Comprehensive metabolomic and lipidomic alterations in response to heat stress during seed germination and seedling growth of Arabidopsis

Abstract

Temperature affects seed germination and seedling growth, which is a critical and complex stage in plant life cycle. However, comprehensive metabolic basis on temperature implicating seed germination and seedling growth remains less known. Here, we applied the high-throughput untargeted metabolomic and advanced shotgun lipidomic approaches to profile the Arabidopsis 182 metabolites and 149 lipids under moderate (22°C, 28°C) and extreme high (34°C, 40°C) temperatures. Our results showed that a typical feature of the metabolism related to organic acids/derivates and amines was obviously enriched at the moderate temperature, which was implicated in many cellular responses towards tricarboxylic acid cycle (TCA), carbohydrates and amino acids metabolism, peptide biosynthesis, phenylpropanoid biosynthesis and indole 3-acetate (IAA) biosynthetic pathway. Whereas, under extreme high temperatures, there was no seed germination, but 148 out of total 182 metabolites were highly enriched, involving in the galactose metabolism, fatty acid degradation, tryptophan/phenylalanine metabolism, and shikimic acid-mediated pathways especially including alkaloids metabolism and glucosinolate/flavone/flavonol biosynthesis. Phosphatidylcholine (PC) and phosphatidylethanolamine (PE) also exhibited the gradually increased tendency from moderate temperatures to extreme high temperatures; whereas phosphatidylserine (PS), phosphatidic acid (PA), phosphatidylglycerol (PG), monogalactosyldiacylglycerol (MGDG), digalactosyldiacylglycerol (DGDG) and sulfoquinovosyldiacylglycerol (SQDG) were contrary to decrease. Another typical feature of the distinguished metabolites between 22°C and 28°C, the TCA, disaccharides, nucleotides, polypeptides, SQDG and the biosynthesis of fatty acids and glucobrassicin-mediated IAA were obviously decreased at 28°C, while amino acids, trisaccharides, PE, PC, PA, PS, MGDG, DGDG and diacylglycerol (DAG) preferred to enrich at 28°C, which characterized the alteration of metabolites and lipids during fast seedling growth. Taking together, our results provided the comprehensive metabolites phenotyping, revealed the characteristics of metabolites necessary for seed germination and/or seedling growth under different temperatures, and provided insights into the different metabolic regulation of metabolites and lipid homeostasis for seed germination and seedling growth.

Keywords: arabidopsis; heat stress; lipidome; metabolite; phenotyping; seed germination; seedling growth.

Figure 1

Global analysis of metabolite and lipids for Arabidopsis seeds germination/seedling growth under four temperatures conditions. (A) The observation of seeds germinated at 22°C, 28°C, 34°C and 40°C, respectively. Col_22 means seeds germinated at 22°C, Col_28 means seeds germinated at 28°C, Col_34 means seeds germinated at 34°C, Col_40 means seeds germinated at 40°C. (B) Principal component analysis of the inter-group metabolic differences during Arabidopsis seeds germination/seedling growth under four temperatures conditions. (C) Principal component analysis of the inter-group lipids differences during Arabidopsis seeds germination/seedling growth under four temperatures conditions. (D) Significantly distinct metabolites between moderate (22°C, 28°C), and extreme high temperatures (34°C, 40°C) were analyzed by volcano plot. X-axis was Log2 fold change and the y-axis was –Log10 P value. The red dots on the left indicated that metabolites of seeds germination at moderate temperature significantly reduced level, compared with the extreme high temperature. The red dots on the right indicated metabolites level increased significantly. Blue dots represented metabolites with only P<0.05 and green dots represent metabolites with only FC>1.5. Gray dots referred to all other identified metabolites with no significant changes between moderate and extreme high temperatures. (E) Analysis of metabolic pathways based on the differences of metabolites between moderate (22°C, 28°C) and extreme high temperatures (34°C, 40°C). Bioinformatic analysis was performed using the MetDNA tools at http://metdna.zhulab.cn/.

Figure 2

Comparative analysis of metabolites for Arabidopsis seeds germination/seedling growth between moderate and extreme high temperatures. (A) Heatmap showing Z score scaled the metabolites with higher level at moderate temperature (22°C, 28°C), compared with those at the extreme high temperature (34°C, 40°C). The relative enrichment of each metabolite in different samples were arranged in rows, and the relative levels of all metabolites in each sample were distributed in columns. The grids in heatmap represented metabolites, and shades of color represented relative level of metabolites. Red and blue colors indicate an increase and a decrease of metabolite level, respectively, and white color means no change. Col_22 means seeds germinated at 22°C, Col_28 means seeds germinated at 28°C, Col_34 means seeds germinated at 34°C, Col_40 means seeds germinated at 40°C. (B) Heatmap showing Z score scaled the metabolites with the highest level at 28°C, compared with other temperature conditions. The relative enrichment of each metabolite in different samples were arranged in rows, and the relative levels of all metabolites in each sample were distributed in columns. The grids in heatmap represented metabolites, and shades of color represented relative level of metabolites. Red and blue colors indicate an increase and a decrease of metabolite level, respectively, and white color means no change. Col_22 means seeds germinated at 22°C, Col_28 means seeds germinated at 28°C, Col_34 means seeds germinated at 34°C, Col_40 means seeds germinated at 40°C.

Figure 3

Comparative analysis of lipidome for Arabidopsis seeds germination/seedling growth under four temperature conditions. (A) The distinguished alteration of lipids between moderate (22°C, 28°C) and extreme high temperatures (34°C, 40°C) were analyzed by volcano plot. X-axis was Log2 fold change and the y-axis was –Log10 P value. The red dots on the left indicated that lipids of seeds germination at moderate temperature significantly reduced level, compared with the extreme high temperature. The red dots on the right indicated lipids level significantly increased. Blue dots represented metabolites with only P<0.05 and green dots represent metabolites with only FC>1.5. Gray dots referred to all other identified metabolites with no significant changes between moderate and extreme high temperatures. Phosphatidic acid (PA), Phosphatidylcholine (PC), Phosphatidylethanolamine (PE), Phosphatidylserine (PS), Phosphatidylinositol (PI), Phosphatidylglycerol (PG), Diacylglycerol (DAG), Triacylglycerol (TAG), Sulfoquinovosyldiacylglycerol (SQDG), Monogalactosyldiacylglycerol (MGDG), Digalactosyldiacylglycerol (DGDG). (B) Heatmap showing Z score scaled the lipids alteration at moderate (22°C, 28°C) and the extreme high temperatures (34°C, 40°C). The relative enrichment of each lipid in different samples were arranged in rows, and the relative levels of all lipids in each sample were distributed in columns. The grids in heatmap represented lipids, and shades of color represented relative level of lipids. Red and blue colors indicate an increase and a decrease of lipid level, respectively, and white color means no change. Col_22 means seeds germinated at 22 °C, Col_28 means seeds germinated at 28°C, Col_34 means seeds germinated at 34 °C, Col_40 means seeds germinated at 40 °C. (C–G) Relative levels of phospholipid classes (small figure on the left of block diagram) and levels of different molecular species (small figure on the right of block diagram) under four temperature conditions. The FC difference was calculated as extreme high temperature group vs. moderate temperature group. Phospholipids shown as PC (B), PE (C), PS (D), PI (E) and ceramide (F). Phosphatidylcholine (PC), Phosphatidylethanolamine (PE), Phosphatidylserine (PS), Phosphatidylinositol (PI). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

Comparative analysis of phosphatidic acid and glycerol lipids for Arabidopsis seed germination/seedling growth under four temperature conditions. (A) Heatmap showing Z score scaled the alteration of phosphatidic acid and glycerol lipids at moderate (22°C, 28°C) and the extreme high temperatures (34°C, 40°C). The relative enrichment of each lipid in different samples were arranged in rows, and the relative levels of all lipids in each sample were distributed in columns. The grids in heatmap represented lipids, and shades of color represented relative level of lipids. Red and blue colors indicate an increase and a decrease of lipid level, respectively, and white color means no change. Col_22 means seeds germinated at 22°C, Col_28 means seeds germinated at 28°C, Col_34 means seeds germinated at 34°C, Col_40 means seeds germinated at 40°C. (B–D) Relative classed level of phosphatidic acid and glycerol lipids (small figure on the left of block diagram) and levels of different molecular species (small figure on the right of block diagram) under four temperature conditions. The FC difference was calculated as extreme high temperature group vs. moderate temperature group. Phospholipids are shown as PA (B), DAG (C) and TAG (D). Phosphatidic acid (PA), Diacylglycerol (DAG), Triacylglycerol (TAG). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 5

Comparative analysis of phosphatidylglycerol and galactolipids for Arabidopsis seed germination/seedling growth under four temperature conditions. (A) Heatmap showing Z score scaled the alteration of phosphatidylglycerol and galactolipids at moderate (22°C, 28°C) and the extreme high temperatures (34°C, 40°C). The relative enrichment of each metabolite in different samples were arranged in rows, and the relative levels of all metabolites in each sample were distributed in columns. The grids in heatmap represented metabolites, and shades of color represented relative level of metabolites. Red and blue colors indicate an increase and a decrease of metabolite level, respectively, and white color means no change. Col_22 means seeds germinated at 22 °C, Col_28 means seeds germinated at 28°C, Col_34 means seeds germinated at 34 °C, Col_40 means seeds germinated at 40 °C. (B–D) Lipid classes content differences of phospholipids under four different temperature (small figure on the left of block diagram) and changes in lipid content of different molecular species between low temperature group and high temperature group (small figure on the right of block diagram). The FC difference was calculated as high temperature group vs. low temperature group. Lipids shown here are PG (B), MGDG (C) and DGDG (D). Phosphatidylglycerol (PG), Monogalactosyldiacylglycerol (MGDG), Digalactosyldiacylglycerol (DGDG). Data are presented as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6

Distinguished analysis of metabolites and lipids for Arabidopsis seedling growth between 22°C and 28°C. (A) Distinct metabolites between 22°C and 28°C were analyzed by volcano plot. X-axis was Log₂ fold change and the y-axis was –Log₁₀ P value. The red dots on the left indicated that metabolites level of seeds germination at 22°C significantly reduced, compared with 28°C. The red dots on the right indicated metabolites level significantly increased. Blue dots represented metabolites with only P<0.05 and green dots represent metabolites with only FC>1.5. Gray dots referred to all other identified metabolites with no significant changes between 22°C and 28°C. (B) Distinct lipids between 22°C and 28°C were analyzed by volcano plot. X-axis was Log₂ fold change and the y-axis was –Log₁₀ P value. The red dots on the left indicated that lipids of seeds germination at 22°C significantly reduced level, compared with 28°C. The red dots on the right indicated lipids level significantly increased. Blue dots represented metabolites with only P<0.05 and green dots represent metabolites with only FC>1.5. Gray dots referred to all other identified metabolites with no significant changes between 22°C and 28°C. (C) Heatmap showing Z score scaled the alteration of metabolites between 22°C and 28°C. The relative enrichment of each metabolite in different samples were arranged in rows, and the relative levels of all metabolites in each sample were distributed in columns. The grids in heatmap represented metabolites, and shades of color represented relative level of metabolites. Red and blue colors indicate an increase and a decrease of metabolite level, respectively, and white color means no change. Col_22 means seeds germinated at 22 °C, Col_28 means seeds germinated at 28°C. (D) Heatmap showing Z score scaled the alteration of lipids between 22°C and 28°C. The relative enrichment of each lipid in different samples were arranged in rows, and the relative levels of all lipids in each sample were distributed in columns. The grids in heatmap represented lipids, and shades of color represented relative level of lipids. Red and blue colors indicate an increase and a decrease of lipid level, respectively, and white color means no change. Col_22 means seeds germinated at 22 °C, Col_28 means seeds germinated at 28°C.

Figure 7

A model illustrating the main alteration of metabolites and lipids during seed germination under heat stress. The metabolites and lipids are implicated in TCA cycle, amino acids metabolism, sugar metabolism, lipids metabolism, secondary metabolites, phytohormone SA and auxin signaling, mRNA transcription during seed germination and seedling growth under heat stress. The metabolites shown in red font indicated significantly increased levels, and shown in blue font indicated the decreased levels under the temperatures of 34°C and 40°C, compared with those under the temperatures of 22°C and 28°C. Lipids were presented as number of carbon atoms and unsaturation degrees. Numbers in bracket were the unsaturation degrees of fatty acyl chains.