Seed germination was carried out according to the previous study (Boyes et al., 2001). Briefly, seeds of Arabidopsis thaliana ecotype Columbia (Col) were surface-sterilized and kept in the dark at 4°C for 3 days. The seeds were then sown onto filter papers with pre-soaked sterile water, and placed at 22 °C under light for 3 hours before transferring to the dark chamber for another 21 hours at 22°C, 28°C, 34°C and 40°C, respectively.

Total metabolites and lipids from 50 mg samples with whole tissue were extracted with methyl tert-butyl ether (MTBE) and methanol (MeOH). Briefly, 50 mg plant materials were mixed with 225 μL of ice-cold MeOH and homogenized for three times. The homogenate was then sonicated in an ice bath for 10 min. Added 750 μL pre-cooled MTBE and vortexed the mixture for 30 s and then kept on ice for 1 hour. The samples were vortexed for 30 s after adding 188 μL deionized water, and centrifuged at 14000 g for 10 min at 4°C. All experimental steps were performed on ice. The upper organic phase (800 μL) was carefully transferred to new tubes. The lower phase (200 μL) was transferred to additional new tubes. Repeat the above extraction steps twice. Finally, the three extractions were combined and dried with nitrogen gas.

The upper phase dried extract was dissolved in chloroform: methanol: 300 mmol/L ammonium acetate (300: 665: 35; v: v: v) for lipids analysis. The lower phase dried extract was dissolved in methanol: water (1:1) for metabolites analysis.

The Shimadzu Nexera UHPLC system equipped with the 6600 QTOF mass spectrometer (AB Sciex) was applied for untargeted metabolomics analysis. 1 uL aliquots of sample was injected into a Waters Acquity UPLC T3 column (100mm×2.1mm, 1.7 μm) maintained at 45°C by gradient elution. Mobile phase A was deionized water with 5 mM ammonium acetate (NH₄AC) and 0.02% formic acid, and phase B was acetonitrile: water (95:5, v/v) with 5 mM NH₄AC and 0.02% formic acid. The flow rate was 0.3 mL/min. The ion source parameters were set as follows: scan range, 50-1200 Da; source temperature, 300°C; curtain gas, 35; collision gas, medium; two ion source gases, 60; ion spray voltage, 5500/-4500V; DP, 60; CE, 35 ± 15.

The shotgun lipidomic analysis was carried out according to the previous study (Wang et al., 2016). Briefly, Shimadzu CBM-20A lite HPLC system coupled with a 6500 QTrap triple quadruple mass spectrometer was used to detect lipids. The mobile phase was methanol: dichloromethane (1:1, v/v) with 10 mM NH₄AC. 50 μL aliquots of sample solution was injected and the flow gradient was performed with 7 μL/min for 6 min, and then 30 μL/min for 2 min and 7 μL/min for 1 min. MS/MS was triggered by an inclusion list encompassing corresponding MS mass ranges scanned in 1 Da increments. Both MS and MS/MS data were combined to monitor PA, PG, PI, MAG, DAG, TAG, MGDG and DGDG ions as ammonium adducts; PC, PE, PS and Cer as protonated ions; SQDG as deprotonated anions ( Supplemental Table S1 ). The ion source parameters were set as follows: source temperature, 200°C; curtain gas, 20; collision gas, medium; two ion source gases, 15/20; ion spray voltage, 5500V; entrance potential, 10V.

The raw data of metabolomics was preprocessed by MetDNA2 (http://metdna.zhulab.cn/), identified according to the database, and some of them were confirmed by ZenoTOF 7600 (AB Sciex). The relative amount was comparatively quantified according to the peak intensity. The data of shotgun lipidomics was processed by LipidView Software (AB Sciex) for identification and quantification.

The data matrix of identified metabolites and lipids was further analyzed by univariate and multivariate statistical methods through website https://cloud.oebiotech.cn/ and R. Significantly altered variables were defined by the following criteria: P value < 0.05, FDR < 0.05 and Fold change > 1.5.

Seeds were germinated at 22°C, 28°C, 34°C and 40°C, respectively. As shown in Figure 1A , both 22°C and 28°C were suitable for seed germination, while the seeds were not germinated at either 34°C or 40°C. The hypocotyl of seedlings was longer at 28 °C than that at 22°C. Total 182 metabolic molecules were identified by using high-throughput untargeted metabolomic approach, which covered the metabolism of amino acids, amines, peptides, carbohydrates, lipids, nucleosides, organic acids, indoles, benzene/derivatives and flavones ( Supplementary Data 1 ).

For shotgun analysis, we innovatively introduced specific ion pairs, and thus identified both glycolipids and glycerides in one injection, which further developed the previous approaches of ESI/MS for glycolipids and LC/MS for glycerides, respectively. Total 149 lipids including 13 PAs, 13 PCs, 18 PEs, 11 PGs, 2 PIs, 4 PSs, two subclasses of glycerides (22 DAGs, 25 TAGs), three subclasses of galactosyldiacylglycerides (17 MGDGs, 24 DGDGs, 1 SQDGs) and 2 ceramides (Cer 34:1, Cer 38:1) were identified ( Supplementary Data 2 ).

Principal component analysis (PCA) was applied to analyze the metabolites which drove the group separation. PC1 and PC2 captured 73.6% and 10.9% of the total variance in the metabolites of FW-normalized dataset, respectively ( Figure 1B ). For lipids, PC1 and PC2 captured 51.4% and 20.3% of the total variance in the FW-normalized dataset, respectively ( Figure 1C ). The analysis showed that all samples were definitely divided into two clusters, grown at 22°C and 28°C (moderate temperature), and grown at 34°C and 40°C (extreme high temperature), respectively, which demonstrated that the metabolites and lipids were obviously distinct between moderate and extreme high temperatures.

The volcano plot further exhibited 148 metabolites significantly altered between moderate and extreme high temperatures (red points in Figure 1D , Supplementary Data 3 ). A total of 94 molecules dramatically increased in seeds grown at extreme high temperature, which included carbohydrates (D-Gal alpha 1->6D-Gal alpha 1->6D-Glucose, maltotriose, mannobiose, stachyose, raffinose), flavonoids (rutin, quercitrin, quercetin) and tryptophan metabolic derivatives (kynurenic acid, tryptamine) ( Supplementary Data 3 ), whereas 54 metabolites significantly decreased at these extreme high temperatures. Specifically, the phenylpropanoid biosynthesis (sinapate, 4-hydroxycinnamic acid, spermidine), TCA (succinate, citrate, mevalonic acid) tended to have low levels at 34°C and 40°C ( Supplementary Data 3 ). However, metabolites, such as amino acids, indoles, benzoic acids and their derivatives involving in different metabolic pathways changed variously ( Figure 1D , Supplementary Data 3 ).

Based on the differential metabolites analyzed by volcano analysis, we further performed the pathway enrichment analysis to investigate the important roles of altered metabolites during seedling establishment under the moderate and extreme high temperatures ( Figure 1E ). Of all these significant pathways, phenylpropanoid biosynthesis and phenylalanine metabolism showed obvious differences. Galactose metabolism associated with sugar metabolism during seed germination was also obviously altered. Other energy metabolic pathways such as TCA, glucosinolate biosynthesis pathway, glyoxylate and dicarboxylate metabolism pathways were also obviously different between 22~28°C and 34~40°C ( Figure 1E ). Multiple amino acid metabolic pathways including alanine, aspartate and glutamate metabolism, tyrosine metabolism, lysine degradation, tryptophan metabolism, valine, leucine and isoleucine degradation/biosynthesis, phenylalanine, tyrosine and tryptophan biosynthesis and beta-alanine metabolism were altered between 22~28°C and 34~40°C ( Figure 1E ), which suggested that amino acid metabolism played vital roles in maintaining metabolic homeostasis during temperature stress. The distinguished alteration of fatty acid degradation pathway indicated that lipid metabolism significantly acted on seed germination and seedling growth ( Figure 1E ). The shikimic acid-mediated metabolic pathways including flavonoid/flavonol biosynthesis and terpenoids were also implicated in temperature responses ( Figure 1E ).

To further investigate the significance of metabolites at different temperatures, the hierarchical heatmap analysis Z score scaled the fold changes for metabolites. The 148 out of total 182 metabolites were enriched at extreme high temperature, compared with moderate temperature ( Supplementary Figures 1, 2 ), which involved the metabolomic pathways of galactose metabolism (sucrose, raffinose, stachyose, D-Gal alpha 1->6D-Gal alpha 1->6D-Glucose), tryptophan metabolism (tryptophan, tryptamine, kynurenic acid, 3-indoleacetonitrile), vitamins metabolism (thiamin diphosphate, 2-alpha-hydroxyethy-thiamine diphosphate, thiamine), glucosinolate, and phenylalanine, tyrosine and tryptophan biosynthesis (methionine, tryptophan, phenylalanine), flavone and flavonol biosynthesis (quercetin, quercitrin, rutin), valine, leucine and isoleucine degradation (thiamin diphosphate, 2-Methylpropanoyl-CoA, 2-Methyl-1-hydroxybutyl-ThPP), lysine degradation (lysine, butanoyl-CoA, N6-Acetyl-L-lysine), and fatty acid degradation (butanoyl-CoA, hexanoyl-CoA) ( Supplementary Figures 1, 2 ). These results demonstrated that many metabolic pathways, especially the pathways of secondary metabolites, were implicated in the response to extreme high temperature during seedling establishment.

As shown in Figure 2A , there only 33 metabolites mostly belonging to organic acids/derivates and amines were obviously enriched at 22°C and 28°C, compared to the extreme high temperature, mainly involving in TCA cycle (citrate, succinate, isocitrate), beta-alanine metabolism (spermidine, thiamine, methyltyramine, propynoic acid), phenylalanine metabolism (3-hydroxyphenylacetate, 2-hydroxyphenylacetate), purine metabolism (adenine, adenosine), tyrosine metabolism (4-hydroxycinnamic acid, 3-methoxy-4-hydroxyphenylglycolaldehyde, 3-hydroxyphenylacetate), phenylpropanoid biosynthesis (spermidine, sinapate, 4-hydroxycinnamic acid), carbonhydrates metabolism (D-xylose, acetylglucosamine-1-phosphate) and peptide biosynthesis (kyotorphin). Compared with 22°C, there had 17 metabolites enriched only at 28°C, which were mainly amino acids/derivates including asparagine, valine, tyrosine, glutamine, isoleucine, histidine and threonine, N5-ethyle-L-glutamine and N5-phenyl-L-glutamine, were especially involved in the pathways of cyanoamino acid metabolism (tyrosine, asparagine, isoleucine), glyoxylate and dicarboxylate metabolism (glutamine), ABC transporters (glutamine, histidine) and tyrosine-mediated biosynthesis of various secondary metabolites including phenylpropanoid biosynthesis, glucosinolate biosynthesis (also isoleucine), isoquinoline alkaloid biosynthesis, betalain biosynthesis, ubiquinone and other terpenoid-quinone biosynthesis ( Figure 2B ).

By using the approach of untargeted metabolomics, some metabolites involving plant hormone biosynthesis were also discovered in this study, especially the biosynthesis of auxin, salicylic acid (SA) and ethylene. All the precursor and intermediates for auxin biosynthesis were detected, which showed that the precursor (tryptophan) and intermediates of the two bypasses including tryptamine, indole-3-acetonitrile, indole-3-pyruvate and indole-3-acetyl-beta-1-D-glucoside to form IAA were enriched at 34°C and 40°C ( Supplementary Figure 1 ); whereas, indole-3-acetaldehyde from tryptamine to form IAA was enriched at 22°C and 28°C ( Figure 2A ), indicating that three pathways of IAA biosynthesis responded to different temperature conditions and the bypass from intermediate indole-3-acetaldehyde might be a major pathway for promoting seed germination at moderate temperature. The biosynthesis of indole-3-acetonitrile from glucobrassicin was a unique trait of members of the Brassicales and evidenced to be also used for synthesis of Arabidopsis IAA under drought stress (Hornbacher et al., 2022). In our study, glucobrassicin was higher at 22°C, and indole-3-acetonitrile accumulated at 28°C, which might indicate that this pathway was also used to synthesize the IAA under increased temperature. The intermediates for SA biosynthesis (4-hydroxycinnamic acid, 4-methylsalicylate) was obviously enriched at 22°C and 28°C ( Supplemental Figures S1 ; Figure 2A ), which suggested that SA might be involved in promoting the seed germination at moderate temperature. The intermediates for ethylene biosynthesis, S-methyl-5’-thioadenosine and adenine, were obviously enriched at 22°C and 28°C, and their levels were higher at 22°C than at 28°C ( Figure 2A ), which suggested that 22°C would be optimum for Arabidopsis seed germination, compared with 28°C, although all the seeds were germinated at both temperatures.

Phospholipids function not only as membrane structure but also as messengers to play vital roles for plant development. The volcano plot was used to analyze the significant differences between 22~28°C and 34~40°C, and 104 lipid species were obviously altered among 149 lipid species identified ( Figure 3A ).

The hierarchical heatmap analysis Z score scaled the fold changes of phospholipids, which showed that most species of PEs (34:2, 34:3, 36:6, 38:3, 38:4, 38:5, 38:6, 40:2, 42:2) and PGs species (34:0, 34:2, 34:3, 34:4, 36:3, 36:5), and all the species of PSs (34:2, 34:3, 40:2, 40:3) and PAs (32:0, 32:1, 34:2, 34:3, 34:4, 36:2, 36:3, 36:4, 36:5, 36:6, 38:2, 38:3, 38:4) tended to have high levels at moderate temperatures); whereas, other species of PEs and most PCs species tended to have high levels at extreme high temperature ( Figures 3B , 4A , 5A ) which indicated that phospholipids were distinguished in response to temperatures. The total lipids classes, PC, PE, PS, PA and PG, showed the significant differences between moderate and extreme high temperature; whereas, PIs and ceramides didn’t exhibit the obvious differences in response to temperatures ( Figures 3C–G , 4B , 5B ). PC and PE showed the increased tendency from 22°C and 28°C to 34°C. Oppositely, PS, PA and PG were enriched at 22°C and 28°C. Compared with 22°C, however, both PS and PA were obviously enriched at 28°C. These results suggested the increased tendency of phospholipids in response to temperatures during seed germination and seedling growth. Increased PC during heat stress was suggested to serve as a fatty acyl donors for phospholipid:diacylglycerol acyltransferase 1 (PDAT1) to DAG acylation (Mueller et al., 2017).

The degree of lipid unsaturation was closely related to membrane lipid fluidity, and might affect the physiological efficiency of membrane. The acyl fatty acid (FA) chain length and unsaturation of phospholipids were analyzed in response to moderate and extreme high temperatures, respectively. All the PC species with long FA chain and more unsaturated degree were accumulated at high temperatures; whereas, the high saturated PC species with short FA chain length (PC 32:0 and PC 32:1) tended to be abundant at extreme high temperature ( Figure 3C ). Similarly, the PE species (32:0, 32:1) were also enriched at extreme high temperatures ( Figure 3D ). All the FA chain length and unsaturated degrees of both PS and PI exhibited the similar downward tendency in response to extreme high temperatures ( Figures 3E, F ). All the PA and PG species with long FA chain length and unsaturated degrees were also decreased, especially PA 34:4, 36:4 and PG 34:3, at extreme high temperatures ( Figures 4B , 5B ). These results suggested that the alteration of FA chain length and unsaturated degrees were closed to their head groups of different phospholipids in response to temperatures during seedling establishment.

DAG plays important roles in lipid metabolism, which could generate TAG and were also as substrate to biosynthesize both the phospholipids intermediates PA and galactolipid MDGD. DAG was enriched at 28°C, compared with other temperatures, but DAG did not show significant difference between moderate and extreme high temperatures ( Figures 4A, C ). However, the FA chain of DAG with different unsaturated degrees species showed that DAGs with one double bonds (32:1, 34:1, 36:1) were accumulated at 22°C and 28°C ( Figure 4C ). TAGs were the most abundant in glycerolipids during seedling establishment and were more enriched at extreme high temperatures ( Figure 4D ). The FA chain length and unsaturated degrees analysis showed that the saturated TAGs (52:0) were accumulated at 34~40°C ( Figure 4B ). PDAT1 catalyzed the transfer of a fatty acid from PC to DAG to form TAG and LPC. The seedlings with PDAT1 deficiency had much lower level of TAGs under heat stress, compared to wild-type plants (Mueller et al., 2017). In this study, PC and TAGs accumulation under heat stress ( Figures 3C , 4D ) might be caused by PDAT1 activation.

PG is essential component of the chloroplast membranes and also as substrate to biosynthesize galactolipids, which showed the similar altered tendency to galactolipids ( Figure 5B ). The alteration of galactolipids (MGDGs, DGDGs, SQDGs) were similar to glycerolipids and enriched at moderate temperatures ( Figure 5A ). The lipid unsaturated degrees of galactolipids were all decreased at extreme high temperatures ( Figures 5C, D ). As previously reported, the decreased galactolipids (MGDG and DGDG) might block plastid biogenesis (Yu et al., 2015).

Based on the distinguished profiles of metabolome and lipidome during seed germination and seedling growth at moderate and extreme high temperatures, we further analyzed the metabolomic and lipidomic differences between the moderate temperatures of 22°C and 28°C. A total of 59 metabolites and 50 lipids were significantly altered between 22°C and 28°C, which included 23 amino acids, 10 sugars/derives, 9 organic acids, 5 CoA, 2 purine/derives, 2 nicotine/derives, 1 indole, 1 phenol, 13 PEs, 10 PCs, 7 PAs, 5 MGDGs, 4 DGDGs, 4 DAGs, 3 PGs, 2 PSs, 1 SQDG and 1 ceramide ( Figures 6A, B ).

The sugars, FAs, nucleotides and polypeptides were obviously enriched at 22°C, which were implicated in carbohydrate metabolism (melibiose, epimelibiose, palatinose, isomaltose, mannobiose, 2-alpha-D-glucosyl-D-glucose), TCA (citrate, isocitrate, succinate, thiamin diphosphate), phosphoenolpyruvate pool (N-acylglucosamine 1-phosphate, 2-methylpropanoyl-CoA), FAs metabolism (Octanoyl-CoA, Hexanoyl-CoA, Butanoyl-CoA), nucleotide metabolism (guanosine, adenine), bioactive cyclin dipeptides kyotorphin (l-tyrosyl-l-arginine) and tripeptides (ophthalmate, glutathione) ( Figure 6C ). However, most of amino acids detected including L-type, D-type and D/L-allothreonine preferred to enrich at 28°C ( Figure 6C ).

Lipids including phospholipids, DAGs, galactolipids showed the different enrichment between 22°C and 28°C. The species of SQDG 36:6 and PG 34:4 were especially low at 28°C ( Figure 6D ). Whereas, other species of lipids including PC (32:0, 34:3, 34:4, 36:4, 38:5), most PE (32:1, 32:2, 34:2, 34:2, 34:4, 36:2, 36:3, 36:4, 38:2, 38:3, 38:4, 38:5, 40:2), PA (32:0, 32:1, 34:2, 36:2, 36:3, 38:2, 38:3), and all the PS were higher at 28°C, compared with 22°C ( Figure 6D ). PG (32:0), DAG (32:0, 36:0), MGDG (34:1, 34:2, 34:3, 36:1, 36:2), DGDG (34:2, 36:3, 38:3, 38:4) with low unsaturated degree also showed high levels at 28°C, compared with 22°C ( Figure 6D ). These results suggested that most of lipids tended to increase to promote seedling growth under moderate higher temperature.