the BR pathway, undergo phosphorylation to regulate diverse plant processes (Fridman and Savaldi-Goldstein, 2013; He et al., 2005; Sun et al., 2010; Wang et al., 2002; Yin et al., 2002; Yu et al., 2011). BES/BZR1 activation depends on BIN2 dephosphorylation by the upstream BR receptor BRI1 (He et al., 2002; Yin et al., 2002, 2005), which stabilizes BES1 proteins and enhances their regulatory functions (Kim and Wang, 2010; Belkhadir and Jaillais, 2015). Recent research has shown that BES/BZR1 is dephosphorylated and activated under heat stress, independent of BIN2 activity (Albertos et al., 2022). This study also demonstrates that the heat-induced dephosphorylation of BES/BZR1 is partly due to the inhibitory influence of ABA on PP2C phosphatases (Albertos et al., 2022). Our study, however, suggests an alternative mechanism for heat-induced BES/BZR dephosphorylation in wheat, involving TaSERL2. Specifically, we identified that TaSERL2 overexpression accelerates TaBZR2 degradation (Figures 3, 4 and 7e,f). This degradation is achieved by phosphorylation. TaSERL2 and its effect on TaBZR2 stability diminish under heat stress, allowing non-phosphorylated TaBZR2 to accumulate, thereby enhancing wheat heat tolerance (Figure 7a–f). This mechanism operates independently of BR signalling, highlighting a unique role for the TaSERL2-TaBZR2 in heat stress response. Furthermore, our study demonstrates that MG132, a specific inhibitor of the 26S proteasome, significantly inhibits the degradation of TaBZR2 mediated by TaSERL2 (Figure S19), which indicates that the degradation of TaBZR2 protein by TaSERL2 depends on the 26S proteasome pathway.

SERK family proteins are well-known co-receptors in plants (Ma et al., 2016), interacting with leucine-rich repeat receptor-like proteins (LRR-RLPs) to facilitate signal transmission (Liebrand et al., 2014; Xia et al., 2023). In rice, the SERK family includes 11 members (Singla et al., 2009), with TaSERL2 sharing homology with OsSERL2 (Figure 3a; Figure S8c). Previous studies indicate that OsSERL2, modulated by cold-induced LRR-RLP, mediates chilling stress response in rice by activating MAPK cascades (Xia et al., 2023). In contrast, our findings demonstrate that heat stress inhibits TaSERL2 phosphorylation in wheat (Figure 7c,d), leading to non-phosphorylated TaBZR2 accumulation, which stabilizes TaBZR2 and boosts heat tolerance (Figure 7). This finding indicates that SERL2 functions differently in temperature stress responses, with TaSERL2 weakening heat tolerance in wheat while OsSERL2 promotes chilling tolerance in rice. Interestingly, BRI1's co-receptor, BAK1, another SERK family member, does not interact with TaBZR2 (Figure S9d,e), further supporting TaBZR2's BR-independent regulation. In conclusion, our study uncovers the importance of the TaSERL2-TaBZR2 module in wheat's response to heat stress (Figure 8). By reducing TaSERL2 phosphorylation, TaBZR2 activity is stabilized, improving plant heat stress tolerance. Importantly, this module not only expands the current understanding of plant heat stress response but also offers potential strategies for enhancing wheat resilience through molecular breeding.

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Materials and methods

Plant materials and growth conditions

Six wheat cultivars were chosen, including two heat tolerance cultivars, ZM1860 and ZM7698, two moderate heat-susceptible cultivars, XM26 and ZM1835, and two heat-susceptible cultivars, BN207 and ChineseSpring (CS). Wheat was grown in an artificial climate box controlled at 60% relative humidity, under long-day conditions (light for 14 h/23 °C, dark for 10 h/20 °C) with a light intensity of 200 μmol m⁻² s⁻¹. Wheat seedlings (at the two-leaf heart stage) were exposed to 42 °C for 48 h to stimulate heat stress conditions. The wheat cultivar ‘ZM1860’ was employed to isolate TaBZRs and evaluate their expression patterns under stress conditions at various time intervals (0, 1, 3, 6, 12, 24, and 48 h). Six cultivars were used to isolate TaBZR2-3B and evaluate their expression patterns under stress conditions at various time intervals (0, 1, 3, 6, 12, 24, and 48 h). After each treatment, wheat seedlings were flash-frozen in liquid nitrogen and stored at −80 °C for subsequent analyses.

RNA extraction, RT-qPCR

RNA extractions were conducted using an RNAprep Pure Plant Kit (TIANGEN, Beijing, China). cDNA was produced using the FastKing RT kit (TIANGEN, Beijing, China), and for RT-qPCR, the 2 × SG Fast qPCR Master Mix (Sangon Biotech, Shanghai, China) was employed according to the manufacturer's recommendations. Amplification was performed using a CFX96 Real-Time PCR Detection System (Bio-Rad). The reference gene employed for data analysis was TaActin. Primer pairs are presented in Table S4. Relative expression levels were assessed using the $2^{-\Delta C_{\mathrm{t}}}$ method.

Plasmid construction and plant transformation

To construct candidate gene knockdown constructs, the 400 bp sense and 400 bp anti-sense orientations of TaBZR2 fragments were linked by the 152 bp intron sequence of the maize alcohol dehydrogenase 1 (adh1) gene. The 952-bp fragment was generated by Sangon Biotech. (Shanghai, China). This recombinant DNA was inserted into the pWMB110 vector to produce the pWMB110-TaBZR2-RNAi construct. The open reading frame (ORF) of TaBZR2-3B and TaSERL2-A were cloned from ZM1860 and inserted into pMWB110 using BamHI sites to produce the Ubi: TaBZR2 and Ubi: TaSERL2 construct. All binary vectors with the desired constructs were transferred into strain EHA105 (Zoman Bio, Beijing, China) and transformed into the wheat cultivar ‘Fielder’ or rice cultivar ‘Nipponbare’ via Agrobacterium-mediated transformation.

Phenotype identification in different wheat plants

T3 RNAi, WT, and overexpressing wheat plants were employed to assess heat stress tolerance. A solar greenhouse controlled at 60% relative humidity, and long-day conditions (light for 16 h/25 °C, dark for 8 h/25 °C) were used to grow wheat following 10 days of vernalization at 4 °C. To treat wheat plants at the heading stage, 7 days after flowering, plants in pots were transferred to a growth chamber under a cycle of 16 h of light at 36 °C and 8 h of dark at 30 °C. The segments of the plant shoots were collected on the first day for subsequent proline content analyses. The segments of the plant shoots were collected on the 20th day for subsequent physiological and biochemical index analyses. Other wheat plants were transferred to normal conditions until harvest. Following harvest, agronomic traits of different wheat plant types were characterized and subjected to statistical analysis. All experiments were designed using 12 independent replicates.

To evaluate the heat stress tolerance of different types of transgenic wheat and wild-type (WT) plants in field conditions, a transparent plastic tent was utilized to cover the transgenic plants and WT at 7 days post-flowering. After subjecting the plants to a 20-day heat stress treatment, photographs were captured, and the plants on the ground were collected for subsequent physiological and biochemical analysis. The residual wheat plants were cultivated under standard growth conditions until harvest. Subsequently, agronomic characteristics of various types of wheat plants were assessed and subjected to statistical analysis. All experiments were conducted with 20 independent replicates.

T3 overexpressing rice and wheat plants were employed to assess seeding stage plants heat stress tolerance. Rice plants were grown in an artificial climate box controlled under 70% relative humidity, and long-day conditions (light for 14 h/26 °C, dark for 10 h/23 °C) with a light intensity of 200 μmol m⁻² s⁻¹. To stimulate heat stress conditions, rice seedlings (at the two-leaf heart stage) were exposed to 45 °C for 48 h and transferred to normal conditions for 5 days. Wheat plants were grown in an artificial climate box controlled under 60% relative humidity, and long-day conditions (light for 14 h/23 °C, dark for 10 h/20 °C) with a light intensity of 200 μmol m⁻² s⁻¹. To stimulate heat stress conditions, wheat seedlings (at the two-leaf heart stage) were exposed to 42 °C or 45 °C for 48 h and transferred to normal conditions for 3 days. The complete plants were collected for physiological and biochemical index analyses. All experiments were designed using three independent replicates.

Yeast two-hybrid screening

The full-length sequences TaBZR2 (1-355aa), TaBZR2-N (1–149aa), and various truncates TaBZR2-C (45–355aa; Figure S7) were generated using Tks PrimeGflex™ DNA polymerase (TaKaRa Bio) PCR amplification. Primer combinations and sequences are presented in Table S4. Each fragment was cloned into the pGBKT7 vector using the In-fusion cloning system (TaKaRa Bio). All vectors were transformed into yeast Y2HGold cells. Transformation was conducted using PEG/LiAc yeast as outlined in the Yeast Protocols Handbook PT3024-1 (TaKaRa Bio). Transformants were grown at 30 °C on Synthetic Dropout (SD)/−Trp. The self-activating activity was assessed on SD/−Trp with 100 μg/mL of X-a-gal. Levels of 50–250 ng/mL of aureobasidin A (AbA) were used to suppress reporter gene expression.

TaBZR2-N (aa 1–149) was cloned into the pGBKT7 vector to produce BD-TaBZR2-N as ‘bait’. The sequences of TaSERL2s were cloned into the pGADT7 vector to produce AD-TaSERL2s, as ‘prey’. The selection media SD/−Leu-Trp and SD/−Leu-Trp-His-Ade supplemented with 250 ng/mL of AbA and 100 μg/mL of X-a-gal were employed to monitor yeast cell growth.

Bimolecular fluorescence complimentary (BiFC) assays

Two approaches, wheat protoplast, and tobacco, were employed for BiFC analysis. For BiFC assays in wheat protoplasts, the open reading frame (ORF) of TaBZR2 was cloned into 35S::nYFP, and the ORF of each TaSERL2 was cloned into 35S::cYFP to produce protein fusion constructs. Appropriate pairs of plasmid DNAs were co-transformed into wheat mesophyll protoplasts via PEG-mediated transformation. Following incubation at 23 °C for 20 h, YFP fluorescence in the transformed protoplasts was assessed using a confocal laser scanning microscope (Zeiss LSM 700; Carl Zeiss AG, Oberkochen, Germany).

For BiFC assays in tobacco, the ORF of TaBZR2 was cloned into the pXY106 vector to generate nYFP-TaBZR2 fusion protein, while the ORFs of TaSERLs were cloned into the pXY104 vector to create the TaSERL2-cYFP fusion protein. A. tumefaciens GV3101 was employed in co-infiltration experiments using 3-week-old Nicotiana benthamiana plants. A. tumefaciens strains at an optical density at OD₆₀₀ = 0.3 were used for leaf infiltration. The P19 helper plasmid was included in all combinations at an optical density at OD₆₀₀ = 0.25 and incubated at room temperature for 4 h. Agrobacterium was infiltrated into the leaves of tobacco and cultured for 36 h. YFP signals were observed using a confocal laser scanning microscope (Zeiss LSM 700; Carl Zeiss AG, Oberkochen, Germany).

Luciferase complementary imaging (LCI)

The LCI assays of TaBZR2 and TaSERLs were performed as described previously (Yu et al., 2021). The pCAMBIA1300 n-LUC or pCAMBIA1300 c-LUC vectors were used as negative controls. The candidate protein (TaSERL2s) and bait protein (TaBZR2) were cloned into vectors with nLUC and cLUC tags. Luciferase activity was assessed 48 h later.

In vitro glutathione S-transferase (GST) pull-down assay

To obtain GST-tagged TaBZR2 sequences and His-tagged TaSERL2A/B/D sequences, TaBZR2 was introduced into pGEX4T-1 and TaSERL2s were introduced into pCold-TF, and then into Escherichia coli BL21(DE3). TaBZR2-GST and TaSERL2-His recombinant proteins were purified from the culture supernatant using a His-tag Protein Purification Kit and GST-tag Protein Purification Kit (Beyotime, Beijing, China), respectively. Following purification, the recombinant proteins were quantified using a Bio-Rad protein assay reagent. The protocol for pulldown experiments involving TaBZR2-GST and TaSERL2s-His was described previously (Liu et al., 2022).

Co-immunoprecipitation analysis

The TaSERL2A/B/D-GFP and TaBZR2-Flag fusion constructs were co-transformed into N. benthamiana leaves. Total proteins were isolated and incubated with anti-GFP beads at 4 °C for 4–6 h with gently shaking. Input and eluted protein were electrophoretically separated and specific proteins detected by immunoblotting with anti-GFP (HT801-01; TransGen Biotech, Beijing, China) or anti-Flag (HT101-01) antibodies.

Virus-induced gene silencing (VIGS)

To silence TaSERL2, TaHSFb2a, and TaHSP21, two fragments of the genes were inserted in reverse orientation into the barley stripe mosaic virus RNAγ to produce the recombinant vectors BSMV: TaSERL2(1-2as), BSMV: TaHSFb2a(1-2as) and BSMV: TaHSP21(1-2as). The experiment was conducted as described previously (Cheng et al., 2023).

DNA affinity purification sequencing analysis

We conducted DAP-seq to characterize genes directly targeted by TaBZR2 by following a previously described procedure (Bartlett et al., 2017). The gDNA from ZM1860 wheat was extracted, purified, fragmented, and ligated to a short DNA sequencing adapter to construct a DAP-seq library. The TF TaBZR2 was prepared by in vitro expression, linked to Halo-tag, and co-constructed into an expression vector for purification using Magne Halo-Tag Beads. The Halo-TaBZR2 protein was bound to anti-Halo monoclonal antibody agarose beads (Promega, USA, cat G9211) and incubated with 200 ng of fragmented gDNA for 1 h at room temperature. After incubation, the beads were rinsed to recover DNA. After extracting DAP DNA, the enriched DNAs were fragmented into short fragments using ultrasound. Next, the DNA fragments underwent end repair, 3′A addition, and ligation to Illumina sequencing adapters. DNA fragments with appropriate sizes (usually 100–300 bp, including adapter sequence) were chosen for PCR amplification. The cDNA/DNA/Small RNA libraries were sequenced using the Illumina sequencing platform by Genedenovo Biotechnology Co., Ltd (Guangzhou, China). PCR amplification and sequencing were performed using the Illumina Novaseq 6000 platform. Enrichment sites (peaks) were utilized to identify target genes and recognition motifs of TaBZR2 by mapping read data to the reference genome. Two biological replicates were analysed.

RNA sequencing analysis

Four treatments were set up: WT no-treatment control, OE-TaBZR2 no-treatment control, WT treatment at 42 °C for 6 h, and OE-TaBZR2 treatment at 42 °C for 6 h, with three biological replicates per treatment. Total RNA was extracted using Trizol kit (Invitrogen, Carlsbad, CA, USA). RNA quality was assessed by an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) by detection electrophoresis using rnase-free agarose gels. RNA-seq sequencing was performed by Gidio Bio, Guangzhou. The sequenced data were analysed on the Gideon Bio Omicshare cloud platform.

LUC activity analysis

To characterize transcriptional activity, genes encoding TaBZR2 and TaSERL2A were cloned into pCambia1305-GFP, and target gene promoters were inserted into pGreen0800II. Then, recombinant and control blank vectors were co-expressed in N. benthamiana leaves and grown for 48 h, after which the leaves were removed, and their surfaces were treated with D-luciferin. After 5 min of incubation in the dark, the LUC signal was observed using a plant living imaging system. Total protein in the leaves was isolated using the Plant Total Protein Extraction Kit (CW0891M; Cwbio, Beijing, China), and the proteins were characterized using the Dual-Luciferase Reporter Assay System (Promega, Madison, WI). LUC data were obtained using an automatic microplate reader.

Electrophoretic mobility shift assay

The CDS sequences of TaBZR2 were cloned into pGEX-4T-1 vectors and introduced into Escherichia coli BL21(DE3) chemically competent cells (Zoman Bio, Beijing, China). The cells were incubated at 37 °C until reaching an OD₆₀₀ of 0.6–0.8. Then, 0.5 mM IPTG was added and shaken for 14 h at 16 °C. Proteins were purified using a GST-tag protein purification kit (Zoman Bio, Beijing, China). The purity of the purified protein was assessed using 10% SDS-PAGE gels, and proteins with purity exceeding 90% were used for subsequent tests.

Electrophoretic mobility shift assays (EMSAs) were performed using a Light Shift Chemiluminescent EMSA Kit (Thermo Fisher Scientific, Rockford, IL, USA). The eluted proteins and biotin end-labelled duplex DNA probe were mixed with EMSA binding buffer, and incubated at 25 °C for 30 min. Unlabelled probes in 5-, 20-, and 200-fold molar excesses were used in competition analysis to test the specificity of the binding sequence. The positive control protein was the cAMP receptor protein from E. coli, the biotin end-labelled duplex DNA probe was the coliform lac promoter–operator region, a total of 214 bp, and the negative control was the 214 bp free probe (Hellman and Fried, 2007). The mixture was separated using a 6% polyacrylamide gel, and the DNA was transferred to a nylon membrane (Millipore, USA). The signal was visualized using a LightShift Chemiluminescent EMSA Kit (Thermo Fisher Scientific). The primers utilized are presented in Table S4.

Yeast one-hybrid assay

The coding sequence of TaBZR2 was cloned into the pGADT7 vector, and the putative binding motif, as well as the mutated binding motif, were cloned into the vector pAbAi. pGADT7-TaBZR2 was then co-transferred with pAbAi-motif to Y1HGold. A concentration of 300 ng/mL AbA was utilized to suppress the expression of the reporter gene. SD/-Leu/-Ura medium was employed to select transformants and transfer the positive clones to SD/−Leu/-Ura plates to grow.

LC–MS/MS Analysis

The LC–MS/MS analysis was performed as reported previously (Zhou et al., 2020). Briefly, TaBZR2-GST or TaSERL2-His phosphorylated gel strip was used to analysis. The proteins in the reaction were then digested using trypsin (1:50 trypsin-to-protein ratio, w/w) at 37 °C overnight. Phosphopeptides were enriched for LC–MS/MS analysis with an Orbitrap Fusion Tribrid mass spectrometer (Thermo Fisher Scientific). The MS/MS spectra were analysed with Thermo Proteome Discoverer (Thermo Fisher Scientific, version 2.2), and the identified phosphorylated peptides were manually inspected to ensure confidence in the assignment of phosphorylation sites.

In vitro and in vivo phosphorylation assay

For TaSERL2-mediated in vitro phosphorylation, 5 μg of TaSERL2s-His were incubated with 2 μg of TaBZR2-GST in 50 μL of the reaction buffer (20 mM Tris-Cl (pH 7.5), 10 mM MgCl₂, 1 mM DTT, 100 mM ATP) for 1 h at 25 °C and the reaction was halted by adding SDS loading buffer. Phosphorylation was detected using a 75 μM phos-tag TM Acrylamide AAL-107 assay (F4002; APExBIO, Houston, TX, USA) with anti-GST monoclonal antibody (HT601-02; TransGen, Beijing, China) at 1: 2000 dilution. λ protein phosphatase (λPPase; P0753S; NEB, England) was utilized to confirm that the delayed migration band of TaBZR2 in phos-tag gel was caused by its phosphorylation.

For in vivo phosphorylation, the TaSERL2A/B/D-GFP and TaBZR2-Flag fusion constructs were co-transformed into N. benthamiana leaves. Total protein was extracted using Tissue Protein Extraction Reagent (CW0891M; Cwbio, Beijing, China), and cell debris was removed via two centrifugation steps (15 000 g, 10 min, 4 °C) to collect supernatants. Phosphorylation was detected using a 75 μM phos-tag TM Acrylamide AAL-107 assay (F4002; APExBIO, Houston, TX, USA) with anti-Flag monoclonal antibody (HT801-02; TransGen, Beijing, China) at 1:2000 dilution.

Conflict of interest

The authors declare no conflicts of interest.

Author contributions

WGX coordinated the project, conceived and designed the experiments, and edited the manuscript. XYH, TFY, and CJP performed the experiments and wrote the first draft. YHF, YHF, YL, JC, and HBD contributed valuable discussions. ZSX and YZM reviewed and revised this paper. All authors have read and approved the final manuscript.

Funding

This research was financially supported by the National Key Research and Development Program of China (2022YFD1200205), National Key Research and Development Program of China (2023YFD1201004), Key Scientific and Technological Project of Henan Province (231100110100), the ‘First-class Project’ of Shennong Laboratory (SN01-2022-01), agricultural biological breeding major projects (2023ZD0402602-06), the earmarked fund for China Agriculture Research System (CARS-03), and the Major Project on Agricultural Bio-breeding of China (2023ZD04026).

Data availability statement

RNA-seq and DAP-seq data were submitted to the Sequence Read Archive of the NCBI under the accession number PRJNA1173349 and PRJNA1173784.

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