Thermal priming enhances heat tolerance in alfalfa (Medicago sativa L.) through activation of multiple metabolic pathways

Thermal priming enhances heat tolerance in alfalfa (Medicago sativa L.) through activation of multiple metabolic pathways | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Thermal priming enhances heat tolerance in alfalfa (Medicago sativa L.) through activation of multiple metabolic pathways Chengmin Jin, Jingliang Sun, Jinrui Zhao, Ziyao Zhang, Han Lu, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7191702/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Dec, 2025 Read the published version in BMC Plant Biology → Version 1 posted 12 You are reading this latest preprint version Abstract Background: Elevated environmental temperatures disrupt plant physiological homeostasis, imposing thermal stress that severely compromises growth and development. While thermal priming - a brief exposure to sublethal high temperature has been shown to enhance subsequent heat stress tolerance in plants, the underlying molecular mechanisms remain poorly characterized. In this study, we employed an integrated physiological, transcriptomic and metabolomic approach to investigate how thermal priming [37℃ for 2 h (P1) followed by 43℃ for 2 h (P2), designated P3] improves heat tolerance in alfalfa ( Medicago sativa L.) compared to unprimed controls (UP) exposed directly to 43℃. Results: Physiological analyses revealed that thermal priming significantly enhanced lodging resistance while increasing superoxide dismutase (SOD) and catalase (CAT) activities and reducing malondialdehyde (MDA) accumulation, indicative of improved oxidative stress management. Transcriptome profiling identified 1,217 upregulated genes in primed plants (P3 vs UP), with 50.2% being activated during the initial priming phase (P1). Cluster analysis demonstrated stage-specific pathway activation: brassinosteroid (BR) signaling, spliceosome activity, glutathione metabolism and fatty acid metabolism pathways were rapidly induced during early priming (P1), while phenylpropanoid biosynthesis was activated later during the second phase (P2). Metabolomic analyses provided further mechanistic insights, showing that thermal priming triggered significant lignin accumulation in stems, enhanced activity of the ascorbate-glutathione (AsA-GSH) cycle with increased antioxidant levels, and elevated content of unsaturated fatty acids including erucic acid, linolenic acid and oleic acid, suggesting membrane lipid remodeling. Conclusions: Our findings demonstrate that thermal priming establishes a multi-faceted defense system in alfalfa through BR-mediated signaling. This coordinated response involves activation of the AsA-GSH cycle for reactive oxygen species (ROS) scavenging, upregulation of phenylpropanoid biosynthesis for structural reinforcement through lignin deposition, accumulation of unsaturated fatty acids to maintain membrane stability, and enhancement of spliceosome activity to ensure proper processing of heat-responsive transcripts. The sequential activation of these pathways during the priming phases creates a 'stress memory' that prepares plants for subsequent heat challenges. These insights advance our understanding of thermal priming mechanisms and provide potential targets for improving crop heat tolerance through molecular breeding strategies. Thermal priming Alfalfa Multivariate analysis Brassinosteroid Spliceosome Metabolic pathway Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Background Thermal priming, the process by which brief exposure to non-lethal high temperatures enhances subsequent heat stress tolerance, represents a critical adaptive strategy in plants [1]. This phenomenon has been demonstrated to improve thermotolerance through coordinated physiological, biochemical, and molecular responses [2]. Evidence from multiple species confirms the protective effects of thermal priming: in rice, priming at 28℃ maintained membrane integrity and biomass accumulation while reducing ROS and MDA levels during heat stress [3]. Similarly, brassica plants primed at 40℃ exhibited increased soluble sugars, CAT and peroxidase (POD) activities, along with reduced membrane damage when exposed to 45℃ stress [4]. Other studies have documented enhanced photosynthetic efficiency in azalea [5], improved antioxidant capacity in cape gooseberry [6], and preserved photosynthetic function in grapes following thermal priming [7]. At the molecular level, thermal priming induces sustained expression of small heat shock proteins (sHSPs) such as HSP21, HSP22, and HSP17.6C, with primed plants showing more rapid and robust induction of these molecular chaperones [8]. In Arabidopsis , priming establishes epigenetic memory through JMJ-mediated demethylation of H3K27me3 at HSP loci, enabling faster transcriptional responses to subsequent heat stress [8]. These findings collectively demonstrate that thermal priming enhances thermotolerance through multiple mechanisms including photosynthetic protection, osmotic adjustment, and antioxidant defense. Alfalfa ( Medicago sativa L.), the "king of forages," represents a globally important perennial legume valued for its nutritional quality, palatability, and environmental benefits in soil improvement and ecological restoration [9]. While adapted to warm climates (optimal growth at 18–22℃), rising global temperatures now pose significant challenges to alfalfa production [10]. Heat stress impairs alfalfa at multiple developmental stages, reducing seed germination rates, compromising photosynthetic efficiency, disrupting water relations, and inducing oxidative damage through ROS accumulation [11]. These physiological disturbances lead to decreased accumulation of critical nutrients including dry matter, carbohydrates, and starch [12], with yield reductions estimated at 17% per 1℃ temperature increase [13]. Despite extensive documentation of thermal priming effects across plant species, the specific mechanisms underlying this phenomenon in alfalfa remain poorly understood. This study employed the heat-tolerant cultivar 'Sanditi' and aimed to characterize morphological and physiological responses to thermal priming under heat stress; to identify differentially expressed genes and metabolic pathways through transcriptomic and metabolomic analyses; and to elucidate the molecular basis of priming-enhanced thermotolerance. Our integrated approach provides novel insights into alfalfa's adaptive strategies against recurrent high-temperature stress and establishes a framework for improving heat tolerance in this critical forage crop. Results Screening of heat-resistant alfalfa varieties To identify suitable germplasm for thermal priming studies, we evaluated the heat tolerance of 12 alfalfa varieties using a comprehensive membership function approach. This analysis incorporated multiple physiological indicators including chlorophyll content, MDA accumulation, electrolyte leakage (EL), and key antioxidant enzyme activities (POD, SOD, CAT) under 37℃ heat stress (Fig. 1 , Table S1 ). The screening revealed significant variability in thermotolerance among cultivars, with membership scores ranging from 0.244 to 0.763. 'Sanditi' emerged as the most heat-resistant variety (score: 0.763), demonstrating superior maintenance of chlorophyll content, reduced oxidative damage (lower MDA and EL), and enhanced antioxidant enzyme activities compared to other cultivars. In contrast, cultivar '343' showed the poorest performance (score: 0.244). Based on these results, we selected 'Sanditi' for all subsequent thermal priming experiments due to its robust heat stress responses. Effect of thermal priming on alfalfa growth morphology To investigate the impact of thermal priming on alfalfa growth phenotypes, seedlings were subjected to the heat priming (Fig. 2 A). The results showed that both non-primed (UP) and primed (P3) groups exhibited wilting and lodging following heat stress, whereas control (CK) plants maintained normal morphology. Notably, P3 group demonstrated significantly reduced lodging severity (P < 0.05) and less pronounced leaf wilting compared to UP group (Fig. 2 B). These findings suggest that thermal priming can effectively mitigate heat stress-induced morphological impairments in alfalfa. To assess the physiological impact of thermal priming on alfalfa, we measured chlorophyll content, MDA levels, soluble protein content, and antioxidant enzyme activities (SOD, POD, CAT) in seedlings from the CK (control), UP (unprimed + heat stress), and P3 (primed + heat stress) groups. Chlorophyll content and soluble protein levels were significantly higher in P3 than UP (Fig. 3 A, C), suggesting that priming helps maintain photosynthetic efficiency and protein stability under stress. MDA content, a marker of lipid peroxidation, significantly increased in both UP and P3 groups compared to CK, confirming heat-induced membrane damage. However, P3 exhibited 18% lower MDA levels than UP (P < 0.05), indicating that thermal priming mitigates heat stress-induced membrane damage (Fig. 3 B). SOD, POD, and CAT activities were also elevated in UP and P3 compared to CK. P3 showed a more pronounced increase in SOD and CAT but reduced POD activity when compared to UP (Fig. 3 D-F), indicating that thermal priming selectively enhances key ROS-scavenging enzymes, reducing oxidative damage. To evaluate ROS generation and cellular integrity under heat stress, we performed histochemical staining. Nitrotetrazolium blue chloride (NBT), diaminobenzidine (DAB) and Evans blue (EB) staining revealed lighter staining in P3 vs. UP, demonstrating that thermal priming reduces ROS accumulation and cell damage (Fig. 3 G). Transcriptome profiling of thermal priming effects To investigate the molecular mechanisms underlying thermal exercise-induced heat tolerance in alfalfa, we performed transcriptome sequencing on leaves from five experimental groups: CK, UP, P1, P2, and P3. The sequencing generated a total of 101.15 GB of high-quality clean data, with each sample producing at least 6 GB of data. All samples exhibited Q30 scores exceeding 94% and GC content ranging from 42.11–42.95%, confirming the high quality of the transcriptome sequencing data (Table S2 ). Differential gene expression analysis (|FoldChange| ≥ 1 and FDR < 0.05) identified: P1 vs CK: 4,891 differentially expressed genes (DEGs; 2,495 up-regulated and 2,396 down-regulated); P2 vs CK: 10,299 DEGs (5,664 up-regulated and 4,635 down-regulated); P3 vs CK: 8,778 DEGs (4,286 up-regulated and 4,492 down-regulated); UP vs CK: 8,130 DEGs (3,828 up-regulated and 4,302 down-regulated) (Fig. 4 A-C). These results clearly demonstrate that thermal exercise significantly alters gene expression profiles in alfalfa under heat stress conditions. Gene expression patterns during thermal priming To elucidate the specific effects of thermal priming on alfalfa heat tolerance, we analyzed 1,217 differentially expressed genes (DEGs) that were uniquely up-regulated in the P3 group. These DEGs were categorized based on their temporal expression patterns during the thermal exercise regimen. Totally 305 DEGs (25.1%) showed initial upregulation during P1 phase, 301 DEGs (24.7%) exhibited subsequent upregulation during P2 phase, and 661 DEGs (50.2%) demonstrated late-stage upregulation during P3 phase (Fig. 4 C-D). Notably, 49.8% of the total up-regulated DEGs (606 genes) were activated during the thermal priming period (prior to P3), while the remaining 50.2% showed delayed response. This biphasic pattern suggests both immediate and progressive transcriptional responses to thermal conditioning. Expression patterns of DEGs in alfalfa after thermal priming treatment To systematically characterize the dynamic transcriptional responses to thermal exercise, we performed Mfuzz clustering analysis of all differentially expressed genes (DEGs) across treatment groups, identifying 16 distinct expression pattern clusters (Fig. 5 ). Compared with the UP group, the upregulated genes in the P3 group were significantly enriched in class 3, class 4, class 13 and class 14. After P1 treatment, the expression level of DEGs in class 3, class 4 and class 14 was up-regulated, while after P2 treatment, the expression level of DEGs in class 4 was down-regulated, but further up-regulated in class 3 and class 14. DEGs in class 13 was up-regulated only after P3 treatment. After KEGG enrichment analysis of DEGs in the above four classes, it was found that they were all enriched in plant hormone signal transduction, spliceosome pathway, phenylpropanoid biosynthesis pathway, glutathione metabolism pathway and fatty acid biosynthesis pathway (Fig. 6 ). These results demonstrate that thermal exercise induces phase-dependent transcriptional reprogramming, with coordinated activation of stress-responsive metabolic and signaling pathways that collectively enhance alfalfa's heat tolerance. Expression patterns of brassinosteroid-related DEGs Through classification and statistical analysis of "plant hormone signal transduction"-related genes in clusters 3, 4, 13, and 14, we observed significant enrichment of BR-related genes across all clusters (Fig. 7 A). BRs are a class of steroid-structured phytohormones that play crucial roles in promoting plant growth and enhancing stress resistance to drought, high temperature, and low temperature conditions. Among BR compounds, brassinolide (BL) exhibits the highest biological activity. BL biosynthesis originates from campesterol (CR) through a series of enzymatic reactions (Fig. 7 B). The BR receptor is the membrane-localized receptor-like protein kinase BRI1. BR binding to BRI1 triggers a phosphorylation cascade that activates downstream transcription factors (e.g., BES1/BZR1). These transcription factors subsequently translocate to the nucleus to regulate expression of target genes, thereby modulating plant growth, development, and stress responses [14–15]. To elucidate the molecular regulatory mechanisms of BR in thermal priming responses, we systematically analyzed expression patterns of genes involved in BR biosynthesis and signaling pathways across different treatment groups. As illustrated in Fig. 7 B, the BR biosynthetic pathway involves key enzymatic steps including sterol hydroxylation, side chain modification, and oxidation, mediated by genes encoding: sterol C-23 hydroxylase (CPD), sterol side chain modification enzyme (ROT3), cytochrome P450 family protein (CYP92A6), 2-Cys peroxidase (BAS1), dwarfing-related protein (DWF4), and C-6 oxidase (BR6ox1). Expression profiling revealed that while CPD and ROT3 were upregulated following P1 treatment, other biosynthetic genes (including DWF4 and BR6ox1 ) showed transcriptional suppression. Notably, despite downregulation of most BR biosynthetic genes after P1 treatment, key BR signaling components such as BRI1 , BAK1 , and BZR1 were upregulated (Fig. 7 C). These findings demonstrate that the BR-BRI1-BES1 signaling pathway plays a pivotal role in thermal priming-induced heat tolerance enhancement in alfalfa. Effect of thermal priming on spliceosome pathway The spliceosome pathway is a core mechanism in plant gene expression regulation, playing an irreplaceable role in plant growth, development, environmental adaptation, and response to external signals [16–17]. To investigate the molecular regulatory mechanisms of the spliceosome pathway under thermal priming, we systematically analyzed the composition and expression patterns of spliceosome-related genes (Fig. 8 A) across different groups (class 3, 4, 13, and 14). Expression profiling revealed upregulation of key spliceosome factors, including SNRP70 (involved in 5' splice site recognition by U1 snRNP) and P68 (which unwinds the U1-5' splice site duplex) (Fig. 8 B). U2 snRNP is responsible for identifying the branch point sequence (BPS) within the intron region. U2B facilitates the anchoring of U2 snRNP to precursor mRNA via nonspecific binding of the SF3B complex to the BPS. As shown in Fig. 8 B, both U2B and SF3B exhibited significant upregulation. Similarly, within the U4/U6. U5 tri-snRNP complex, genes associated with structural maintenance and mature mRNA splicing were markedly upregulated. These findings demonstrate that thermal priming induces significant upregulation of spliceosome pathway-related genes. Effect of thermal priming on phenylpropanoid biosynthesis pathway Lignin, a key component of the secondary cell wall, provides structural rigidity and mechanical strength to plants, facilitating water transport and enhancing resistance to adverse environmental conditions [18]. KEGG enrichment analysis revealed significant upregulation of lignin biosynthesis-related genes following thermal exercise (Fig. 9 A). The upregulation of key phenylpropanoid pathway genes may promote the accumulation of secondary metabolites, including phenols and lignin. Lignin content measurements in alfalfa leaves and stems demonstrated that the P3 group exhibited significantly higher lignin accumulation compared to the UP group. Under heat stress, both the UP and P3 groups showed increased lignin content relative to the CK group, with the P3 group displaying the highest levels (Fig. 9 B-C). These findings indicate that thermal priming enhances lignin synthesis in alfalfa under heat stress, leading to greater stem lignin deposition, improved mechanical strength, and enhanced lodging resistance. Effect of thermal priming on glutathione metabolic pathway The glutathione metabolic pathway plays a crucial role in plant stress responses, with glutathione S-transferase (GST) serving as the key enzyme that catalyzes the initial step of glutathione (GSH) conjugation [19]. As a vital antioxidant, glutathione exhibits both antioxidant and detoxification properties. Furthermore, it participates in the ASA-GSH cycle, forming an essential component of the non-enzymatic antioxidant system and representing a primary pathway for ROS scavenging in plants. Enrichment analysis revealed significant upregulation of genes involved in glutathione metabolism, including GST and glutathione peroxidase (GPX), following thermal exercise. As shown in Fig. 10 .A, antioxidant-related genes such as GST and GPX are rapidly activated in P1 stage of hot start. Dehydroascorbic acid reductase (DHAR) and glutathione-S-transferase Pi (GSTP) were activated in the following P2 and P3 stages. This immediate response suggests that alfalfa rapidly initiates its antioxidant defense mechanisms to counteract heat stress-induced damage. Notably, the expression of these antioxidant enzyme genes further increased after heat stress, indicating that thermal exercise not only triggers the initial antioxidant response but also enhances the plant's capacity to withstand subsequent heat stress. Comparative analysis of biochemical components showed that under heat stress conditions, both UP and P3 groups exhibited increased levels of GSH, glutathione reductase (GR), DHAR, monodehydroascorbate reductase (MDHAR), and ascorbate peroxidase (APX) compared to the CK group, while demonstrating lower ASA content. Importantly, the P3 group showed higher concentrations of GSH, GR, DHAR, MDHAR, ASA, and APX than the UP group (Fig. 10 B-G). These findings demonstrate that thermal priming induces the expression of GST , DHAR , and related genes, leading to increased accumulation of antioxidants including GSH and APX. This response promotes efficient operation of the ASA-GSH cycle, enhances overall antioxidant capacity, and consequently improves alfalfa's thermotolerance when facing subsequent high-temperature stress. Effect of thermal priming on fatty acid composition and content Fatty acids serve as fundamental components of cell membranes, where their saturation degree and carbon chain length critically influence membrane fluidity. These properties are essential for plant growth, physiological metabolism, and abiotic stress resistance [20]. To investigate whether thermal priming enhances plant thermotolerance through fatty acid (FA) modulation, we analyzed free fatty acid profiles using gas chromatography-tandem mass spectrometry (GC-MS). Our analysis identified 17 free fatty acids across P1, P2, P3, UP, and CK groups (Table S3 ). The composition revealed that palmitic acid (6.7%-11% of total FAs) was predominated in saturated fatty acids (SFA), followed by stearic acid (1.3%-2.3%). Among the monounsaturated fatty acids (MUFA), oleic acid had the highest content, followed by erucic acid, accounting for 1.7%-4.1% of total fatty acids. Among polyunsaturated fatty acids (PUFA), the highest content was α-linolenic acid, accounting for 74.6%-80.6% of total fatty acids, followed by linoleic acid, accounting for 5.3%-8.1% of total fatty acids. Notably, the P3 group exhibited significantly higher total FA content (600.52 ± 61.70 µg/g) versus control (402.52 ± 2.73 µg/g), P1 (448.11 ± 9.58 µg/g), P2 (561.69 ± 49.66 µg/g), and UP (424.52 ± 19.62 µg/g) groups. KEGG enrichment analysis of P3vs UP metabolites showed significant enrichment in unsaturated fatty acid biosynthesis, metabolic pathways, and fatty acid biosynthesis (Fig. 11 A-B). Transcriptional data further indicated upregulation of FAD genes across all groups. According to |log2FC| >0.6, P < 0.05, the differential metabolites in P3 vs UP group were screened (Table 1). The main differential metabolites are α-linolenic acid, erucic acid, oleic acid, arachidic acid and docosanoic acid. Among unsaturated fatty acids, the contents of α-linolenic acid, oleic acid and erucic acid in P3 group increased by 52.6%, 147.1% and 107.5% respectively compared with those in UP group, showing an upward trend. Among linear saturated fatty acids, arachidonic acid and docosanoic acid in P3 group were 52.5% and 51.6% of those in UP group, respectively, showing a downward trend. While SFA levels remained stable, MUFAs and PUFAs increased markedly, particularly PUFAs. This suggests thermal priming preferentially enhances unsaturated fatty acid synthesis, potentially improving membrane fluidity and heat resistance in alfalfa. Table.1 The content of different fatty acids in P3 vs UP group Fatty acid name Type P3 (µg/g) UP (µg/g) P-value log2FC Type α-linolenic acid Polyunsaturated fatty acid 483.783 ± 49.726a 317.118 ± 1.437c 0.022 0.609 UP erucic acid Monounsaturated fatty acids 0.944 ± 0.008 0.455 ± 0.027 0.018 1.053 UP oleic acid Monounsaturated fatty acids 23.972 ± 3.315 9.696 ± 3.315 0.014 1.305 UP arachidic acid Linear saturated fatty acid 0.426 ± 0.026 0.811 ± 0.101 0.014 -0.929 DOWN docosanoic acid Linear saturated fatty acid 0.304 ± 0.053 0.589 ± 0.031 0.049 -0.954 DOWN Validation of RNA-seq data by quantitative RT-PCR (RT-qPCR) To validate the reliability of our RNA-Seq results, we selected seven key genes involved in phenylpropanoid biosynthesis and glutathione metabolism pathways for RT-qPCR analysis. The RT-qPCR results demonstrated excellent consistency with the RNA-Seq data (Fig. 12 ), confirming the accuracy of our transcriptome sequencing findings. This strong correlation between both analytical methods validates the differential gene expression patterns observed in response to thermal exercise. Discussion As a critical physiological response mechanism to environmental temperature fluctuations, thermal priming enables plants to systematically adjust their physiological processes following exposure to high temperatures, thereby enhancing their capacity to withstand subsequent heat stress. This adaptive response exhibits temporal persistence, as plants retain elevated thermotolerance even after ambient temperatures normalize. The underlying mechanisms involve complex biochemical and molecular reprogramming, including gene expression modulation, metabolic pathway adjustments, and signal transduction activation. Oxidative stress and antioxidant defense Heat stress disrupts cellular redox homeostasis, leading to ROS accumulation. While ROS function as signaling molecules regulating growth and stress responses [21], excessive ROS induces oxidative damage to DNA, proteins, and lipids, ultimately impairing cell viability [22]. Plants counteract ROS via enzymatic (SOD, POD, CAT) and non-enzymatic (GSH, ASA) antioxidant systems [23]. In this study, heat-stressed alfalfa exhibited increased SOD, POD, and CAT activities. Notably, thermally primed (P3) seedlings displayed further elevation in SOD and CAT activities but reduced POD activity compared to unprimed (UP) plants, suggesting that thermal priming preferentially scavenges superoxide radicals (O 2− ) and H₂O₂, thereby diminishing POD demand. Consequently, P3 seedlings maintained lower ROS levels and superior growth under heat stress, underscoring the role of thermal priming in enhancing oxidative stress resilience. Brassinosteroid (BR) signaling and heat tolerance As a key phytohormone, BR enhances plant thermotolerance. Prior studies demonstrate that BR elevates photosynthetic efficiency (Fv/Fm) and stomatal conductance in heat-stressed rice [24]. BR activates the expression of HSFA2 , DREB2A , and HSP s via BZR1-mediated repression of ERF49 in Arabidopsis [25]. In addition, BR promotes dry matter and nutrient translocation in maize under heat stress [26]. BR regulates HSP17.6A via histone acetyltransferase HAC1 to sustain proteostasis [27]. Our transcriptomic analysis revealed that thermal priming potentiates BR signaling by upregulating receptor genes ( BRI1 / BAK1 ), enhancing receptor density and signal sensitivity even without elevated BR levels. The transcription factor BZR1, a central BR pathway regulator, was upregulated, directly activating downstream thermotolerance genes, including HSPs and genes involved in mitigating ROS toxicity. BR signaling pathway can regulate several downstream metabolic pathways and secondary metabolic pathways. Liu et al. ( 2023 ) found that BR treatment increased the activities of PAL and 4CL in watermelon seedlings under zinc stress and induced lignin accumulation [28]. Under salt stress, the contents of phenolic compounds, flavonoids and lignin were increased by BR in order to reduce the damage in Ornamental Gourd [29]. Guo et al. ( 2024 ) found that BR can increase lignin synthesis-related genes such as PAL and lignin deposition in Ginkgo biloba [30]. In Korean pine, BR can also improve PAL activity and lignin content in cells through phenylpropanoid biosynthesis [31]. Zhou et al. ( 2018 ) found that BR in grapes can enhance AsA-GSH cycle by increasing the activities of MDHAR, GR, APX and DHAR, and the contents of antioxidant ascorbic acid (MA) and dehydroascorbic acid (DHA), thus reducing the damage of plants under Cu stress [32]. Niu et al. ( 2025 ) found that under low temperature stress, BR promoted the accumulation of phenols such as GSH and hesperidin in jujube fruit, maintained the quality and reduced chilling injury [33]. Dong et al. ( 2025 ) found that BR treatment can significantly up-regulate genes such as FAD2 , FAD3 and LOX in grapes, maintain the proportion of unsaturated fatty acids in membrane lipids, and realize the stability of membrane structure [34]. Li et al. ( 2022 ) found that BR can also increase the accumulation of butyric acid, octanoic acid, decanoic acid, linoleic acid and other substances in grapes, and improve their low temperature resistance [35]. In this study, thermal priming activated these pathways, increasing metabolites (e.g., lignin, phenolics, unsaturated fatty acids) that collectively bolster heat tolerance in alfalfa. Phenylpropanoid pathway and lignin deposition Phenylpropanoids (e.g., flavonoids, lignin) are pivotal for stress adaptation [36]. Phenylpropionic acids are mainly derivatives of cinnamic acid, including caffeic acid, ferulic acid, mustard acid, etc. Because of their excellent ability of scavenging free radicals, they are considered as the main antioxidants to resist oxidative damage and are necessary for plants to adapt to biotic and abiotic stresses [37]. Lignin is one of the important products in the biosynthesis of phenylpropanoid. It can not only enhance the mechanical strength of plants and the hardness of cell walls, but also have many biological functions such as resisting the invasion of adverse external environment and diverting water transport in tissues [38]. Lignin is synthesized by phenylpropanoid biosynthesis with phenylalanine as the substrate. PAL converts phenylalanine into cinnamic acid, which is then reduced by 4CL, CCR, CAD and other enzymes in turn to generate corresponding coenzyme A, and finally the catalytic intermediate coenzyme A is converted into corresponding lignin monomer [39]. Under heat stress, phenylpropanoids are considered as markers of heat stress in plants [40]. For example, phenylpropionic acids and flavonoids in carrot cells protect plants from heat stress by inhibiting ROS formation [41]. Paupière et al. ( 2021 ) found that HSFb1 induced the accumulation of phenylpropanoid metabolites in tomatoes, which enhanced the heat resistance [42]. In this study, the expression levels of PAL, COMT, F5H, CCR and 4CL related to lignin biosynthesis increased significantly after thermal priming. Compared with CK, it was observed that the expression of lignin synthesis-related genes decreased in the early stage of thermal exercise, which may be because high temperature interfered with enzyme activity and transcription factor binding ability, thus inhibiting the normal expression of related genes. At P3 stage after thermal priming, the expression of lignin synthesis-related genes was significantly higher than that of UP group without thermal priming. This shows that through thermal priming, plants may start the adaptive mechanism, enhance their structural stability and stress resistance, and up-regulate the expression of lignin synthesis-related genes, thus promoting lignin synthesis and improving the resistance to high temperature. The quantitative results of lignin content showed that the lignin content in alfalfa stems increased, which is also the reason why plants after thermal priming showed stronger lodging resistance. In addition, the determination of lignin content in seedling stems of P1 and P2 treatment points during thermal priming showed that alfalfa began to synthesize lignin at the early stage of thermal priming and accumulated continuously. In a word, thermal priming promotes the synthesis and accumulation of lignin in alfalfa stems, and effectively improves the lodging resistance of alfalfa seedlings. AsA-GSH cycle and redox balance The ascorbate-glutathione (AsA-GSH) cycle represents a crucial antioxidant defense system in plants, functioning to scavenge ROS through coordinated action of antioxidants (AsA and GSH) and enzymes including APX, GPX, and glutathione reductase (GR) [43]. In this cycle, APX catalyzes the oxidation of AsA to eliminate harmful ROS such as superoxide radicals (O 2− ) and hydrogen peroxide (H₂O₂), while GR, DHAR, and MDHAR regenerate reduced AsA and GSH to sustain the cycle's activity [44]. Extensive research has established a positive correlation between AsA-GSH cycle activity and plant stress tolerance. For instance, strawberry seedlings exposed to combined high temperature and high light stress exhibited enhanced GR, DHAR, and MDHAR activities, facilitating AsA-GSH regeneration and consequently improving stress resilience [45]. Similarly, Li et al. ( 2020 ) demonstrated that the halophyte Suaeda salsa upregulates GR under salt stress to boost GSH production and alleviate oxidative damage [46]. Our experimental findings demonstrate that thermal priming significantly enhances the AsA-GSH cycle's efficiency in alfalfa seedlings. Quantitative analyses revealed elevated levels of both enzymatic components (APX, GR, DHAR, MDHAR) and non-enzymatic antioxidants (AsA, GSH) in primed plants. This coordinated upregulation enables more effective ROS detoxification and redox homeostasis maintenance, thereby reducing oxidative stress damage under high temperature conditions. The enhanced AsA-GSH cycle activity represents a key mechanism through which thermal priming confers improved thermotolerance in alfalfa seedlings. Fatty acid remodeling and membrane stability As a key component of biofilm, cork and plant epidermis wax, fatty acids are not only the basic raw materials for building cell and tissue structures, but also effectively maintain the material exchange regulation and barrier function between organisms and the external environment by forming hydrophobic barriers, and also play an important role in resisting abiotic stresses [47]. At higher temperature, plants reduce the phase transition temperature by increasing unsaturated fatty acids in membrane lipids, so as to keep the cell membrane liquid-crystalline at high temperature and avoid the loss of cell function caused by the solidification or rupture of membrane structure, so as to alleviate the influence of high temperature stress. For example, the deletion of C18:1 and C18:2 in accD-C794 mutant in Arabidopsis thaliana leads to heat sensitivity, which indicates that unsaturated fatty acids play an important role in heat stress tolerance [48]. This ability mainly depends on the regulation of fatty acid desaturase (FAD) on unsaturated fatty acid level [49]. Beisson et al. ( 2007 ) found that in Arabidopsis thaliana , the mutant with FAD3 gene deletion showed serious membrane damage at 42℃, and the linolenic acid content in chloroplast membrane lipid decreased by 60%, accompanied by a three-fold increase in MDA accumulation [50]. Chen et al. ( 2021 ) found that under the continuous stress of 38℃, the leaf EL of the rice OsFAD3 overexpression line decreased by 45% compared with that of the wild type, indicating that the membrane permeability was effectively maintained [51]. Our transcriptome analysis showed that thermal priming increases the expression of FAD gene, causes the accumulation of polyunsaturated fatty acids and maintains the stability of membrane system. High temperature changes of membrane lipid from liquid crystal state to gel state, which destroys the membrane structure. The three double bonds of α-linolenic acid (C18:3) make the membrane have high fluidity, which can reduce the phase transition temperature of the membrane and delay the curing of membrane lipid induced by high temperature [52]. You et al. ( 2024 ) found that the decrease of α-linolenic acid content in rice would destroy the integrity and stability of cell membrane system under heat stress [53]. Ibrahim et al. (2019) found that after tomato seeds were soaked at 50℃ for 2 hours, the α-linolenic acid content increased and the germination ability was significantly improved [54]. Tang et al. ( 2024 ) found that α-linolenic acid can also produce jasmonic acid (JA) through lipoxygenase (LOX) pathway, and then activate the expression of HSFs and HSPs through JA, thus alleviating high temperature injury [55]. In this study, the content of α-linolenic acid was the highest in P3 group, which was significantly increased by 52.6% compared with UP group. The results showed that thermal priming could reduce the injury of alfalfa seedlings at high temperature by increasing the content of α-linolenic acid. As a monounsaturated fatty acid, the accumulation of oleic acid may improve the thermal stability by lowering the lipid transformation temperature of the membrane. Hou et al. ( 2006 ) found that soybean seeds increased oleic acid content at high temperature to adapt to the temperature increase [56]. Li et al. ( 2023 ) found that in soybean GmPDCT1 / GmPDCT2 silent mutant, the transformation from oleic acid to linoleic acid was blocked, and the SOD activity and POD activity of leaves increased by 45% and 60% respectively under salt stress [57]. In this study, the oleic acid content of P3 group increased by 147.1% compared with that of UP group, which indicated that it played an important role in improving the heat resistance of alfalfa under high temperature stress. The synthesis of erucic acid (C22:1) is based on oleic acid-CoA. Through continuous condensation, reduction, dehydration and reduction, two carbon units are added each time, and erucic acid is generated after three rounds of extension. As a long-chain monounsaturated fatty acid, erucic acid may help plants maintain the stability of membrane structure under high temperature stress by changing the fluidity of cell membrane lipids. There was no significant difference in saturated fatty acid content between P3 group and CK group, but it was significantly lower than P2 group. It is speculated that the membrane fluidity can be optimized by increasing the content of saturated fatty acids and balancing the ratio of saturated and unsaturated fatty acids in alfalfa during thermal exercise to avoid excessive liquefaction of the membrane at high temperature. However, in the face of high temperature stress again, alfalfa mainly reduces the damage caused by high temperature by increasing the content of unsaturated fatty acids. Spliceosome activation and transcriptional plasticity Spliceosome are dynamic complexes composed of snRNP (such as U1, U2, U4/U6, U5) and non-snRNP proteins. Spliceosome pathway is not only a molecular switch of gene expression in plants, but also a key hub connecting genetic information and phenotypic plasticity. By precisely regulating alternative splicing and constitutive splicing, it gives plants molecular flexibility to cope with complex environments [58]. For example, under low temperature stress, SME1 ensures the correct splicing of many mRNA precursors in Arabidopsis thaliana [59]. In addition, He et al. ( 2023 ) found that under heat stress, the activity and composition of splicing also change significantly [60]. Gu et al. ( 2024 ) found that the spliceosome pathway in Chinese cabbage was significantly up-regulated under high temperature stress [61]. Lee et al. ( 2017 ) found that SF1 participated in the regulation of flowering and heat tolerance by participating in the splicing of the precursor of HsfA2 [62]. Rosenkranz et al. ( 2021 ) found that five SR genes in tomato were up-regulated by high temperature induction to optimize transcription and splicing efficiency [63]. In this study, SNRP70 which recognizes the 5' splicing site on the precursor mRNA in U1 snRNP, P68 which unwinds the duplex of U1-5' splicing site, and Y2B, SF3B, SRF and other proteins which recognize and bind the branching sequence in U2 snRNP can make more U1 bind to the 5' splicing site faster, and U2 bind to the branching point faster, thus accelerating the early assembly of splices and allowing cells to process more precursor mRNA in a unit time. Up-regulation of key genes in U4/U6. U5 tri-snRNP complex makes the supply of the protein complex more abundant, which can accelerate the transformation of splice from "pre-splice" to "mature splice" and push splicing into the catalytic stage. In this study, after thermal priming, these spliceosome pathway-related genes were up-regulated rapidly after P1 treatment, which significantly increased the output rate of mature mRNA, and many proteins with different functions were synthesized rapidly. These protein participate in the regulation of antioxidant enzyme system and enhance the ability of plants to remove ROS; or participate in the remodeling of cell membrane components to maintain the fluidity and integrity of cell membrane at high temperature. The rapid response of this post-transcriptional regulation mechanism is the key for plants to obtain high temperature adaptability during thermal priming. After heat-priming, the splicing process of precursor mRNA is accelerated by pre-activating the genes related to the spliceosome pathway, so that mature mRNA and functional protein can be synthesized rapidly. In the face of extreme high temperature, these gene expression products mobilized in advance quickly play a role in helping plants to effectively adapt to high temperature, which embodies the important significance of thermal priming to give plants survival advantages in adversity by stimulating post-transcriptional regulation. Conclusion This study demonstrates that heat priming enhances alfalfa's thermotolerance by elevating chlorophyll content, soluble protein levels, and the activities of SOD and CAT, while reducing ROS accumulation and MDA content under high-temperature stress. Transcriptome analysis revealed that differentially expressed genes in P3 vs UP were significantly enriched in hormone signaling pathways, primary metabolite biosynthesis, and secondary metabolite biosynthesis, with a strong association with BR signaling. Mechanistically, heat priming exerts its protective effects through BR-mediated metabolic regulation and the phenylpropanoid biosynthesis pathway, leading to increased lignin synthesis in stems, thereby improving lodging resistance. Heat priming also boosts the AsA-GSH cycle, elevating antioxidant enzyme activity and non-enzymatic antioxidant levels to mitigate ROS-induced damage, and promotes unsaturated fatty acid synthesis, maintaining membrane fluidity under heat stress. On the other hand, heat priming facilitates mRNA splicing and export by upregulating the spliceosome pathway, ensuring efficient gene expression under stress. Through these synergistic mechanisms (Fig. 13 ), heat priming enables alfalfa to establish a robust thermotolerance defense system, ultimately enhancing its heat resistance. Materials and methods Plant materials and growing conditions The experiment was conducted in the College of Environment and Resources of Dalian Minzu University in May 2023. Twelve commercial alfalfa varieties (Table S4 ) were purchased as experimental materials, and the seeds were soaked in the dark for 24 hours to accelerate germination. Seeding the germinated seeds into flowerpots (10cm×10cm×10cm), wherein the substrate is peat soil, vermiculite and pearl salt, which are stirred evenly at 2:1:1, and each pot is seeded with 8–10 alfalfa seeds, and each variety is planted in 10 pots, and then cultured in a solar greenhouse (average temperature 25.0 ± 2℃, relative humidity 60 ± 5%). During the cultivation period, water should be poured every 3 days to keep the soil moisture sufficient. After 14 days to the three-leaf stage, alfalfa seedlings with similar growth and no diseases and pests were selected for the next experiment. Heat-resistance screening By simulating the high temperature environment in an incubator, leaves of 12 alfalfa varieties under high temperature stress were collected to determine physiological and biochemical indexes, and the heat resistance of different alfalfa varieties was comprehensively evaluated by membership function method [64]. After accelerating germination, alfalfa was moved to a constant temperature light incubator. The control group was cultured at 25℃, and the experimental group was treated with heat stress at 37℃. After 4 hours' treatment, leaves of alfalfa seedlings under heat stress (HT) and control (CK) were collected for determination of chlorophyll content, antioxidant enzyme activity, electrolyte permeability and MDA content. All assays were performed in three independent replicates. Finally, the relative heat tolerance of alfalfa varieties was evaluated by membership function method. Thermal priming treatment Heat-resistant alfalfa 'Sanditi' was used as the experimental material. After 12 days of cultivation, it was treated with high temperature stress as shown in Fig. 2 .A, and it was divided into control group CK (Control), thermal exercise group P (Primed) and heat control group UP (unprimed) without heat priming. The plants were subjected to heat stress in a light incubator. The light intensity was 12000 lux, the light period was 16h light /8h darkness, and the relative humidity was 60%. Pots are randomly placed to reduce the influence of different positions. In the thermal exercise group, P first exercised at 37℃ for 2 hours, then recovered at 23℃ for 2 hours, then exercised at 43℃ for 2 hours, and then recovered at 23℃ for 2 days. Finally, P group and UP group were subjected to heat stress at 43℃ for 4 hours. Collect leaves at P1, P2, P3, CK, and UP, respectively, and were frozen with liquid nitrogen and stored in a refrigerator at -80℃ for subsequent experiments. Physiological index determination Chlorophyll content in alfalfa leaves was determined by acetone-ethanol mixed extraction [65]. The content of MDA was determined by thiobarbituric acid colorimetry [66]. The activity of SOD was determined by nitroblue tetrazole photochemical reduction method [67]. The activity of POD was determined by guaiacol method [68]. The activity of CAT was determined by ultraviolet absorption method [69]. EL was measured by conductivity meter [70]. Coomassie brilliant blue method was used to determine the content of soluble protein. The leaves were infiltrated with NBT, DAB [71] and EB [72] respectively, and the histochemical staining was carried out to detect the accumulation of superoxide anion, hydrogen peroxide and cell death in alfalfa leaves. All the above experiments were carried out in three biological repetitions. Transcriptome sequencing and data analysis A total of 15 samples (5 treatments×3 biological replicates) were collected for Qualcomm quantitative transcriptome sequencing detection. The experimental process included RNA extraction and detection, cDNA library construction and computer sequencing [73]. The total RNA of alfalfa leaves was extracted by Tiangen RNAprep pure plant kit (centrifugal column), and the applied methods and steps strictly followed the instructions of the kit. The absorbance of A260/A280 is measured by ultra-micro spectrophotometer to ensure that the purity of total RNA is between 1.8 and 2.1 to avoid pollution. By utilizing the polyA tail characteristics of eukaryotic mRNA, mRNA was enriched using Oligo (dT) magnetic beads. After treatment with fragmentation buffer, one strand cDNA was synthesized by reverse transcription using random hexamers. During the second strand synthesis, dUTP was used instead of dTTP to construct a chain specific library. Subsequently, end repair, A-tail addition, and adapter connection were performed, followed by magnetic bead purification and screening of a library containing 250–350 bp insertion fragments. After Qubit quantification, Qsep400 fragment analysis, and Q-PCR calibration, 150 bp double ended sequencing was performed on the Illumina platform. The sequencing data was subjected to fastp quality control to remove reads containing adapters, N ratios > 10%, or low-quality bases (Q ≤ 20) > 50%. Using HISAT to align clean reads to the reference genome and StringTie for predicting new genes. Calculate the logarithmic ratio of genes through featureCounts and convert it into FPKM values to quantify expression levels. Differential analysis was performed using DESeq2, and after Benjamini&Hochberg correction, differentially expressed genes were screened using the corrected P-value and log 2 fold change threshold. KEGG and GO enrichment analysis were performed based on hypergeometric tests [74]. RT-qPCR analysis. In order to verify the accuracy of RNA-seq sequencing, seven differentially expressed genes speculated to be related to thermal exercise to improve the heat tolerance of alfalfa were randomly selected for RT-qPCR verification. The seven differentially expressed genes are PAL (Phenylalanine Ammonia-Lyase), F5H (Fertilize 5-hydroxylase), 4CL (4-coumarate Coenzyme A Ligase), Hsf2b (Heat stress transcription factor b-2b), CCR (Cinnamoyl-coa reduction), GST (Glutathione-s-transfer) and Peroxidase. Using β-Actin as the reference gene, the gene-specific primers were designed by PrimerPremer5.0, and the primer sequence is shown in Table S5 . RT-qPCR was performed on ABI/Thermo Fisher. Each gene was analyzed in three biological samples, and each biological sample was repeated three times. The relative expression of the target gene was measured by 2 −ΔΔCt [75]. Metabolite content detection Lignin content in leaves and stems of alfalfa was determined by using lignin content detection kit (Shanghai Liquid Quality Detection Technology Co., Ltd.). Assay of ASA-GSH activity Using commercial kits (Shanghai Liquid Quality Testing Technology Co., Ltd.), the contents of reduced glutathione and ascorbic acid were determined, and the activities of APX, glutathione reductase (GR), monodehydroascorbic acid reductase (MDHAR), dehydroascorbic acid reductase (DHAR) and other enzymes were determined. Detection of free fatty acids by proteomics Thaw the sample on ice, take 50 mg to EP tube, add 700 µl of extract, vortex at 2500 r/min for 10min, then perform ultrasonic treatment at 4℃ for 15min, centrifuge at 4200 rpm for 5min, transfer 500 µl of supernatant to a glass bottle containing 200 µl 0.05 M sodium hydroxide methanol solution, and blow dry with nitrogen. The residue was redissolved with 400 µl 0.4 M sodium hydroxide methanol solution and incubated at 70℃ for 10 min. After cooling, add 500 µl dichloromethane and 200 µl double distilled water, vortex at 2500 r/min for 5 min, centrifuge at 4200 rpm for 5 min, take 300 µl supernatant to a new bottle containing 300 µl 2 M methanol hydrochloric acid solution, and react at 70℃ for 20 min for methyl esterification. After cooling, add 500 µl n-hexane and 300 µl double distilled water, vortex at 2500 r/min for 5 min, and centrifuge at 4200 rpm for 5 min, and collect the supernatant containing fatty acid methyl ester into the injection bottle for GC-MS analysis [75–78]. R language prcomp function, the data is standardized by unit variance and unsupervised principal component analysis is carried out. Taking |Log2FC| as the threshold, the metabolites were annotated in combination with KEGG compound database, mapped to KEGG pathway, and then enriched and analyzed by MSEA. Data statistics. Analysis of variance (ANOVA) was performed using IBM SPSS Statistics 22. Plots were made using Origin 2018. Declarations Acknowledgements Not applicable. Author contributions JH designed the experiments. JCM, SJL, ZJR, ZZY, LH, ZJX performed the experiments and data analysis, and drafted the manuscript. All authors read and approved the final manuscript. Funding This work was supported by the central finance forestry science and technology extension demonstration project [2023]TG05. Availability of data and materials Raw data was deposited in NCBI database under SRA accession: PRJNA1294583(https://submit.ncbi.nlm.nih.gov/subs/sra/SUB15477709/overview). Ethics approval and consent to participate This experiment does not involve human experiments and animal experiments. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References Yao XH, Li YL, Chen J, Zhou ZX, Wen Y, Fang K, Yang FB, Li TT, Zhang DW, Lin HH. Brassinosteroids enhance BES1-required thermomemory in Arabidopsis thaliana . 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Supplementary Files Additionalfile1.xlsx Additional file 1: Table S1 Comprehensive evaluation results of heat tolerance of 12 alfalfa varieties Additionalfile2.xlsx Additional file 2: Table S2 Transcriptome sequencing data Additionalfile3.xlsx Additional file 3: Table S3 Fatty acid content test results Additionalfile4.xlsx Additional file 4: Table S4 Name and source of alfalfa varieties Additionalfile5.xlsx Additional file 5: Table S5 Primer sequences Cite Share Download PDF Status: Published Journal Publication published 09 Dec, 2025 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Revision requested 03 Oct, 2025 Reviews received at journal 29 Sep, 2025 Reviews received at journal 28 Sep, 2025 Reviewers agreed at journal 25 Sep, 2025 Reviewers agreed at journal 23 Sep, 2025 Reviewers agreed at journal 20 Sep, 2025 Reviewers agreed at journal 08 Aug, 2025 Reviewers invited by journal 03 Aug, 2025 Editor invited by journal 01 Aug, 2025 Editor assigned by journal 01 Aug, 2025 Submission checks completed at journal 01 Aug, 2025 First submitted to journal 22 Jul, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7191702","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":495839173,"identity":"ab6c1819-81b1-4039-9d11-2bb38d14499e","order_by":0,"name":"Chengmin Jin","email":"","orcid":"","institution":"Dalian Minzu University","correspondingAuthor":false,"prefix":"","firstName":"Chengmin","middleName":"","lastName":"Jin","suffix":""},{"id":495839174,"identity":"39874eb0-6eb5-43af-a923-23f6ba45799a","order_by":1,"name":"Jingliang Sun","email":"","orcid":"","institution":"Dalian Minzu University","correspondingAuthor":false,"prefix":"","firstName":"Jingliang","middleName":"","lastName":"Sun","suffix":""},{"id":495839175,"identity":"0e4b0a58-c551-4364-9629-09e815054459","order_by":2,"name":"Jinrui Zhao","email":"","orcid":"","institution":"Dalian Minzu University","correspondingAuthor":false,"prefix":"","firstName":"Jinrui","middleName":"","lastName":"Zhao","suffix":""},{"id":495839176,"identity":"f3bee109-041c-44a4-a7eb-e75dcb62f66b","order_by":3,"name":"Ziyao Zhang","email":"","orcid":"","institution":"Dalian Minzu University","correspondingAuthor":false,"prefix":"","firstName":"Ziyao","middleName":"","lastName":"Zhang","suffix":""},{"id":495839177,"identity":"6c5d3595-8428-4a59-af34-bc05402532c6","order_by":4,"name":"Han Lu","email":"","orcid":"","institution":"Dalian Minzu University","correspondingAuthor":false,"prefix":"","firstName":"Han","middleName":"","lastName":"Lu","suffix":""},{"id":495839178,"identity":"d407e42a-0a2b-4c13-9065-3dd41adafd76","order_by":5,"name":"Jixiang Zou","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAtklEQVRIiWNgGAWjYFCCA4wPeArALAOitTAb8BiQpoWBTYI0LfKNZ8wq3hjcSWxgb94mwVBzh7AWxoZjaTfnGDxLbOA5VibBcOwZYS3MDIeP3eYxOJzYIJFjJsHYcJiwFjaGg23FYC3yb4jUwgO0hRliCw+RWoDOT5acY3DYuI0nrdgi4RgRWuRnnDH88KbisGw/++GNNz7UEKGFQeIAhGYDEQlEaGBg4G8gStkoGAWjYBSMZAAAdDw4NeQeNd0AAAAASUVORK5CYII=","orcid":"","institution":"Dalian Minzu University","correspondingAuthor":true,"prefix":"","firstName":"Jixiang","middleName":"","lastName":"Zou","suffix":""},{"id":495839179,"identity":"4637cbaf-cc29-44f9-a3da-5fdf8c82d2a4","order_by":6,"name":"Hua Jin","email":"","orcid":"","institution":"Dalian Minzu University","correspondingAuthor":false,"prefix":"","firstName":"Hua","middleName":"","lastName":"Jin","suffix":""}],"badges":[],"createdAt":"2025-07-23 03:38:28","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7191702/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7191702/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-025-07726-w","type":"published","date":"2025-12-09T15:58:27+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":88599234,"identity":"b1f2f778-93bd-4209-a50d-9972aa6a086e","added_by":"auto","created_at":"2025-08-08 07:32:42","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":47082,"visible":true,"origin":"","legend":"\u003cp\u003eComprehensive evaluation of heat tolerance in alfalfa varieties under heat stress conditions. The membership functions of 12 alfalfa indexes were accumulated and the average value was obtained. In order to avoid errors caused by variety differences, Xj, Xmax and Xmin are all calculated by \"relative values\" instead of \"measured values\". Relative value = stress group/control group.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7191702/v1/0057360b340e60fe740229bd.png"},{"id":88599235,"identity":"a654b0b3-07f0-440f-a4d5-1e95fe6b2f00","added_by":"auto","created_at":"2025-08-08 07:32:43","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":506286,"visible":true,"origin":"","legend":"\u003cp\u003eTreatment methodology and phenotypic response. A. Experimental protocols. B. Effect of thermal priming on alfalfa growth. Control: seedlings prior triggering; unprimed: Seedlings are triggered by heat stress; primed: Seedlings are subjected to heat priming and heat stress triggering.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7191702/v1/5c07f7eaa66933f8f1bef3d4.png"},{"id":88599257,"identity":"941e1908-f484-409a-8aaf-7d26c734fa5a","added_by":"auto","created_at":"2025-08-08 07:32:44","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":239768,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of thermal priming on physiological index of alfalfa leaves under heat stress conditions. A. Chlorophyll content. B.MDA content. C. Soluble protein content. D. SOD activity. E. CAT activity. F. POD activity. G. Histochemical staining. Data were expressed as the mean ± standard error of three independent biological replicates. Different letters indicated significant differences of P \u0026lt; 0.05 according to Duncan test.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7191702/v1/3321303769edcbf8112401ca.png"},{"id":88600300,"identity":"c7153a1f-1671-4a21-9b1d-572340803985","added_by":"auto","created_at":"2025-08-08 07:40:43","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":273458,"visible":true,"origin":"","legend":"\u003cp\u003eNumber of differentially expressed genes in different treatment groups. A. Venn diagram of total DEGs in each treatment group. B. Number of DEGs in each treatment group. C. Venn diagram of upregulated DEGs in each treatment group. D. Expression patterns of upregulated DEGs in the P3 vs UP group.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7191702/v1/96afe5fc4cd5684c3adcea19.png"},{"id":88599241,"identity":"b241268c-bc04-4366-929b-64a49312d745","added_by":"auto","created_at":"2025-08-08 07:32:43","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":536293,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression patterns of DEGs under different treatments in leaves. Sub class represents gene groups with the same changing trend, and the number after total represents the number of genes in each group. The cluster centers are marked as the black lines.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7191702/v1/0f637bef651b97daac435923.png"},{"id":88599248,"identity":"b43c2b96-bbd4-4eb3-a918-97e37caf528d","added_by":"auto","created_at":"2025-08-08 07:32:43","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":229536,"visible":true,"origin":"","legend":"\u003cp\u003eKEGG enrichment analysis of differentially expressed genes in each module. A. Class 3. B. Class 4. C. Class 13. D. Class 14.\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7191702/v1/755b6488095a33c04c701075.png"},{"id":88600309,"identity":"3517322a-8293-473e-b9e1-895543f9707c","added_by":"auto","created_at":"2025-08-08 07:40:44","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":229740,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of thermal priming on hormone signal transduction pathway. A. Hormone signal statistics in each module. B. Brassinolide synthesis pathway. C. Brassinolide signal transduction pathway.\u003c/p\u003e","description":"","filename":"image7.png","url":"https://assets-eu.researchsquare.com/files/rs-7191702/v1/3ded8c2c3379d2865759c4fe.png"},{"id":88600307,"identity":"d985a353-71b3-45f0-a969-f0548f086226","added_by":"auto","created_at":"2025-08-08 07:40:44","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":314944,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of thermal priming on RNA splicing pathway. A. Spliceosome pathway B. Gene expression of various components in the spliceosome pathway.\u003c/p\u003e","description":"","filename":"image8.png","url":"https://assets-eu.researchsquare.com/files/rs-7191702/v1/b19c0c2705a40314db2d911f.png"},{"id":88599252,"identity":"33a9adf9-a481-4200-89f4-ff3b6046ba29","added_by":"auto","created_at":"2025-08-08 07:32:43","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":105032,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of thermal priming on phenylpropanoid biosynthesis pathway. A. Thermal map of lignin synthesis pathway. B. Lignin content in leaves. C. Lignin content in roots. Data were expressed as the mean ± standard error of three independent biological replicates. Different letters indicated significant differences of P \u0026lt; 0.05 according to Duncan test.\u003c/p\u003e","description":"","filename":"image9.png","url":"https://assets-eu.researchsquare.com/files/rs-7191702/v1/1086ed8dda7f9efc63d9c47f.png"},{"id":88600628,"identity":"953fbc6b-ed9d-4b48-bb27-d7f1b718c7eb","added_by":"auto","created_at":"2025-08-08 07:48:44","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":202382,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of thermal priming on glutathione metabolism pathway. A. Thermal map of glutathione metabolic pathway. B. GSH content. C. GR activity. D. DHAR activity. E. MDHAR activity. F. ASA content. G. APX activity. Data were expressed as the mean ± standard error of three independent biological replicates. Different letters indicated significant differences of P \u0026lt; 0.05 according to Duncan test.\u003c/p\u003e","description":"","filename":"image10.png","url":"https://assets-eu.researchsquare.com/files/rs-7191702/v1/2687814b0e8085ac9619fbf2.png"},{"id":88599253,"identity":"6fd9eb93-08f4-4f8f-aa00-883e2f7c0eb9","added_by":"auto","created_at":"2025-08-08 07:32:43","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":157828,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of thermal priming on fatty acid content. A. Dotplot of KEGG enrichment analysis of differential metabolites. B. Barplot of KEGG enrichment analysis of differential metabolites. C. Thermal map of fatty acid desaturase gene expression.\u003c/p\u003e","description":"","filename":"image11.png","url":"https://assets-eu.researchsquare.com/files/rs-7191702/v1/c13d42d305b187dc6755a8fc.png"},{"id":88599258,"identity":"a4ab7234-3c49-48aa-a2ae-a4f04e314d7a","added_by":"auto","created_at":"2025-08-08 07:32:44","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":20977,"visible":true,"origin":"","legend":"\u003cp\u003eValidation of gene expression by RT-qPCR analysis.\u003c/p\u003e","description":"","filename":"image12.png","url":"https://assets-eu.researchsquare.com/files/rs-7191702/v1/92db3649d80dd57784a70ef9.png"},{"id":88599263,"identity":"dbab4da1-9e28-4c80-a8f6-5a0e0dbc32bc","added_by":"auto","created_at":"2025-08-08 07:32:44","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":184151,"visible":true,"origin":"","legend":"\u003cp\u003eHypothetical model of improving heat tolerance of alfalfa by thermal exercise. The green arrow indicates upward adjustment and the red arrow indicates downward adjustment.\u003c/p\u003e","description":"","filename":"image13.png","url":"https://assets-eu.researchsquare.com/files/rs-7191702/v1/0873b7b75d1f9379911b682f.png"},{"id":98245250,"identity":"43a7257c-e7a7-48d0-b4c1-ab314431874b","added_by":"auto","created_at":"2025-12-15 16:17:28","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3943314,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7191702/v1/51f46db1-c0ed-48d1-9c8e-7c56289c5220.pdf"},{"id":88600303,"identity":"e0c97d03-3163-46e2-bca5-b9bda69f79d1","added_by":"auto","created_at":"2025-08-08 07:40:43","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":10842,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 1: \u003c/strong\u003eTable S1 Comprehensive evaluation results of heat tolerance of 12 alfalfa varieties\u003c/p\u003e","description":"","filename":"Additionalfile1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7191702/v1/ff7ab1c527e57ee048bb559d.xlsx"},{"id":88600306,"identity":"70ee7ba5-bd9e-4259-a047-7c005e49666e","added_by":"auto","created_at":"2025-08-08 07:40:44","extension":"xlsx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":10662,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 2: \u003c/strong\u003eTable S2 Transcriptome sequencing data\u003c/p\u003e","description":"","filename":"Additionalfile2.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7191702/v1/f54441827df1550f47aa246e.xlsx"},{"id":88599262,"identity":"f03bf927-acc7-4b43-95de-69a3f26e5e29","added_by":"auto","created_at":"2025-08-08 07:32:44","extension":"xlsx","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":12132,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 3: \u003c/strong\u003eTable S3 Fatty acid content test results\u003c/p\u003e","description":"","filename":"Additionalfile3.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7191702/v1/e3c682f2f72b188cdbb2f915.xlsx"},{"id":88599255,"identity":"9d2e67e0-32a0-4f5a-a47b-5da595803dc4","added_by":"auto","created_at":"2025-08-08 07:32:43","extension":"xlsx","order_by":4,"title":"","display":"","copyAsset":false,"role":"supplement","size":10397,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 4: \u003c/strong\u003eTable S4 Name and source of alfalfa varieties\u003c/p\u003e","description":"","filename":"Additionalfile4.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7191702/v1/2498cbea8ff69fd9e3e6d253.xlsx"},{"id":88599254,"identity":"2d38b191-b322-4074-a82a-5c6c0df948c2","added_by":"auto","created_at":"2025-08-08 07:32:43","extension":"xlsx","order_by":5,"title":"","display":"","copyAsset":false,"role":"supplement","size":10607,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eAdditional file 5: \u003c/strong\u003eTable S5 Primer sequences\u003c/p\u003e","description":"","filename":"Additionalfile5.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-7191702/v1/a1fc1553d3acf286b7625588.xlsx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Thermal priming enhances heat tolerance in alfalfa (Medicago sativa L.) through activation of multiple metabolic pathways","fulltext":[{"header":"Background","content":"\u003cp\u003eThermal priming, the process by which brief exposure to non-lethal high temperatures enhances subsequent heat stress tolerance, represents a critical adaptive strategy in plants [1]. This phenomenon has been demonstrated to improve thermotolerance through coordinated physiological, biochemical, and molecular responses [2]. Evidence from multiple species confirms the protective effects of thermal priming: in rice, priming at 28℃ maintained membrane integrity and biomass accumulation while reducing ROS and MDA levels during heat stress [3]. Similarly, brassica plants primed at 40℃ exhibited increased soluble sugars, CAT and peroxidase (POD) activities, along with reduced membrane damage when exposed to 45℃ stress [4]. Other studies have documented enhanced photosynthetic efficiency in azalea [5], improved antioxidant capacity in cape gooseberry [6], and preserved photosynthetic function in grapes following thermal priming [7].\u003c/p\u003e\u003cp\u003eAt the molecular level, thermal priming induces sustained expression of small heat shock proteins (sHSPs) such as HSP21, HSP22, and HSP17.6C, with primed plants showing more rapid and robust induction of these molecular chaperones [8]. In \u003cem\u003eArabidopsis\u003c/em\u003e, priming establishes epigenetic memory through JMJ-mediated demethylation of H3K27me3 at HSP loci, enabling faster transcriptional responses to subsequent heat stress [8]. These findings collectively demonstrate that thermal priming enhances thermotolerance through multiple mechanisms including photosynthetic protection, osmotic adjustment, and antioxidant defense.\u003c/p\u003e\u003cp\u003eAlfalfa (\u003cem\u003eMedicago sativa\u003c/em\u003e L.), the \"king of forages,\" represents a globally important perennial legume valued for its nutritional quality, palatability, and environmental benefits in soil improvement and ecological restoration [9]. While adapted to warm climates (optimal growth at 18\u0026ndash;22℃), rising global temperatures now pose significant challenges to alfalfa production [10]. Heat stress impairs alfalfa at multiple developmental stages, reducing seed germination rates, compromising photosynthetic efficiency, disrupting water relations, and inducing oxidative damage through ROS accumulation [11]. These physiological disturbances lead to decreased accumulation of critical nutrients including dry matter, carbohydrates, and starch [12], with yield reductions estimated at 17% per 1℃ temperature increase [13].\u003c/p\u003e\u003cp\u003eDespite extensive documentation of thermal priming effects across plant species, the specific mechanisms underlying this phenomenon in alfalfa remain poorly understood. This study employed the heat-tolerant cultivar 'Sanditi' and aimed to characterize morphological and physiological responses to thermal priming under heat stress; to identify differentially expressed genes and metabolic pathways through transcriptomic and metabolomic analyses; and to elucidate the molecular basis of priming-enhanced thermotolerance. Our integrated approach provides novel insights into alfalfa's adaptive strategies against recurrent high-temperature stress and establishes a framework for improving heat tolerance in this critical forage crop.\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cb\u003eScreening of heat-resistant alfalfa varieties\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo identify suitable germplasm for thermal priming studies, we evaluated the heat tolerance of 12 alfalfa varieties using a comprehensive membership function approach. This analysis incorporated multiple physiological indicators including chlorophyll content, MDA accumulation, electrolyte leakage (EL), and key antioxidant enzyme activities (POD, SOD, CAT) under 37℃ heat stress (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e, Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThe screening revealed significant variability in thermotolerance among cultivars, with membership scores ranging from 0.244 to 0.763. 'Sanditi' emerged as the most heat-resistant variety (score: 0.763), demonstrating superior maintenance of chlorophyll content, reduced oxidative damage (lower MDA and EL), and enhanced antioxidant enzyme activities compared to other cultivars. In contrast, cultivar '343' showed the poorest performance (score: 0.244). Based on these results, we selected 'Sanditi' for all subsequent thermal priming experiments due to its robust heat stress responses.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of thermal priming on alfalfa growth morphology\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the impact of thermal priming on alfalfa growth phenotypes, seedlings were subjected to the heat priming (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). The results showed that both non-primed (UP) and primed (P3) groups exhibited wilting and lodging following heat stress, whereas control (CK) plants maintained normal morphology. Notably, P3 group demonstrated significantly reduced lodging severity (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) and less pronounced leaf wilting compared to UP group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). These findings suggest that thermal priming can effectively mitigate heat stress-induced morphological impairments in alfalfa.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo assess the physiological impact of thermal priming on alfalfa, we measured chlorophyll content, MDA levels, soluble protein content, and antioxidant enzyme activities (SOD, POD, CAT) in seedlings from the CK (control), UP (unprimed\u0026thinsp;+\u0026thinsp;heat stress), and P3 (primed\u0026thinsp;+\u0026thinsp;heat stress) groups. Chlorophyll content and soluble protein levels were significantly higher in P3 than UP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA, C), suggesting that priming helps maintain photosynthetic efficiency and protein stability under stress. MDA content, a marker of lipid peroxidation, significantly increased in both UP and P3 groups compared to CK, confirming heat-induced membrane damage. However, P3 exhibited 18% lower MDA levels than UP (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), indicating that thermal priming mitigates heat stress-induced membrane damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB). SOD, POD, and CAT activities were also elevated in UP and P3 compared to CK. P3 showed a more pronounced increase in SOD and CAT but reduced POD activity when compared to UP (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD-F), indicating that thermal priming selectively enhances key ROS-scavenging enzymes, reducing oxidative damage. To evaluate ROS generation and cellular integrity under heat stress, we performed histochemical staining. Nitrotetrazolium blue chloride (NBT), diaminobenzidine (DAB) and Evans blue (EB) staining revealed lighter staining in P3 vs. UP, demonstrating that thermal priming reduces ROS accumulation and cell damage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eG).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eTranscriptome profiling of thermal priming effects\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo investigate the molecular mechanisms underlying thermal exercise-induced heat tolerance in alfalfa, we performed transcriptome sequencing on leaves from five experimental groups: CK, UP, P1, P2, and P3. The sequencing generated a total of 101.15 GB of high-quality clean data, with each sample producing at least 6 GB of data. All samples exhibited Q30 scores exceeding 94% and GC content ranging from 42.11\u0026ndash;42.95%, confirming the high quality of the transcriptome sequencing data (Table \u003cspan refid=\"MOESM2\" class=\"InternalRef\"\u003eS2\u003c/span\u003e). Differential gene expression analysis (|FoldChange| \u0026ge; 1 and FDR\u0026thinsp;\u0026lt;\u0026thinsp;0.05) identified: P1 vs CK: 4,891 differentially expressed genes (DEGs; 2,495 up-regulated and 2,396 down-regulated); P2 vs CK: 10,299 DEGs (5,664 up-regulated and 4,635 down-regulated); P3 vs CK: 8,778 DEGs (4,286 up-regulated and 4,492 down-regulated); UP vs CK: 8,130 DEGs (3,828 up-regulated and 4,302 down-regulated) (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA-C). These results clearly demonstrate that thermal exercise significantly alters gene expression profiles in alfalfa under heat stress conditions.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eGene expression patterns during thermal priming\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo elucidate the specific effects of thermal priming on alfalfa heat tolerance, we analyzed 1,217 differentially expressed genes (DEGs) that were uniquely up-regulated in the P3 group. These DEGs were categorized based on their temporal expression patterns during the thermal exercise regimen. Totally 305 DEGs (25.1%) showed initial upregulation during P1 phase, 301 DEGs (24.7%) exhibited subsequent upregulation during P2 phase, and 661 DEGs (50.2%) demonstrated late-stage upregulation during P3 phase (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC-D). Notably, 49.8% of the total up-regulated DEGs (606 genes) were activated during the thermal priming period (prior to P3), while the remaining 50.2% showed delayed response. This biphasic pattern suggests both immediate and progressive transcriptional responses to thermal conditioning.\u003c/p\u003e\u003cp\u003e\u003cb\u003eExpression patterns of DEGs in alfalfa after thermal priming treatment\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo systematically characterize the dynamic transcriptional responses to thermal exercise, we performed Mfuzz clustering analysis of all differentially expressed genes (DEGs) across treatment groups, identifying 16 distinct expression pattern clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e). Compared with the UP group, the upregulated genes in the P3 group were significantly enriched in class 3, class 4, class 13 and class 14. After P1 treatment, the expression level of DEGs in class 3, class 4 and class 14 was up-regulated, while after P2 treatment, the expression level of DEGs in class 4 was down-regulated, but further up-regulated in class 3 and class 14. DEGs in class 13 was up-regulated only after P3 treatment. After KEGG enrichment analysis of DEGs in the above four classes, it was found that they were all enriched in plant hormone signal transduction, spliceosome pathway, phenylpropanoid biosynthesis pathway, glutathione metabolism pathway and fatty acid biosynthesis pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e).\u003c/p\u003e\u003cp\u003eThese results demonstrate that thermal exercise induces phase-dependent transcriptional reprogramming, with coordinated activation of stress-responsive metabolic and signaling pathways that collectively enhance alfalfa's heat tolerance.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eExpression patterns of brassinosteroid-related DEGs\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThrough classification and statistical analysis of \"plant hormone signal transduction\"-related genes in clusters 3, 4, 13, and 14, we observed significant enrichment of BR-related genes across all clusters (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eA). BRs are a class of steroid-structured phytohormones that play crucial roles in promoting plant growth and enhancing stress resistance to drought, high temperature, and low temperature conditions. Among BR compounds, brassinolide (BL) exhibits the highest biological activity. BL biosynthesis originates from campesterol (CR) through a series of enzymatic reactions (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB). The BR receptor is the membrane-localized receptor-like protein kinase BRI1. BR binding to BRI1 triggers a phosphorylation cascade that activates downstream transcription factors (e.g., BES1/BZR1). These transcription factors subsequently translocate to the nucleus to regulate expression of target genes, thereby modulating plant growth, development, and stress responses [14\u0026ndash;15]. To elucidate the molecular regulatory mechanisms of BR in thermal priming responses, we systematically analyzed expression patterns of genes involved in BR biosynthesis and signaling pathways across different treatment groups. As illustrated in Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eB, the BR biosynthetic pathway involves key enzymatic steps including sterol hydroxylation, side chain modification, and oxidation, mediated by genes encoding: sterol C-23 hydroxylase (CPD), sterol side chain modification enzyme (ROT3), cytochrome P450 family protein (CYP92A6), 2-Cys peroxidase (BAS1), dwarfing-related protein (DWF4), and C-6 oxidase (BR6ox1). Expression profiling revealed that while \u003cem\u003eCPD\u003c/em\u003e and \u003cem\u003eROT3\u003c/em\u003e were upregulated following P1 treatment, other biosynthetic genes (including \u003cem\u003eDWF4\u003c/em\u003e and \u003cem\u003eBR6ox1\u003c/em\u003e) showed transcriptional suppression. Notably, despite downregulation of most BR biosynthetic genes after P1 treatment, key BR signaling components such as \u003cem\u003eBRI1\u003c/em\u003e, \u003cem\u003eBAK1\u003c/em\u003e, and \u003cem\u003eBZR1\u003c/em\u003e were upregulated (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eC). These findings demonstrate that the BR-BRI1-BES1 signaling pathway plays a pivotal role in thermal priming-induced heat tolerance enhancement in alfalfa.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of thermal priming on spliceosome pathway\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe spliceosome pathway is a core mechanism in plant gene expression regulation, playing an irreplaceable role in plant growth, development, environmental adaptation, and response to external signals [16\u0026ndash;17]. To investigate the molecular regulatory mechanisms of the spliceosome pathway under thermal priming, we systematically analyzed the composition and expression patterns of spliceosome-related genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eA) across different groups (class 3, 4, 13, and 14). Expression profiling revealed upregulation of key spliceosome factors, including SNRP70 (involved in 5' splice site recognition by U1 snRNP) and P68 (which unwinds the U1-5' splice site duplex) (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB). U2 snRNP is responsible for identifying the branch point sequence (BPS) within the intron region. U2B facilitates the anchoring of U2 snRNP to precursor mRNA via nonspecific binding of the SF3B complex to the BPS. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003eB, both U2B and SF3B exhibited significant upregulation. Similarly, within the U4/U6. U5 tri-snRNP complex, genes associated with structural maintenance and mature mRNA splicing were markedly upregulated. These findings demonstrate that thermal priming induces significant upregulation of spliceosome pathway-related genes.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of thermal priming on phenylpropanoid biosynthesis pathway\u003c/b\u003e\u003c/p\u003e\u003cp\u003eLignin, a key component of the secondary cell wall, provides structural rigidity and mechanical strength to plants, facilitating water transport and enhancing resistance to adverse environmental conditions [18]. KEGG enrichment analysis revealed significant upregulation of lignin biosynthesis-related genes following thermal exercise (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eA). The upregulation of key phenylpropanoid pathway genes may promote the accumulation of secondary metabolites, including phenols and lignin. Lignin content measurements in alfalfa leaves and stems demonstrated that the P3 group exhibited significantly higher lignin accumulation compared to the UP group. Under heat stress, both the UP and P3 groups showed increased lignin content relative to the CK group, with the P3 group displaying the highest levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003eB-C). These findings indicate that thermal priming enhances lignin synthesis in alfalfa under heat stress, leading to greater stem lignin deposition, improved mechanical strength, and enhanced lodging resistance.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of thermal priming on glutathione metabolic pathway\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe glutathione metabolic pathway plays a crucial role in plant stress responses, with glutathione S-transferase (GST) serving as the key enzyme that catalyzes the initial step of glutathione (GSH) conjugation [19]. As a vital antioxidant, glutathione exhibits both antioxidant and detoxification properties. Furthermore, it participates in the ASA-GSH cycle, forming an essential component of the non-enzymatic antioxidant system and representing a primary pathway for ROS scavenging in plants. Enrichment analysis revealed significant upregulation of genes involved in glutathione metabolism, including GST and glutathione peroxidase (GPX), following thermal exercise. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003e.A, antioxidant-related genes such as \u003cem\u003eGST\u003c/em\u003e and \u003cem\u003eGPX\u003c/em\u003e are rapidly activated in P1 stage of hot start. Dehydroascorbic acid reductase (DHAR) and glutathione-S-transferase Pi (GSTP) were activated in the following P2 and P3 stages. This immediate response suggests that alfalfa rapidly initiates its antioxidant defense mechanisms to counteract heat stress-induced damage. Notably, the expression of these antioxidant enzyme genes further increased after heat stress, indicating that thermal exercise not only triggers the initial antioxidant response but also enhances the plant's capacity to withstand subsequent heat stress.\u003c/p\u003e\u003cp\u003eComparative analysis of biochemical components showed that under heat stress conditions, both UP and P3 groups exhibited increased levels of GSH, glutathione reductase (GR), DHAR, monodehydroascorbate reductase (MDHAR), and ascorbate peroxidase (APX) compared to the CK group, while demonstrating lower ASA content. Importantly, the P3 group showed higher concentrations of GSH, GR, DHAR, MDHAR, ASA, and APX than the UP group (Fig.\u0026nbsp;\u003cspan refid=\"Fig10\" class=\"InternalRef\"\u003e10\u003c/span\u003eB-G). These findings demonstrate that thermal priming induces the expression of \u003cem\u003eGST\u003c/em\u003e, \u003cem\u003eDHAR\u003c/em\u003e, and related genes, leading to increased accumulation of antioxidants including GSH and APX. This response promotes efficient operation of the ASA-GSH cycle, enhances overall antioxidant capacity, and consequently improves alfalfa's thermotolerance when facing subsequent high-temperature stress.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eEffect of thermal priming on fatty acid composition and content\u003c/b\u003e\u003c/p\u003e\u003cp\u003eFatty acids serve as fundamental components of cell membranes, where their saturation degree and carbon chain length critically influence membrane fluidity. These properties are essential for plant growth, physiological metabolism, and abiotic stress resistance [20]. To investigate whether thermal priming enhances plant thermotolerance through fatty acid (FA) modulation, we analyzed free fatty acid profiles using gas chromatography-tandem mass spectrometry (GC-MS). Our analysis identified 17 free fatty acids across P1, P2, P3, UP, and CK groups (Table \u003cspan refid=\"MOESM3\" class=\"InternalRef\"\u003eS3\u003c/span\u003e). The composition revealed that palmitic acid (6.7%-11% of total FAs) was predominated in saturated fatty acids (SFA), followed by stearic acid (1.3%-2.3%). Among the monounsaturated fatty acids (MUFA), oleic acid had the highest content, followed by erucic acid, accounting for 1.7%-4.1% of total fatty acids. Among polyunsaturated fatty acids (PUFA), the highest content was α-linolenic acid, accounting for 74.6%-80.6% of total fatty acids, followed by linoleic acid, accounting for 5.3%-8.1% of total fatty acids. Notably, the P3 group exhibited significantly higher total FA content (600.52\u0026thinsp;\u0026plusmn;\u0026thinsp;61.70 \u0026micro;g/g) versus control (402.52\u0026thinsp;\u0026plusmn;\u0026thinsp;2.73 \u0026micro;g/g), P1 (448.11\u0026thinsp;\u0026plusmn;\u0026thinsp;9.58 \u0026micro;g/g), P2 (561.69\u0026thinsp;\u0026plusmn;\u0026thinsp;49.66 \u0026micro;g/g), and UP (424.52\u0026thinsp;\u0026plusmn;\u0026thinsp;19.62 \u0026micro;g/g) groups.\u003c/p\u003e\u003cp\u003eKEGG enrichment analysis of P3vs UP metabolites showed significant enrichment in unsaturated fatty acid biosynthesis, metabolic pathways, and fatty acid biosynthesis (Fig.\u0026nbsp;\u003cspan refid=\"Fig11\" class=\"InternalRef\"\u003e11\u003c/span\u003eA-B). Transcriptional data further indicated upregulation of \u003cem\u003eFAD\u003c/em\u003e genes across all groups. According to |log2FC| \u0026gt;0.6, P\u0026thinsp;\u0026lt;\u0026thinsp;0.05, the differential metabolites in P3 vs UP group were screened (Table\u0026nbsp;1). The main differential metabolites are α-linolenic acid, erucic acid, oleic acid, arachidic acid and docosanoic acid. Among unsaturated fatty acids, the contents of α-linolenic acid, oleic acid and erucic acid in P3 group increased by 52.6%, 147.1% and 107.5% respectively compared with those in UP group, showing an upward trend. Among linear saturated fatty acids, arachidonic acid and docosanoic acid in P3 group were 52.5% and 51.6% of those in UP group, respectively, showing a downward trend. While SFA levels remained stable, MUFAs and PUFAs increased markedly, particularly PUFAs. This suggests thermal priming preferentially enhances unsaturated fatty acid synthesis, potentially improving membrane fluidity and heat resistance in alfalfa.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTable.1 The content of different fatty acids in P3 vs UP group\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e\u003ccolgroup cols=\"7\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eFatty acid name\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eType\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\"\u003e\u003cp\u003eP3 (\u0026micro;g/g)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUP (\u0026micro;g/g)\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\"\u003e\u003cp\u003eP-value\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c6\"\u003e\u003cp\u003elog2FC\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c7\"\u003e\u003cp\u003eType\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eα-linolenic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePolyunsaturated fatty acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e483.783\u0026thinsp;\u0026plusmn;\u0026thinsp;49.726a\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e317.118\u0026thinsp;\u0026plusmn;\u0026thinsp;1.437c\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.022\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e0.609\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eUP\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eerucic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMonounsaturated fatty acids\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.944\u0026thinsp;\u0026plusmn;\u0026thinsp;0.008\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.455\u0026thinsp;\u0026plusmn;\u0026thinsp;0.027\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.018\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1.053\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eUP\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eoleic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eMonounsaturated fatty acids\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e23.972\u0026thinsp;\u0026plusmn;\u0026thinsp;3.315\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e9.696\u0026thinsp;\u0026plusmn;\u0026thinsp;3.315\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.014\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e1.305\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eUP\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003earachidic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLinear saturated fatty acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.426\u0026thinsp;\u0026plusmn;\u0026thinsp;0.026\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.811\u0026thinsp;\u0026plusmn;\u0026thinsp;0.101\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.014\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e-0.929\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eDOWN\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003edocosanoic acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003eLinear saturated fatty acid\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003e0.304\u0026thinsp;\u0026plusmn;\u0026thinsp;0.053\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003e0.589\u0026thinsp;\u0026plusmn;\u0026thinsp;0.031\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e\u003cp\u003e0.049\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\".\" colname=\"c6\"\u003e\u003cp\u003e-0.954\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c7\"\u003e\u003cp\u003eDOWN\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003e\u003cb\u003eValidation of RNA-seq data by quantitative RT-PCR (RT-qPCR)\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo validate the reliability of our RNA-Seq results, we selected seven key genes involved in phenylpropanoid biosynthesis and glutathione metabolism pathways for RT-qPCR analysis. The RT-qPCR results demonstrated excellent consistency with the RNA-Seq data (Fig.\u0026nbsp;\u003cspan refid=\"Fig12\" class=\"InternalRef\"\u003e12\u003c/span\u003e), confirming the accuracy of our transcriptome sequencing findings. This strong correlation between both analytical methods validates the differential gene expression patterns observed in response to thermal exercise.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eAs a critical physiological response mechanism to environmental temperature fluctuations, thermal priming enables plants to systematically adjust their physiological processes following exposure to high temperatures, thereby enhancing their capacity to withstand subsequent heat stress. This adaptive response exhibits temporal persistence, as plants retain elevated thermotolerance even after ambient temperatures normalize. The underlying mechanisms involve complex biochemical and molecular reprogramming, including gene expression modulation, metabolic pathway adjustments, and signal transduction activation.\u003c/p\u003e\u003cp\u003e\u003cb\u003eOxidative stress and antioxidant defense\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHeat stress disrupts cellular redox homeostasis, leading to ROS accumulation. While ROS function as signaling molecules regulating growth and stress responses [21], excessive ROS induces oxidative damage to DNA, proteins, and lipids, ultimately impairing cell viability [22]. Plants counteract ROS via enzymatic (SOD, POD, CAT) and non-enzymatic (GSH, ASA) antioxidant systems [23]. In this study, heat-stressed alfalfa exhibited increased SOD, POD, and CAT activities. Notably, thermally primed (P3) seedlings displayed further elevation in SOD and CAT activities but reduced POD activity compared to unprimed (UP) plants, suggesting that thermal priming preferentially scavenges superoxide radicals (O\u003csup\u003e2\u0026minus;\u003c/sup\u003e) and H₂O₂, thereby diminishing POD demand. Consequently, P3 seedlings maintained lower ROS levels and superior growth under heat stress, underscoring the role of thermal priming in enhancing oxidative stress resilience.\u003c/p\u003e\u003cp\u003e\u003cb\u003eBrassinosteroid (BR) signaling and heat tolerance\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs a key phytohormone, BR enhances plant thermotolerance. Prior studies demonstrate that BR elevates photosynthetic efficiency (Fv/Fm) and stomatal conductance in heat-stressed rice [24]. BR activates the expression of \u003cem\u003eHSFA2\u003c/em\u003e, \u003cem\u003eDREB2A\u003c/em\u003e, and \u003cem\u003eHSP\u003c/em\u003es via BZR1-mediated repression of ERF49 in \u003cem\u003eArabidopsis\u003c/em\u003e [25]. In addition, BR promotes dry matter and nutrient translocation in maize under heat stress [26]. BR regulates HSP17.6A via histone acetyltransferase HAC1 to sustain proteostasis [27]. Our transcriptomic analysis revealed that thermal priming potentiates BR signaling by upregulating receptor genes (\u003cem\u003eBRI1\u003c/em\u003e/\u003cem\u003eBAK1\u003c/em\u003e), enhancing receptor density and signal sensitivity even without elevated BR levels. The transcription factor BZR1, a central BR pathway regulator, was upregulated, directly activating downstream thermotolerance genes, including HSPs and genes involved in mitigating ROS toxicity.\u003c/p\u003e\u003cp\u003eBR signaling pathway can regulate several downstream metabolic pathways and secondary metabolic pathways. Liu et al. (\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) found that BR treatment increased the activities of PAL and 4CL in watermelon seedlings under zinc stress and induced lignin accumulation [28]. Under salt stress, the contents of phenolic compounds, flavonoids and lignin were increased by BR in order to reduce the damage in Ornamental Gourd [29]. Guo et al. (\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) found that BR can increase lignin synthesis-related genes such as \u003cem\u003ePAL\u003c/em\u003e and lignin deposition in Ginkgo biloba [30]. In Korean pine, BR can also improve PAL activity and lignin content in cells through phenylpropanoid biosynthesis [31]. Zhou et al. (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2018\u003c/span\u003e) found that BR in grapes can enhance AsA-GSH cycle by increasing the activities of MDHAR, GR, APX and DHAR, and the contents of antioxidant ascorbic acid (MA) and dehydroascorbic acid (DHA), thus reducing the damage of plants under Cu stress [32]. Niu et al. (\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) found that under low temperature stress, BR promoted the accumulation of phenols such as GSH and hesperidin in jujube fruit, maintained the quality and reduced chilling injury [33]. Dong et al. (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2025\u003c/span\u003e) found that BR treatment can significantly up-regulate genes such as \u003cem\u003eFAD2\u003c/em\u003e, \u003cem\u003eFAD3\u003c/em\u003e and \u003cem\u003eLOX\u003c/em\u003e in grapes, maintain the proportion of unsaturated fatty acids in membrane lipids, and realize the stability of membrane structure [34]. Li et al. (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2022\u003c/span\u003e) found that BR can also increase the accumulation of butyric acid, octanoic acid, decanoic acid, linoleic acid and other substances in grapes, and improve their low temperature resistance [35]. In this study, thermal priming activated these pathways, increasing metabolites (e.g., lignin, phenolics, unsaturated fatty acids) that collectively bolster heat tolerance in alfalfa.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhenylpropanoid pathway and lignin deposition\u003c/b\u003e\u003c/p\u003e\u003cp\u003ePhenylpropanoids (e.g., flavonoids, lignin) are pivotal for stress adaptation [36]. Phenylpropionic acids are mainly derivatives of cinnamic acid, including caffeic acid, ferulic acid, mustard acid, etc. Because of their excellent ability of scavenging free radicals, they are considered as the main antioxidants to resist oxidative damage and are necessary for plants to adapt to biotic and abiotic stresses [37]. Lignin is one of the important products in the biosynthesis of phenylpropanoid. It can not only enhance the mechanical strength of plants and the hardness of cell walls, but also have many biological functions such as resisting the invasion of adverse external environment and diverting water transport in tissues [38]. Lignin is synthesized by phenylpropanoid biosynthesis with phenylalanine as the substrate. PAL converts phenylalanine into cinnamic acid, which is then reduced by 4CL, CCR, CAD and other enzymes in turn to generate corresponding coenzyme A, and finally the catalytic intermediate coenzyme A is converted into corresponding lignin monomer [39]. Under heat stress, phenylpropanoids are considered as markers of heat stress in plants [40]. For example, phenylpropionic acids and flavonoids in carrot cells protect plants from heat stress by inhibiting ROS formation [41]. Paupi\u0026egrave;re et al. (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) found that HSFb1 induced the accumulation of phenylpropanoid metabolites in tomatoes, which enhanced the heat resistance [42]. In this study, the expression levels of PAL, COMT, F5H, CCR and 4CL related to lignin biosynthesis increased significantly after thermal priming. Compared with CK, it was observed that the expression of lignin synthesis-related genes decreased in the early stage of thermal exercise, which may be because high temperature interfered with enzyme activity and transcription factor binding ability, thus inhibiting the normal expression of related genes. At P3 stage after thermal priming, the expression of lignin synthesis-related genes was significantly higher than that of UP group without thermal priming. This shows that through thermal priming, plants may start the adaptive mechanism, enhance their structural stability and stress resistance, and up-regulate the expression of lignin synthesis-related genes, thus promoting lignin synthesis and improving the resistance to high temperature. The quantitative results of lignin content showed that the lignin content in alfalfa stems increased, which is also the reason why plants after thermal priming showed stronger lodging resistance. In addition, the determination of lignin content in seedling stems of P1 and P2 treatment points during thermal priming showed that alfalfa began to synthesize lignin at the early stage of thermal priming and accumulated continuously. In a word, thermal priming promotes the synthesis and accumulation of lignin in alfalfa stems, and effectively improves the lodging resistance of alfalfa seedlings.\u003c/p\u003e\u003cp\u003e\u003cb\u003eAsA-GSH cycle and redox balance\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe ascorbate-glutathione (AsA-GSH) cycle represents a crucial antioxidant defense system in plants, functioning to scavenge ROS through coordinated action of antioxidants (AsA and GSH) and enzymes including APX, GPX, and glutathione reductase (GR) [43]. In this cycle, APX catalyzes the oxidation of AsA to eliminate harmful ROS such as superoxide radicals (O\u003csup\u003e2\u0026minus;\u003c/sup\u003e) and hydrogen peroxide (H₂O₂), while GR, DHAR, and MDHAR regenerate reduced AsA and GSH to sustain the cycle's activity [44]. Extensive research has established a positive correlation between AsA-GSH cycle activity and plant stress tolerance. For instance, strawberry seedlings exposed to combined high temperature and high light stress exhibited enhanced GR, DHAR, and MDHAR activities, facilitating AsA-GSH regeneration and consequently improving stress resilience [45]. Similarly, Li et al. (\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2020\u003c/span\u003e) demonstrated that the halophyte \u003cem\u003eSuaeda salsa\u003c/em\u003e upregulates GR under salt stress to boost GSH production and alleviate oxidative damage [46]. Our experimental findings demonstrate that thermal priming significantly enhances the AsA-GSH cycle's efficiency in alfalfa seedlings. Quantitative analyses revealed elevated levels of both enzymatic components (APX, GR, DHAR, MDHAR) and non-enzymatic antioxidants (AsA, GSH) in primed plants. This coordinated upregulation enables more effective ROS detoxification and redox homeostasis maintenance, thereby reducing oxidative stress damage under high temperature conditions. The enhanced AsA-GSH cycle activity represents a key mechanism through which thermal priming confers improved thermotolerance in alfalfa seedlings.\u003c/p\u003e\u003cp\u003e\u003cb\u003eFatty acid remodeling and membrane stability\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAs a key component of biofilm, cork and plant epidermis wax, fatty acids are not only the basic raw materials for building cell and tissue structures, but also effectively maintain the material exchange regulation and barrier function between organisms and the external environment by forming hydrophobic barriers, and also play an important role in resisting abiotic stresses [47]. At higher temperature, plants reduce the phase transition temperature by increasing unsaturated fatty acids in membrane lipids, so as to keep the cell membrane liquid-crystalline at high temperature and avoid the loss of cell function caused by the solidification or rupture of membrane structure, so as to alleviate the influence of high temperature stress. For example, the deletion of C18:1 and C18:2 in \u003cem\u003eaccD-C794\u003c/em\u003e mutant in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e leads to heat sensitivity, which indicates that unsaturated fatty acids play an important role in heat stress tolerance [48]. This ability mainly depends on the regulation of fatty acid desaturase (FAD) on unsaturated fatty acid level [49]. Beisson et al. (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2007\u003c/span\u003e) found that in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e, the mutant with \u003cem\u003eFAD3\u003c/em\u003e gene deletion showed serious membrane damage at 42℃, and the linolenic acid content in chloroplast membrane lipid decreased by 60%, accompanied by a three-fold increase in MDA accumulation [50]. Chen et al. (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) found that under the continuous stress of 38℃, the leaf EL of the rice \u003cem\u003eOsFAD3\u003c/em\u003e overexpression line decreased by 45% compared with that of the wild type, indicating that the membrane permeability was effectively maintained [51]. Our transcriptome analysis showed that thermal priming increases the expression of \u003cem\u003eFAD\u003c/em\u003e gene, causes the accumulation of polyunsaturated fatty acids and maintains the stability of membrane system.\u003c/p\u003e\u003cp\u003eHigh temperature changes of membrane lipid from liquid crystal state to gel state, which destroys the membrane structure. The three double bonds of α-linolenic acid (C18:3) make the membrane have high fluidity, which can reduce the phase transition temperature of the membrane and delay the curing of membrane lipid induced by high temperature [52]. You et al. (\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) found that the decrease of α-linolenic acid content in rice would destroy the integrity and stability of cell membrane system under heat stress [53]. Ibrahim et al. (2019) found that after tomato seeds were soaked at 50℃ for 2 hours, the α-linolenic acid content increased and the germination ability was significantly improved [54]. Tang et al. (\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) found that α-linolenic acid can also produce jasmonic acid (JA) through lipoxygenase (LOX) pathway, and then activate the expression of \u003cem\u003eHSFs\u003c/em\u003e and \u003cem\u003eHSPs\u003c/em\u003e through JA, thus alleviating high temperature injury [55]. In this study, the content of α-linolenic acid was the highest in P3 group, which was significantly increased by 52.6% compared with UP group. The results showed that thermal priming could reduce the injury of alfalfa seedlings at high temperature by increasing the content of α-linolenic acid. As a monounsaturated fatty acid, the accumulation of oleic acid may improve the thermal stability by lowering the lipid transformation temperature of the membrane. Hou et al. (\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e2006\u003c/span\u003e) found that soybean seeds increased oleic acid content at high temperature to adapt to the temperature increase [56]. Li et al. (\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) found that in soybean \u003cem\u003eGmPDCT1\u003c/em\u003e/\u003cem\u003eGmPDCT2\u003c/em\u003e silent mutant, the transformation from oleic acid to linoleic acid was blocked, and the SOD activity and POD activity of leaves increased by 45% and 60% respectively under salt stress [57]. In this study, the oleic acid content of P3 group increased by 147.1% compared with that of UP group, which indicated that it played an important role in improving the heat resistance of alfalfa under high temperature stress. The synthesis of erucic acid (C22:1) is based on oleic acid-CoA. Through continuous condensation, reduction, dehydration and reduction, two carbon units are added each time, and erucic acid is generated after three rounds of extension. As a long-chain monounsaturated fatty acid, erucic acid may help plants maintain the stability of membrane structure under high temperature stress by changing the fluidity of cell membrane lipids. There was no significant difference in saturated fatty acid content between P3 group and CK group, but it was significantly lower than P2 group. It is speculated that the membrane fluidity can be optimized by increasing the content of saturated fatty acids and balancing the ratio of saturated and unsaturated fatty acids in alfalfa during thermal exercise to avoid excessive liquefaction of the membrane at high temperature. However, in the face of high temperature stress again, alfalfa mainly reduces the damage caused by high temperature by increasing the content of unsaturated fatty acids.\u003c/p\u003e\u003cp\u003e\u003cb\u003eSpliceosome activation and transcriptional plasticity\u003c/b\u003e\u003c/p\u003e\u003cp\u003eSpliceosome are dynamic complexes composed of snRNP (such as U1, U2, U4/U6, U5) and non-snRNP proteins. Spliceosome pathway is not only a molecular switch of gene expression in plants, but also a key hub connecting genetic information and phenotypic plasticity. By precisely regulating alternative splicing and constitutive splicing, it gives plants molecular flexibility to cope with complex environments [58]. For example, under low temperature stress, SME1 ensures the correct splicing of many mRNA precursors in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e [59]. In addition, He et al. (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2023\u003c/span\u003e) found that under heat stress, the activity and composition of splicing also change significantly [60]. Gu et al. (\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2024\u003c/span\u003e) found that the spliceosome pathway in Chinese cabbage was significantly up-regulated under high temperature stress [61]. Lee et al. (\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2017\u003c/span\u003e) found that SF1 participated in the regulation of flowering and heat tolerance by participating in the splicing of the precursor of HsfA2 [62]. Rosenkranz et al. (\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e2021\u003c/span\u003e) found that five \u003cem\u003eSR\u003c/em\u003e genes in tomato were up-regulated by high temperature induction to optimize transcription and splicing efficiency [63]. In this study, SNRP70 which recognizes the 5' splicing site on the precursor mRNA in U1 snRNP, P68 which unwinds the duplex of U1-5' splicing site, and Y2B, SF3B, SRF and other proteins which recognize and bind the branching sequence in U2 snRNP can make more U1 bind to the 5' splicing site faster, and U2 bind to the branching point faster, thus accelerating the early assembly of splices and allowing cells to process more precursor mRNA in a unit time. Up-regulation of key genes in U4/U6. U5 tri-snRNP complex makes the supply of the protein complex more abundant, which can accelerate the transformation of splice from \"pre-splice\" to \"mature splice\" and push splicing into the catalytic stage. In this study, after thermal priming, these spliceosome pathway-related genes were up-regulated rapidly after P1 treatment, which significantly increased the output rate of mature mRNA, and many proteins with different functions were synthesized rapidly. These protein participate in the regulation of antioxidant enzyme system and enhance the ability of plants to remove ROS; or participate in the remodeling of cell membrane components to maintain the fluidity and integrity of cell membrane at high temperature. The rapid response of this post-transcriptional regulation mechanism is the key for plants to obtain high temperature adaptability during thermal priming. After heat-priming, the splicing process of precursor mRNA is accelerated by pre-activating the genes related to the spliceosome pathway, so that mature mRNA and functional protein can be synthesized rapidly. In the face of extreme high temperature, these gene expression products mobilized in advance quickly play a role in helping plants to effectively adapt to high temperature, which embodies the important significance of thermal priming to give plants survival advantages in adversity by stimulating post-transcriptional regulation.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study demonstrates that heat priming enhances alfalfa's thermotolerance by elevating chlorophyll content, soluble protein levels, and the activities of SOD and CAT, while reducing ROS accumulation and MDA content under high-temperature stress. Transcriptome analysis revealed that differentially expressed genes in P3 vs UP were significantly enriched in hormone signaling pathways, primary metabolite biosynthesis, and secondary metabolite biosynthesis, with a strong association with BR signaling. Mechanistically, heat priming exerts its protective effects through BR-mediated metabolic regulation and the phenylpropanoid biosynthesis pathway, leading to increased lignin synthesis in stems, thereby improving lodging resistance. Heat priming also boosts the AsA-GSH cycle, elevating antioxidant enzyme activity and non-enzymatic antioxidant levels to mitigate ROS-induced damage, and promotes unsaturated fatty acid synthesis, maintaining membrane fluidity under heat stress. On the other hand, heat priming facilitates mRNA splicing and export by upregulating the spliceosome pathway, ensuring efficient gene expression under stress. Through these synergistic mechanisms (Fig.\u0026nbsp;\u003cspan refid=\"Fig13\" class=\"InternalRef\"\u003e13\u003c/span\u003e), heat priming enables alfalfa to establish a robust thermotolerance defense system, ultimately enhancing its heat resistance.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cp\u003e\u003cb\u003ePlant materials and growing conditions\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThe experiment was conducted in the College of Environment and Resources of Dalian Minzu University in May 2023. Twelve commercial alfalfa varieties (Table \u003cspan refid=\"MOESM4\" class=\"InternalRef\"\u003eS4\u003c/span\u003e) were purchased as experimental materials, and the seeds were soaked in the dark for 24 hours to accelerate germination. Seeding the germinated seeds into flowerpots (10cm\u0026times;10cm\u0026times;10cm), wherein the substrate is peat soil, vermiculite and pearl salt, which are stirred evenly at 2:1:1, and each pot is seeded with 8\u0026ndash;10 alfalfa seeds, and each variety is planted in 10 pots, and then cultured in a solar greenhouse (average temperature 25.0\u0026thinsp;\u0026plusmn;\u0026thinsp;2℃, relative humidity 60\u0026thinsp;\u0026plusmn;\u0026thinsp;5%). During the cultivation period, water should be poured every 3 days to keep the soil moisture sufficient. After 14 days to the three-leaf stage, alfalfa seedlings with similar growth and no diseases and pests were selected for the next experiment.\u003c/p\u003e\u003cp\u003e\u003cb\u003eHeat-resistance screening\u003c/b\u003e\u003c/p\u003e\u003cp\u003eBy simulating the high temperature environment in an incubator, leaves of 12 alfalfa varieties under high temperature stress were collected to determine physiological and biochemical indexes, and the heat resistance of different alfalfa varieties was comprehensively evaluated by membership function method [64]. After accelerating germination, alfalfa was moved to a constant temperature light incubator. The control group was cultured at 25℃, and the experimental group was treated with heat stress at 37℃. After 4 hours' treatment, leaves of alfalfa seedlings under heat stress (HT) and control (CK) were collected for determination of chlorophyll content, antioxidant enzyme activity, electrolyte permeability and MDA content. All assays were performed in three independent replicates. Finally, the relative heat tolerance of alfalfa varieties was evaluated by membership function method.\u003c/p\u003e\u003cp\u003e\u003cb\u003eThermal priming treatment\u003c/b\u003e\u003c/p\u003e\u003cp\u003eHeat-resistant alfalfa 'Sanditi' was used as the experimental material. After 12 days of cultivation, it was treated with high temperature stress as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e.A, and it was divided into control group CK (Control), thermal exercise group P (Primed) and heat control group UP (unprimed) without heat priming. The plants were subjected to heat stress in a light incubator. The light intensity was 12000 lux, the light period was 16h light /8h darkness, and the relative humidity was 60%. Pots are randomly placed to reduce the influence of different positions. In the thermal exercise group, P first exercised at 37℃ for 2 hours, then recovered at 23℃ for 2 hours, then exercised at 43℃ for 2 hours, and then recovered at 23℃ for 2 days. Finally, P group and UP group were subjected to heat stress at 43℃ for 4 hours. Collect leaves at P1, P2, P3, CK, and UP, respectively, and were frozen with liquid nitrogen and stored in a refrigerator at -80℃ for subsequent experiments.\u003c/p\u003e\u003cp\u003e\u003cb\u003ePhysiological index determination\u003c/b\u003e\u003c/p\u003e\u003cp\u003eChlorophyll content in alfalfa leaves was determined by acetone-ethanol mixed extraction [65]. The content of MDA was determined by thiobarbituric acid colorimetry [66]. The activity of SOD was determined by nitroblue tetrazole photochemical reduction method [67]. The activity of POD was determined by guaiacol method [68]. The activity of CAT was determined by ultraviolet absorption method [69]. EL was measured by conductivity meter [70]. Coomassie brilliant blue method was used to determine the content of soluble protein. The leaves were infiltrated with NBT, DAB [71] and EB [72] respectively, and the histochemical staining was carried out to detect the accumulation of superoxide anion, hydrogen peroxide and cell death in alfalfa leaves. All the above experiments were carried out in three biological repetitions.\u003c/p\u003e\u003cp\u003e\u003cb\u003eTranscriptome sequencing and data analysis\u003c/b\u003e\u003c/p\u003e\u003cp\u003eA total of 15 samples (5 treatments\u0026times;3 biological replicates) were collected for Qualcomm quantitative transcriptome sequencing detection. The experimental process included RNA extraction and detection, cDNA library construction and computer sequencing [73]. The total RNA of alfalfa leaves was extracted by Tiangen RNAprep pure plant kit (centrifugal column), and the applied methods and steps strictly followed the instructions of the kit. The absorbance of A260/A280 is measured by ultra-micro spectrophotometer to ensure that the purity of total RNA is between 1.8 and 2.1 to avoid pollution.\u003c/p\u003e\u003cp\u003eBy utilizing the polyA tail characteristics of eukaryotic mRNA, mRNA was enriched using Oligo (dT) magnetic beads. After treatment with fragmentation buffer, one strand cDNA was synthesized by reverse transcription using random hexamers. During the second strand synthesis, dUTP was used instead of dTTP to construct a chain specific library. Subsequently, end repair, A-tail addition, and adapter connection were performed, followed by magnetic bead purification and screening of a library containing 250\u0026ndash;350 bp insertion fragments. After Qubit quantification, Qsep400 fragment analysis, and Q-PCR calibration, 150 bp double ended sequencing was performed on the Illumina platform.\u003c/p\u003e\u003cp\u003eThe sequencing data was subjected to fastp quality control to remove reads containing adapters, N ratios\u0026thinsp;\u0026gt;\u0026thinsp;10%, or low-quality bases (Q\u0026thinsp;\u0026le;\u0026thinsp;20)\u0026thinsp;\u0026gt;\u0026thinsp;50%. Using HISAT to align clean reads to the reference genome and StringTie for predicting new genes. Calculate the logarithmic ratio of genes through featureCounts and convert it into FPKM values to quantify expression levels. Differential analysis was performed using DESeq2, and after Benjamini\u0026amp;Hochberg correction, differentially expressed genes were screened using the corrected P-value and log\u003csub\u003e2\u003c/sub\u003e fold change threshold. KEGG and GO enrichment analysis were performed based on hypergeometric tests [74].\u003c/p\u003e\u003cp\u003e\u003cb\u003eRT-qPCR analysis.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn order to verify the accuracy of RNA-seq sequencing, seven differentially expressed genes speculated to be related to thermal exercise to improve the heat tolerance of alfalfa were randomly selected for RT-qPCR verification. The seven differentially expressed genes are \u003cem\u003ePAL\u003c/em\u003e (Phenylalanine Ammonia-Lyase), \u003cem\u003eF5H\u003c/em\u003e (Fertilize 5-hydroxylase), \u003cem\u003e4CL\u003c/em\u003e (4-coumarate Coenzyme A Ligase), \u003cem\u003eHsf2b\u003c/em\u003e (Heat stress transcription factor b-2b), \u003cem\u003eCCR\u003c/em\u003e (Cinnamoyl-coa reduction), \u003cem\u003eGST\u003c/em\u003e (Glutathione-s-transfer) and Peroxidase. Using β-Actin as the reference gene, the gene-specific primers were designed by PrimerPremer5.0, and the primer sequence is shown in Table \u003cspan refid=\"MOESM5\" class=\"InternalRef\"\u003eS5\u003c/span\u003e. RT-qPCR was performed on ABI/Thermo Fisher. Each gene was analyzed in three biological samples, and each biological sample was repeated three times. The relative expression of the target gene was measured by 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e [75].\u003c/p\u003e\u003cp\u003e\u003cb\u003eMetabolite content detection\u003c/b\u003e\u003c/p\u003e\u003cp\u003eLignin content in leaves and stems of alfalfa was determined by using lignin content detection kit (Shanghai Liquid Quality Detection Technology Co., Ltd.). Assay of ASA-GSH activity Using commercial kits (Shanghai Liquid Quality Testing Technology Co., Ltd.), the contents of reduced glutathione and ascorbic acid were determined, and the activities of APX, glutathione reductase (GR), monodehydroascorbic acid reductase (MDHAR), dehydroascorbic acid reductase (DHAR) and other enzymes were determined.\u003c/p\u003e\u003cp\u003e\u003cb\u003eDetection of free fatty acids by proteomics\u003c/b\u003e\u003c/p\u003e\u003cp\u003eThaw the sample on ice, take 50 mg to EP tube, add 700 \u0026micro;l of extract, vortex at 2500 r/min for 10min, then perform ultrasonic treatment at 4℃ for 15min, centrifuge at 4200 rpm for 5min, transfer 500 \u0026micro;l of supernatant to a glass bottle containing 200 \u0026micro;l 0.05 M sodium hydroxide methanol solution, and blow dry with nitrogen. The residue was redissolved with 400 \u0026micro;l 0.4 M sodium hydroxide methanol solution and incubated at 70℃ for 10 min. After cooling, add 500 \u0026micro;l dichloromethane and 200 \u0026micro;l double distilled water, vortex at 2500 r/min for 5 min, centrifuge at 4200 rpm for 5 min, take 300 \u0026micro;l supernatant to a new bottle containing 300 \u0026micro;l 2 M methanol hydrochloric acid solution, and react at 70℃ for 20 min for methyl esterification. After cooling, add 500 \u0026micro;l n-hexane and 300 \u0026micro;l double distilled water, vortex at 2500 r/min for 5 min, and centrifuge at 4200 rpm for 5 min, and collect the supernatant containing fatty acid methyl ester into the injection bottle for GC-MS analysis [75\u0026ndash;78].\u003c/p\u003e\u003cp\u003eR language prcomp function, the data is standardized by unit variance and unsupervised principal component analysis is carried out. Taking |Log2FC| as the threshold, the metabolites were annotated in combination with KEGG compound database, mapped to KEGG pathway, and then enriched and analyzed by MSEA.\u003c/p\u003e\u003cp\u003e\u003cb\u003eData statistics.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eAnalysis of variance (ANOVA) was performed using IBM SPSS Statistics 22. Plots were made using Origin 2018.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eJH designed the experiments. JCM, SJL, ZJR, ZZY, LH, ZJX performed the experiments and data analysis, and drafted the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the central finance forestry science and technology extension demonstration project [2023]TG05.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRaw data was deposited in NCBI database under SRA accession:\u003c/p\u003e\n\u003cp\u003ePRJNA1294583(https://submit.ncbi.nlm.nih.gov/subs/sra/SUB15477709/overview).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis experiment does not involve human experiments and animal experiments.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eYao XH, Li YL, Chen J, Zhou ZX, Wen Y, Fang K, Yang FB, Li TT, Zhang DW, Lin HH. 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Plant Cell Rep. 2017;36(7):1083\u0026ndash;95.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRosenkranz RRE, Bachiri S, Vraggalas S, Keller M, Simm S, Schleiff E, Fragkostefanakis S. Identification and regulation of tomato serine/arginine-rich proteins under high temperatures. Front Plant Sci 2021, 12.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eWassie M, Zhang WH, Zhang Q, Ji K, Chen L. Effect of heat stress on growthand physiological traits of alfalfa (\u003cem\u003eMedicago sativa\u003c/em\u003e L.) and a comprehensive evaluation for heat tolerance. Agronomy-Basel. 2019;9:597.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eRao YK, Chou C-H, Tzeng Y-M. A simple and rapid method for identification and determination of cordycepin in \u003cem\u003eCordyceps militaris\u003c/em\u003e by capillary electrophoresis. Anal Chim Acta. 2006;566(2):253\u0026ndash;8.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eDani A, SisWoyo T. 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Total and free fatty acids analysis in milk and dairy fat. In: Separations. vol. 6; 2019.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eCiali\u0026egrave; Rosso M, Stilo F, Mascrez S. Shelf-life evolution of the fatty acid fingerprint in high-quality Hazelnuts (\u003cem\u003eCorylus avellana\u003c/em\u003e. L) harvested different geographical region Foods. 2021,;10(3):685. .\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eShi D, Han T, Chu X. An isocaloric moderately high-fat diet extends lifespan in male rats and \u003cem\u003eDrosophila\u003c/em\u003e. Cell Metabol. 2021;33(3):581\u0026ndash;97.\u003c/span\u003e\u003c/li\u003e\u003cli\u003e\u003cspan\u003eKatoh K, Katoh Y, Kubo A. Serum free fatty acid changes caused by high expression of Stearoyl-CoA Desaturase 1 in tumor tissues are early diagnostic markers for ovarian cancer[J]. Cancer Res Commun. 2023;3(9):1840\u0026ndash;52.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Thermal priming, Alfalfa, Multivariate analysis, Brassinosteroid, Spliceosome, Metabolic pathway","lastPublishedDoi":"10.21203/rs.3.rs-7191702/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7191702/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground: \u003c/strong\u003eElevated environmental temperatures disrupt plant physiological homeostasis, imposing thermal stress that severely compromises growth and development. While thermal priming - a brief exposure to sublethal high temperature has been shown to enhance subsequent heat stress tolerance in plants, the underlying molecular mechanisms remain poorly characterized. In this study, we employed an integrated physiological, transcriptomic and metabolomic approach to investigate how thermal priming [37℃ for 2 h (P1) followed by 43℃ for 2 h (P2), designated P3] improves heat tolerance in alfalfa (\u003cem\u003eMedicago sativa \u003c/em\u003eL.) compared to unprimed controls (UP) exposed directly to 43℃.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults: \u003c/strong\u003ePhysiological analyses revealed that thermal priming significantly enhanced lodging resistance while increasing superoxide dismutase (SOD) and catalase (CAT) activities and reducing malondialdehyde (MDA) accumulation, indicative of improved oxidative stress management. Transcriptome profiling identified 1,217 upregulated genes in primed plants (P3 vs UP), with 50.2% being activated during the initial priming phase (P1). Cluster analysis demonstrated stage-specific pathway activation: brassinosteroid (BR) signaling, spliceosome activity, glutathione metabolism and fatty acid metabolism pathways were rapidly induced during early priming (P1), while phenylpropanoid biosynthesis was activated later during the second phase (P2). Metabolomic analyses provided further mechanistic insights, showing that thermal priming triggered significant lignin accumulation in stems, enhanced activity of the ascorbate-glutathione (AsA-GSH) cycle with increased antioxidant levels, and elevated content of unsaturated fatty acids including erucic acid, linolenic acid and oleic acid, suggesting membrane lipid remodeling.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions: \u003c/strong\u003eOur findings demonstrate that thermal priming establishes a multi-faceted defense system in alfalfa through BR-mediated signaling. This coordinated response involves activation of the AsA-GSH cycle for reactive oxygen species (ROS) scavenging, upregulation of phenylpropanoid biosynthesis for structural reinforcement through lignin deposition, accumulation of unsaturated fatty acids to maintain membrane stability, and enhancement of spliceosome activity to ensure proper processing of heat-responsive transcripts. The sequential activation of these pathways during the priming phases creates a 'stress memory' that prepares plants for subsequent heat challenges. These insights advance our understanding of thermal priming mechanisms and provide potential targets for improving crop heat tolerance through molecular breeding strategies.\u003c/p\u003e","manuscriptTitle":"Thermal priming enhances heat tolerance in alfalfa (Medicago sativa L.) through activation of multiple metabolic pathways","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-08-08 07:32:38","doi":"10.21203/rs.3.rs-7191702/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-10-03T15:52:40+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-29T07:09:55+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-09-28T17:59:51+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"4471233409288833743705335571217710127","date":"2025-09-25T14:38:30+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"304755114729055311080159709392246500062","date":"2025-09-23T14:19:57+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"151747129905253045731616582906246689413","date":"2025-09-20T06:31:27+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"25607815031109627960846714900616768222","date":"2025-08-08T04:37:23+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-08-03T04:06:14+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2025-08-01T22:46:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-08-01T09:32:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-08-01T09:28:53+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2025-07-23T03:33:56+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"ae654a72-5648-4775-871b-89eb2f5a1e50","owner":[],"postedDate":"August 8th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2025-12-15T16:14:08+00:00","versionOfRecord":{"articleIdentity":"rs-7191702","link":"https://doi.org/10.1186/s12870-025-07726-w","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2025-12-09 15:58:27","publishedOnDateReadable":"December 9th, 2025"},"versionCreatedAt":"2025-08-08 07:32:38","video":"","vorDoi":"10.1186/s12870-025-07726-w","vorDoiUrl":"https://doi.org/10.1186/s12870-025-07726-w","workflowStages":[]},"version":"v1","identity":"rs-7191702","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7191702","identity":"rs-7191702","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}