Galactolipid and Phospholipid Profile and Proteome Alterations in Soybean Leaves at the Onset of Salt Stress

Abstract

Salinity is a major environmental factor that constrains soybean yield and grain quality. Given our past observations using the salt-sensitive soybean (Glycine max [L.] Merr.) accession C08 on its early responses to salinity and salt-induced transcriptomic modifications, the aim of this study was to assess the lipid profile changes in this cultivar before and after short-term salt stress, and to explore the adaptive mechanisms underpinning lipid homeostasis. To this end, lipid profiling and proteomic analyses were performed on the leaves of soybean seedlings subjected to salt treatment for 0, 0.5, 1, and 2 h. Our results revealed that short-term salt stress caused dynamic lipid alterations resulting in recycling for both galactolipids and phospholipids. A comprehensive understanding of membrane lipid adaption following salt treatment was achieved by combining time-dependent lipidomic and proteomic data. Proteins involved in phosphoinositide synthesis and turnover were upregulated at the onset of salt treatment. Salinity-induced lipid recycling was shown to enhance jasmonic acid and phosphatidylinositol biosyntheses. Our study demonstrated that salt stress resulted in a remodeling of membrane lipid composition and an alteration in membrane lipids associated with lipid signaling and metabolism in C08 leaves.

Keywords: Glycine max; lipid profiling; lipid signaling; membrane lipids; metabolism; salinity.

FIGURE 1

Changes in lipid acyl species under salt stress in leaves of soybean (Glycine max [L.] Merr.) accession C08. Heatmaps showing the log₂-fold changes in each affected lipid acyl species at 0.5, 1, and 2 h of salt treatment. PA as lipid metabolic intermediates in the endoplasmic reticulum (ER) are derived from 16:0, 18:0-ACP, and 18:1-ACP plastidial de novo fatty acid synthesis. Total lipids were extracted from leaves of 14-day-old C08 seedlings grown in 1/2 strength Hoagland solution and treated with or without 0.9% (w/v) NaCl. The lipid molecular species were quantified by tandem mass spectrometry. Scale bar represents log₂-fold changes. *, significant difference at p ≤ 0.05; **, significant difference at p ≤ 0.01 using the Student’s t-test. The raw data is provided in Supplementary Table 1. ACP, acyl carrier protein; DGDG, digalactosyldiacylglycerol; LysoPC, lysophosphatidylcholine; MGDG, monogalactosyldiacylglycerol; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine.

FIGURE 2

Impact of salt treatment for 0, 0.5, 1, and 2 h on leaf proteome of soybean (Glycine max [L.] Merr.) accession C08. Total leaf protein was extracted and analyzed by tandem mass spectrometry from 14-day-old C08 seedlings grown in 1/2 strength Hoagland solution and treated with or without 0.9% (w/v) NaCl. (A) PCA of proteome profiles. Normalized protein abundance was Log₂ transformed and used as input data, centring and scaling was performed prior than calculating principle components. The first and second principal components were visualized in dot plot, and the variation was labeled in the brackets. (B) Venn diagram of identified proteins in each time point. (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of identified proteins from each time point. The color gradient represents the value of a false discovery rate, with a cut-off ≤0.05. The size of each circle represents the input number of proteins in the enriched pathway. The protein lists of enriched pathways are provided in Supplementary Table 4.

FIGURE 3

Effect of salt treatment on the soybean (Glycine max [L.] Merr.) accession C08 leaf proteome. (A) Heatmap displays the log₂-fold changes in abundance of significant differentially expressed proteins (DEPs) (stress vs. normal control), adjusted p-value ≤ 0.05. The union set was used to generate the plot. (B) Gene ontology (GO) enrichment of DEPs at each time point compared to 0 h sample. Three categories of GO terms including “cellular component,” “biological process” (P), and “molecular function” (F) were used, only items in the last two were found enriched. Dashed line indicates FDR = 0.05. The protein lists of enriched items are provided in Supplementary Table 6.

FIGURE 4

Proteins with differential abundance related to various metabolic pathways in the chloroplasts and peroxisomes during salt stress in leaves of soybean (Glycine max [L.] Merr.) accession C08. Each significant differentially expressed protein is presented as heatmaps with shades of red or blue according to the scale bar. Scale bar indicates log₂-fold changes of protein abundance (stress vs. control) with an adjusted p-value ≤ 0.05. *, significant difference at p ≤ 0.05; **, significant difference at p ≤ 0.01 using ANOVA test. For tandem mass spectrometry, the total leaf protein was extracted from leaves of 14-day-old C08 seedlings grown in 1/2 strength Hoagland solution and treated with or without 0.9% (w/v) NaCl. Differential protein abundances were calculated by label-free quantification of MS peptide signals. AccB, biotin carboxyl carrier protein of acetyl-CoA carboxylase; ACP, acyl carrier protein; DHAR3, dehydroascorbate reductase 3; 12, 13-EOT, 12, 13-epoxyoctadeca-9, 11, 15-trienoic acid; 13-HPOT, 13 hydroperoxyoctadeca-9, 11, 15-trienoic acid; JA, jasmonic acid; 13-LOX, 13-lipoxygenases; OPR3, 12-oxophytodienoate reductase 3; PAP, plastid lipid-associated protein.

FIGURE 5

Proteins with differential abundance involved in the tricarboxylic acid (TCA) cycle, glycolysis and fatty acid β-oxidation pathway during salt stress in leaves of soybean (Glycine max [L.] Merr.) accession C08. Each significant differentially expressed protein is presented as heatmaps with shades of red or blue according to the scale bar. Scale bar indicates log₂-fold changes of protein abundance (stress vs. control) with an adjusted p-value ≤ 0.05. *, significant difference at p ≤ 0.05; **, significant difference at p ≤ 0.01 using ANOVA test. Black arrow, reaction between two intermediates; dashed arrow, several steps in the reaction. For tandem mass spectrometry, the total leaf protein was extracted from leaves of 14-day-old C08 seedlings grown in 1/2 strength Hoagland solution and treated with or without 0.9% (w/v) NaCl. Differential protein abundances were calculated by label-free quantification of MS peptide signals. PDH E1, pyruvate dehydrogenase E1 component.

FIGURE 6

Proteins with differential abundance involved in phospholipid metabolic pathways during salt stress in leaves of soybean (Glycine max [L.] Merr.) accession C08. Each significant differentially expressed protein is presented as heatmaps with shades of red or blue according to the scale bar. Scale bar indicates log₂-fold changes of protein abundance (stress vs. control) with an adjusted p-value ≤ 0.05. ^∗, significant difference at p ≤ 0.05; ^∗∗, significant difference at p ≤ 0.01 using ANOVA test. For tandem mass spectrometry, the total leaf protein was extracted from leaves of 14-day-old C08 seedlings grown in 1/2 strength Hoagland solution and treated with or without 0.9% (w/v) NaCl. Differential protein abundances were calculated by label-free quantification of MS peptide signals. CDP-choline, cytidine diphosphate choline; CDP-DAG, cytidine diphosphate diacylglycerol; PA, phosphatidic; CTP, choline-phosphate cytidylyltransferase; DAG, diacylglycerol; G6P, glucose 6-phosphate; Ins3P, myo–inositol–3–phosphate; IMP, inositol monophosphatase; MIPS, myo-inositol-1-phosphate synthase; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; PI(4,5)P₂, phosphatidylinositol 4, 5-bisphosphate; PI4P, phosphatidylinositol 4-phosphate; PI4PK5, phosphatidylinositol-4-phosphate 5-kinase; PI-PLC, phosphoinositide phospholipase C; PIS, phosphatidylinositol synthase; PS, phosphatidylserine; PS decarboxylase, phosphatidylserine decarboxylase.