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International Journal of Molecular Sciences logoLink to International Journal of Molecular Sciences
. 2022 Oct 13;23(20):12247. doi: 10.3390/ijms232012247

Metabolic Regulation and Lipidomic Remodeling in Relation to Spermidine-induced Stress Tolerance to High Temperature in Plants

Zhou Li 1,2,†, Bizhen Cheng 1,†, Yue Zhao 1, Lin Luo 1, Yan Zhang 1, Guangyan Feng 1, Liebao Han 2, Yan Peng 1,*, Xinquan Zhang 1,*
Editor: Tibor Janda
PMCID: PMC9602532  PMID: 36293104

Abstract

Beneficial effects of spermidine (Spd) on alleviating abiotic stress damage have been explored in plants for hundreds of years, but limited information is available about its roles in regulating lipids signaling and metabolism during heat stress. White clover (Trifolium repens) plants were pretreated with 70 μM Spd and then subjected to high temperature (38/33 °C) stress for 20 days. To further investigate the effect of Spd on heat tolerance, transgenic Arabidopsis thaliana overexpressing a TrSAMS encoding a key enzyme involved in Spd biosynthesis was exposed to high temperature (38/33 °C) stress for 10 days. A significant increase in endogenous Spd content in white clover by exogenous application of Spd or the TrSAMS overexpression in Arabidopsis thaliana could effectively mitigate heat-induced growth retardation, oxidative damage to lipids, and declines in photochemical efficiency and cell membrane stability. Based on the analysis of metabolomics, the amino acids and vitamins metabolism, biosynthesis of secondary metabolites, and lipids metabolism were main metabolic pathways regulated by the Spd in cool-season white clover under heat stress. Further analysis of lipidomics found the TrSAMS-transgenic plants maintained relatively higher accumulations of total lipids, eight phospholipids (PC, phosphatidylcholine; PG, phosphatidylglycerol; PS, phosphatidylserine; CL, cardiolipin; LPA, lysophosphatidic acid; LPC, lyso phosphatidylcholine; LPG, lyso phosphatidylglycerol; and LPI, lyso phosphatidylinositol), one glycoglycerolipid (DGDG, digalactosyl diacylglycerol), and four sphingolipids (Cer, ceramide; CerG2GNAc1, dihexosyl N-acetylhexosyl ceramide; Hex1Cer, hexosyl ceramide; and ST, sulfatide), higher ratio of DGDG: monogalactosyl diacylglycerol (MGDG), and lower unsaturation level than wild-type Arabidopsis thaliana in response to heat stress. Spd-induced lipids accumulation and remodeling could contribute to better maintenance of membrane stability, integrity, and functionality when plants underwent a long period of heat stress. In addition, the Spd significantly up-regulated PIP2 and PA signaling pathways, which was beneficial to signal perception and transduction for stress defense. Current findings provide a novel insight into the function of Spd against heat stress through regulating lipids signaling and reprograming in plants.

Keywords: signaling pathway, heat stress, metabolomics, phospholipids, glycoglycerolipids, sphingolipids, unsaturation index

1. Introduction

For most of plant species adapting to temperate climate, high environmental temperature disturbs metabolic homeostasis, resulting in the retardation of seed germination, plant growth, development, and reproduction [1]. Heat-induced crop failure and other agricultural losses will further increase due to the intensification of global warming [2]. Plants have developed complex defensive strategies to alleviate heat damage including alterations in endogenous hormones or plant growth regulators (PGRs) [3]. Spermidine (Spd) is one of the commonest polyamines acting as a critical PGR for regulation of seed germination, plant growth, and stress tolerance [4]. It has been reported that enhanced Spd biosynthesis and metabolism are positive responses to various abiotic stresses such as heat, drought, and salt stress, since Spd plays a key role in regulating cell signal transduction, osmotic adjustment, antioxidant metabolism, and membrane protection in plants [5,6,7,8]. Significant increase in endogenous Spd content by exogenous application of Spd or transgenic approach could effectively alleviate heat damage owing to enhanced antioxidant capacity in rice (Oryza sativa) leaves [9], starch metabolism in rice seeds [10], photosynthetic performance in tomato (Lycopersicon esculentum) leaves [11], and transcriptional regulation in ripening tomato fruit [12]. Our previous studies also demonstrated that foliar application of the polyamine such as Spd or spermine (Spm) mitigated adverse effects of heat stress in white clover (Trifolium repens) or creeping bentgrass (Agrostis stolonifera) associated with changes in antioxidant defense or heat shock pathways, respectively [13,14].

Oxidative burst and overaccumulation of oxidizing agents damage the cell membrane system, which is one of the major stress symptoms in plants subjected to a prolonged period of heat stress and other abiotic stresses [15,16,17,18]. Therefore, the maintenance of the structural integrity of the membrane is beneficial to the stress defense system [19]. Lipids such as phospholipids (Phls), glycoglycerolipids (Glls), and sphingolipids (Spls) are not only important components of the membrane system, but also exhibit a variety of roles in cell signaling, protein modification, energy storage, and membrane anchoring in plants [20]. The fact that there are more than 200,000 lipid molecular species in biological systems increases the difficulty of lipid research over a long period of time [21]. As a new approach, lipidomics provides a rapid and sensitive strategy to reveal vital function of diverse lipid species in living beings [22]. A recent study demonstrated that a significantly higher polyunsaturated linolenic acid (18:3) content and a lower lipid unsaturation level were in favor of heat adaptation based on comparative lipidomic analysis between heat-tolerant soybean (Glycine max) genotype DS25-1 and heat-susceptible genotype DT97-4290 [23]. General analysis of the lipid profile revealed that enhanced triacylglycerol accumulation and decline in membrane polyunsaturated fatty acids were responsible for better adaptation of Arabidopsis thaliana to heat stress [24]. In addition, Spd in association with changes in lipids has also been reported in plant species. For example, the Spd induced significant increases in phosphatidylinositol 4-phosphate (PIP) and phosphatidylinositol 4,5-bisphosphate (PIP2) in Brassica oleracea seedlings under normal condition [25]. Quick accumulation of stearic acid, linoleic acid, and linolenic acid could be significantly inhibited by exogenous Spd in leaves of cucumber (Cucumis sativus) under salt stress [26]. However, potential roles of Spd in regulating lipid signaling and global lipid compositions in response to environmental stress, especially high temperatures, are not well elucidated in plant species.

Objectives of this study were to investigate effects of exogenous application of Spd on physiological changes and metabolites profile in white clover under heat stress and to further reveal the Spd-regulated lipids signal and remodeling in transgenic Arabidopsis thaliana overexpressing a TrSAMS encoding a S-adenosylmethionine synthetase from white clover involved in Spd biosynthesis in response to heat stress. Current findings facilitated a comprehensive understanding of an underlying mechanism of Spd-mediated stress adaptation associated with the alleviation of growth retardation, enhanced phosphatidylinositol (PI) and phosphatidic acid (PA) signaling pathways, and maintenance of membrane integrity and functionality in plants exposure to high temperature environment.

2. Results

2.1. Stress Tolerance and Metabolomics Regulated by Spermidine in White Clover under Heat Stress

Heat stress significantly inhibited white clover growth, but this adverse effect could be alleviated by the application of Spd under heat stress (Figure 1A). As compared with the control, endogenous Spd content significantly increased under normal and heat stress conditions due to the Spd application (Figure 1B). Shoot length (SL) of Spd-pretreated or non-pretreated plants was significantly inhibited by heat stress; however, Spd-pretreated plants exhibited significantly higher SL than non-pretreated plants under heat stress (Figure 1C). There were no significant differences in root length (RL) among three treatments (C, C + Spd, and H), and the Spd significantly increased the RL under heat stress (Figure 1D). In spite of the Spd application, photochemical efficiency (Fv/Fm) and performance index on absorption basis (PIABS) significantly declined in both of Spd- and non-pretreated plants after 20 days of heat stress, but this declined trend could be significantly alleviated by the exogenous Spd application (Figure 1E,F). Heat-induced increases in malondialdehyde (MDA) content and electrolyte leakage (EL) could also be alleviated significantly by the Spd (Figure 1G,H).

Figure 1.

Figure 1

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Changes in (A) phenotype, (B) endogenous spermidine (Spd) content, (C) shoot length, (D) root length, (E) photochemical efficiency, (F) performance index on absorption basis, (G) malondialdehyde content, and (H) electrolyte leakage in leaf of white clover in response to exogenous Spd and heat stress. Vertical bars indicate ±SE of mean (n = 4) and different letters above column indicate significant differences (p ≤ 0.05) among treatments.

A large number of differentially accumulated metabolites (DAMs) were identified in leaves of white clover in response to the Spd application and heat stress (Supplementary Figure S1). Except for global and overview maps, most of DAMs were involved in biosynthesis of other secondary metabolites, amino acid metabolism, and metabolism of cofactors and vitamins based on the analysis of KEGG function annotation (Supplementary Figure S2). Lipid maps of DAMs showed that most of the identified DAMs were associated with flavonoids, steroids, isoprenoids, and fatty acids and conjugates (Supplementary Figure S3). Volcano plot of DAMs indicated the distribution of DAMs in each group, as the abscissa presented log2 fold change (FC) of DAMs and the ordinate presented significant level (-log10 p-value) (Supplementary Figure S4). A total of 24, 27, 217, and 203 DAMs were identified in the CS vs. C, HS vs. H, H vs. C, ands HS vs. C, respectively (Figure 2A). Propanoic acid, butanoic acid, triacylglycerol (TAG) (18:0-18:1-22:3), valine, serine, pyroglutamic acid, nicotinamide, pyridoxal, abscisic acid, hydroxyecdysome, methyl dihydrojasmonate, and crocetin significantly increased, whereas docosahexaenoic acid, TAG (18:1-18:1-20:1), and oxidized glutathione significantly decreased in the HS vs. H (Figure 2B).

Figure 2.

Figure 2

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Changes in (A) the total numbers of differentially accumulated metabolites (DAMs) in different groups (CS vs. C, HS vs. H, H vs. C, and HS vs. C) and (B) key DAMs in the HS vs H. C, control; CS, control + spermidine; H, heat stress; HS, heat stress + spermidine.

2.2. Endogenous Spermidine Level and Stress Tolerance Regulated by TrSAMS Overexpression in Arabidopsis thaliana under Heat Conditions

Transgenic Arabidopsis overexpressing the TrSAMS (OE4 and OE5) grew bigger than the wild type (WT) under normal condition and also showed better growth under heat stress (Figure 3A). Endogenous Spd contents were significantly higher in the OE4 and OE5 as compared to that in the WT under normal and heat conditions (Figure 3B). Although heat stress decreased fresh weight (FW) and dry weight (DW) of above-ground biomass, leaf length (LL), and leaf width (LW) of WT and transgenic plants, the OE4 and OE5 exhibited significantly higher FW and DW of above-ground biomass, LL, and LW than the WT under normal and heat conditions (Figure 3C,D,F,G). However, there was no significant difference in DW to FW ratio among WT, OE4, and OE5 (Figure 3E). Transgenic plants maintained significantly higher total chlorophyll (Chl), Chl a, Chl b, and Chl a/b ratio than the WT under heat stress (Figure 4A–D). Fv/Fm and PIABS were not significant differences between the WT and transgenic plants under normal condition and declined significantly under heat stress (Figure 4E,F). Transgenic plants exhibited a 43% or 120% increase in Fv/Fm or PIABS compared to the WT under heat stress, respectively (Figure 4E and 4F). Under normal conditions, the TrSAMS overexpression did significantly affect MDA content and EL in leaves. MDA content and EL rose steeply in all plants due to heat damage, but transgenic plants showed 27% and 32% decrease in MDA content or EL than the WT under heat stress, respectively (Figure 4G and 4H).

Figure 3.

Figure 3

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Changes in (A) phenotype, (B) endogenous spermidine content, (C) fresh weight of above-ground biomass, (D) dry weight of above-ground biomass, (E) dry to fresh weight ratio, (F) leaf length, and (G) leaf width of wild type (WT) and transgenic Arabidopsis thaliana overexpressing the TrSAMS (OE4 and OE5) involved in spermidine biosynthesis under normal condition and heat stress. Vertical bars indicate ±SE of mean (n = 4). Different letters above column indicate significant differences (p ≤ 0.05) among treatments under a particular condition (normal condition or heat stress). The asterisk “*” indicates significant differences (p ≤ 0.05) for a particular material (WT, OE4, or OE5) for comparison between normal condition and heat stress.

Figure 4.

Figure 4

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Changes in (A) total chlorophyll content, (B) chlorophyll a content, (C) chlorophyll b content, (D) chlorophyll a/b ratio, (E) photochemical efficiency, (F) performance index on absorption basis, (G) malondialdehyde content, and (H) electrolyte leakage of wild type (WT) and transgenic Arabidopsis thaliana overexpressing the TrSAMS (OE4 and OE5) involved in spermidine biosynthesis under normal condition and heat stress. Vertical bars indicate ±SE of mean (n = 4). Different letters above column indicate significant differences (p ≤ 0.05) among treatments under a particular condition (normal condition or heat stress). The asterisk “*” indicates significant differences (p ≤ 0.05) for a particular material (WT, OE4, or OE5) for comparison between normal condition and heat stress.

2.3. Heat-Map and Principal Component Analysis Based on 31 Lipid Classes in Arabidopsis thaliana in Response to Spermidine and Heat Stress

Heat-map of different lipid classes showed that most of lipid classes were down-regulated in WT and OE4 in response to 3 h or 10 d of heat stress, but PI, PIP2, lysophosphatidic acid (LPA), lyso phosphatidylinositol (LPI), and ceramide (Cer) were significantly up-regulated by heat stress in the OE4 (Figure 5A). First and second principle component explained 41.0% and 29.7% variation among 31 lipid classes in response to the Spd and heat stress in leaves of Arabidopsis thaliana, respectively (Figure 5B). The principal component analysis (PCA) found that the PIP2, ganglioside-disialo trihexosyl ceramide (GD2), Cer, dihexosyl N-acetylhexosyl ceramide (CerG2GNAc1), phosphatidylglycerol (PG), phosphatidylserine (PS), lyso phosphatidylcholine (LPC), lyso phosphatidylethanolamine (LPE), digalactosyl monoacylglycerol (DGMG), and digalactosyl diacylglycerol (DGDG) were the main lipid classes affected by the Spd and heat stress (Figure 5B).

Figure 5.

Figure 5

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Changes in (A) heatmap as demonstrated by log2 (fold change in contents of total lipids, lipid groups, and lipid classes) and (B) principal component analysis (PCA) based on 31 different lipid classes in leaves of wild type (WT) and transgenic Arabidopsis thaliana (OE4) overexpressing the TrSAMS involved in spermidine biosynthesis in response to spermidine and heat stress. The heat map was made based on log2 fold change of heat-stressed plants (3 h and 10 d) as compared to non-stressed plants (0 h).

2.4. Changes in Total Lipids, Phospholipids, Glycoglycerolipids, and Sphingolipids Regulated by Spermidine in Arabidopsis thaliana under Heat Stress

Heat stress index (HSI) of total lipids significantly declined in WT and OE4 from 3 h to 10 d of heat stress, but OE4 exhibited significantly higher HSI of total lipids than WT at 3 h and on the 10th d of heat stress (Figure 6A). OE4 exhibited 36% and 12% increase in HSI of total Phl compared to WT at 3 h and on 10th d of heat stress, respectively (Figure 6A). There was no significant difference in the HSI of total Gll between WT and OE4 at 0 h or on 10th d of heat stress (Figure 6A). OE4 had significantly higher HSI of total Spl than WT at 3 h of heat stress, but no significant difference was detected on 10th d of heat stress (Figure 6A).

Figure 6.

Figure 6

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Changes in heat stress index of (A) total lipids, total phospholipids (Phl), total glycoglycerolipids (Gll), and total sphingolipids (Spl), (B) different Phl classes, and (C) different Gll and Spl classes in leaves of wild type (WT) and transgenic Arabidopsis thaliana (OE4) overexpressing the TrSAMS involved in spermidine biosynthesis during heat stress. Vertical bars indicate ±SE of mean (n = 4) and different letters above column indicate significant differences (p ≤ 0.05) among treatments.

HSI of PA and phosphatidylcholine (PC) were not significantly different between WT and OE4 at 3 h of heat stress, whereas OE4 had significantly higher HSI of PA and lower HIS of PC than WT on the 10th d of heat stress (Figure 6B). OE4 maintained significantly higher or lower HIS of phosphatidylethanolamine (PE) than WT at 3 h and on 10th d of heat stress, respectively (Figure 6B). Although no significant difference in HSI of PG or LPE was detected between OE4 and WT on the 10th d of heat stress, OE4 showed significantly higher HSI of PG and LPE than WT at 3 h of heat stress. HSI of PS, PI, PIP2, and LPC were maintained at a significantly higher level in OE4 as compared to WT during heat stress. OE4 also exhibited significantly higher HSI of PIP, cardiolipin (CL), LPG or LPI at 3 h of heat stress as well as higher HSI of LPA than WT on 10th d of heat stress. There was no significant difference in HSI of phosphatidylinositol triphosphate (PIP3) or mono-lyso cardiolipin (MLCL) between OE4 and WT during heat stress (Figure 6B).

HSI of DGDG, monogalactosyl diacylglycerol (MGDG), and GD2 was not significantly different between WT and OE4 at 3 h or on 10th d of heat stress (Figure 6C). Significantly lower HSI of DGMG and monogalactosyl monoacylglycerol (MGMG) at 3 h of heat stress and significantly lower HSI of MGMG, ceramide phosphate (CerP), dihexosyl ceramide (Hex2Cer), trihexosyl ceramide (Hex3Cer), ganglioside-disialo tetrahexosyl ceramide (GD1a), and ganglioside-disialo dihexosyl ceramide (GD3) on the 10th d of heat stress were detected in WT as compared to that in OE4 (Figure 6C). A 45%, 11%, and 49% increase in the HSI of Cer, hexosyl ceramide (Hex1Cer), and ganglioside-trisialo dihexosyl ceramide (GT3) was detected in OE4 as compared to WT at 3 h of heat stress, respectively (Figure 6C). OE4 also showed significantly higher HSI of CerG2GNAc1 or sulfatide (ST) than WT at 3 h and on 10th d of heat stress (Figure 6C).

2.5. Lipid Molecular Species, Ratios of PC:PE and DGDG:MGDG, and the Unsaturation Index of Lipids Regulated by Spermidine in Arabidopsis thaliana under Heat Stress

Figure 7 shows the changes in lipid molecular species of different lipid classes (PA, PC, PE, PG, PI, CL, LPA, LPC, LPE, LPG, LPI, MLCL, DGDG, MGDG, DGMG, MGMG, Cer, CerP, CerG2GNAc1, GT3, Hex1Cer, Hex2Cer, Hex3Cer, and GD3) when the transgenic plants were compared with the WT (OE4 vs. WT) at 0 h, 3 h, or 10 d of heat stress. Ratio of PC:PE of WT or OE4 did not significantly change during heat stress (Figure 8A). OE4 exhibited a significantly higher ratio of DGDG:MGDG than WT on the 10th d of heat stress (Figure 8B).

Figure 7.

Figure 7

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Changes in lipid molecular species of different lipid classes in leaves when the transgenic Arabidopsis thaliana overexpressing the TrSAMS was compared with the wild-type (WT) Arabidopsis thaliana during heat stress (OE4 vs. WT). Red indicates a significant up-regulation, green indicates a significant down-regulation, and gray indicates no significant change.

Figure 8.

Figure 8

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Changes in (A) the ratio of PC:PE, (B) the ratio of DGDG:MGDG, (C) the unsaturation index of total lipids, (D) the unsaturation index of phospholipids, (E) the unsaturation index of glycoglycerolipids, and (F) the unsaturation index of sphingolipids in leaves of wild type (WT) and transgenic Arabidopsis thaliana overexpressing the TrSAMS involved in spermidine biosynthesis during heat stress. Different letters above column indicate significant differences (p ≤ 0.05) in a particular material (WT or OE4) from 0 h to 10 d. The asterisk “*” indicates significant differences (p ≤ 0.05) between WT and OE4 at one particular time point (0 h, 3 h, or 10 d).

The unsaturation index of total lipids of WT significantly increased at 3 h of heat stress and maintained a significant level after 10 d of heat stress (Figure 8C). However, the unsaturation index of OE4 did alter during heat stress (Figure 8C). Unsaturation index of total Phl of WT and OE4 also did not change significantly when subjected to heat stress for 10 d, but OE4 showed a significantly lower unsaturation index of total Phl than WT at 3 h of heat stress (Figure 8D). Heat stress (3 h and 10 d) induced significant increase in the unsaturation index of total Gll in WT, whereas it did not affect OE4 (Figure 8E). Significantly lower unsaturation index of total Gll was observed in OE4 as compared to that in WT during heat stress (Figure 8E). Unsaturation index of total Spl did not change in WT at 3 h of heat stress, but significantly decreased after 10 d of heat stress (Figure 8F). Heat stress induced a gradual decline in unsaturation index of total Spl in OE4 during heat stress (Figure 8F). OE4 exhibited significantly lower unsaturation index of total Spl than WT at 3 h of heat stress (Figure 8F).

2.6. Key Genes Involved in PI and PA Metabolic Pathways Regulated by Spermidine in Arabidopsis thaliana under Heat Stress

Heat stress induced significant increases in transcript levels of phosphatidylinositol 4-kinase alpha (AtPI4K-alpha), phosphatidylinositol phosphate 5-kinase 1 (AtPIP5K1), phospholipase C 5 (AtPLC5), diacylglycerol kinase 2 (AtDGK2), and phospholipase D delta (AtPLD-delta) in wild type and transgenic plants (Figure 9). Transgenic plants exhibited significantly higher expression levels of these genes than the wild type in response to heat stress (Figure 9). Figure 10 showed regulatory pathways induced by Spd associated with alterations in metabolic homeostasis, lipids signaling, and lipidomic reprogramming in favor of thermotolerance based on the summary of our current conclusions.

Figure 9.

Figure 9

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Changes in key genes involved in metabolic pathways of phosphoinositide (PI) and phosphatidic acid (PA) regulated by spermidine in leaves of Arabidopsis thaliana during heat stress. Vertical bars indicate ±SE of mean (n = 4) and different letters above column indicate significant differences (p ≤ 0.05) among treatments.

Figure 10.

Figure 10

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Regulatory pathways induced by the spermidine associated with alterations in metabolic homeostasis, lipids signaling, and lipidomic remodeling in plants under heat stress. Red or green characters indicate significant increases or decreases induced by spermidine under heat stress, respectively.

3. Discussion

High temperature stress is one of the major threats for most plants only adapting to temperate climate [27]. Increasing evidence has suggested that Spd exhibits a positive role in alleviating growth retardation, leaf senescence, and photoinhibition against heat stress in various plant species including white clover [9,10,14,28]. These previous studies were consistent with our current findings which showed that a significant increase in endogenous Spd content in white clover by exogenous application of Spd or in transgenic Arabidopsis thaliana overexpressing the TrSAMS enhanced the heat tolerance of white clover or Arabidopsis thaliana through maintaining root and shoot growth, photosynthesis, and cell membrane stability under high temperature condition. Metabolome analysis demonstrated that amino acids and vitamin metabolism, biosynthesis of secondary metabolites, and lipid metabolism were main metabolic pathways affected by the Spd in leaves of white clover suffering from heat stress. Accumulation and conversion of amino acids such as valine, serine, and pyroglutamic acid are adaptive responses to abiotic stress due to their roles in osmotic adjustment and biosynthesis of proteins and secondary metabolites [29,30]. Nicotinamide is a derivative of vitamin B3 that exhibits multiple functions of anti-aging, growth promotion, and stress defense in plants [31,32]. It has been found that foliar application of nicotinamide prevented biomass loss and lipid peroxidation in faba bean (Vicia faba) under salt stress [33]. As an essential component of vitamin B6, pyridoxal is involved in fatty acid metabolism, chlorophyll and auxin biosynthesis, and antioxidant under stressful condition [34,35]. In addition, regulatory roles of secondary metabolites such as abscisic acid (ABA), methyl dihydrojasmonate (MDJ), and hydroxyecdysone in stress tolerance have been reported widely in plants. For example, the ABA could mitigate heat-induced oxidative damage in Arabidopsis thaliana [36] and reed (Phragmites communis) [37]. Exogenous application of hydroxyecdysone protected rice against heat stress [38], and the MDJ could delay drought damage in tall fescue (Festuca arundinacea) [39]. Hence, Spd-induced alleviation of adverse effects in white clover could be related to increased accumulation of amino acids (valine, serine, and pyroglutamic acid), vitamins and their derivative (nicotinamide and pyridoxal), and secondary metabolites (ABA, MDJ, and hydroxyecdysone) under heat stress.

Previous studies have found that alterations in contents and composition of lipids, fatty acids, and TAG have a close association with heat tolerance in plant species [24,40,41]. In the current study, Spd-regulated DAMs included unsaturated fatty acid (propanoic acid and butanoic acid), saturated fatty acid (docosahexaenoic acid), and TAG (18:0-18:1-22:3 and 18:1-18:1-20:1) in leaves of white clover subjected to heat stress. To further elucidate potential association between Spd-regulated heat tolerance and lipids metabolism, lipidomics was conducted in wild or TrSAMS-transgenic Arabidopsis thaliana differing in endogenous Spd content. Results showed that TrSAMS-transgenic plants maintained relatively higher total lipids, total Phl, and total Spl contents as compared to the wild type under heat stress, which indicated that the change in endogenous Spd level indeed induced alteration in lipids metabolism. Heat stress triggers transition of membrane phase from a bilayer to non-bilayer resulting in hyper-fluidity of membranes due to a great deal of accumulation of unsaturated carbonyl groups in membranes [42]. Plants tend to decrease unsaturation levels of lipids to reduce lipid hyper-fluidity in favor of better maintenance of optimal membrane fluidity, integrity, and stability under high temperature, which is a common adaptation mechanism in plant species [43]. In addition, unsaturated lipids are easier to be oxidized than saturated ones under oxidative cellular environments such as high temperature condition [20]. A decline in lipid unsaturation level was advantageous to plants during a long-term heat stress, which has been proved in many plant species such as creeping bentgrass [44], Arabidopsis thaliana [45], rice [46], and soybean [23]. Unsaturation index of total lipids significantly increased in wild-type Arabidopsis thaliana but was kept on a normal level in TrSAMS-transgenic plants. OE4 also exhibited significantly lower unsaturation index of total Phl, Gll, and Spl than wild-type Arabidopsis in response to heat stress. These findings indicate that higher lipids saturation level regulated by Spd could be beneficial to maintain membrane stability and integrity in transgenic plants exposed to heat stress.

Phls are essential components of cellular membranes for forming lipid bilayers and also serve as secondary signaling molecules for stress signal transduction in plant cells exposed to high temperature stress [42]. Heat stress triggers PIP2 signaling pathway through activating key kinases including PI4K and PIP5K, as PI and PIP are two essential intermediates of the pathway. PA signaling generation mainly depends on two pathways: one is that structural Phls such as PC and PE are hydrolyzed by PLD to form the PA, and another is that the hydrolysates that are derived from hydrolytic reactions catalyzed by PLC are phosphorylated by DGK to produce the PA [47]. Both of PIP2 and PA are involved in regulations of membrane trafficking, organization cytoskeleton, gene expression, reactive oxygen species homeostasis, and hormonal response [48]. It has been found that thermotolerance could be enhanced significantly by the exogenous PA application associated with significant up-regulation of heat shock pathway in cool-season tall fescue [49]. Drought priming also improved heat tolerance of tall fescue by significantly increasing PI content throughout a period of heat stress [50]. Better adaption to heat stress could be achieved in heat-tolerant soybean genotype by up-regulating accumulation of PI [23]. In addition, a quick conversion of PI to PIP and then to PIP2 has been proved as one of the important adaptive responses to heat stress in plants [51]. Higher increases in PI, PIP, PIP2, and PA contents as well as transcript levels of genes (AtPI4K-alpha, AtPIP5K1, AtPLC5, AtDGK2, and AtPLD-delta) involved in PI and PA metabolism were detected in the OE4 as compared to wild-type Arabidopsis thaliana during heat stress in our current study, indicating Spd-regulated heat tolerance could be associated with the activation of PIP2 and PA signal pathways.

In addition to the importance of PIP2 and PA signaling pathways, other lipids such as PS, PC, PG, and CL also play critical roles in thermotolerance in plants. PS serves as a major constituent of interior surface of cell membranes involved in the maintenance of bilayer curvature and the biosynthesis of PC or PE [52,53]. PC is an abundant composition of cell membranes affecting membrane integrality and also involved in syntheses of PA, LPA, and LPC for the regulation of stress defensive responses in cells [42]. It is well known that the maintenance of higher PC content or ratio is an important strategy for plants adapting to heat stress due to its bilayer-forming characteristics [23,50]. As a main component of thylakoid membranes, PG exhibits positive role in stabilizing structure and function of chloroplasts. Significant increase in PG content conferred survival advantage in a transgenic wheat (Triticum aestivum) associated with less damage to thylakoid membrane and better photosynthetic capacity under heat stress [54]. Being different from the PG, the CL is mainly located in mitochondrial membranes and acts as a maintainer of functional integrity and dynamics of mitochondria in plants. Previous studies have proved that a loss-of-function cls Arabidopsis thaliana mutant which could not synthetize the CL exhibited impaired growth and was also more sensitive to heat stress than WT [55,56]. In response to heat stress, a significant increase in endogenous Spd content improved the accumulation of PS, PC, PG, and CL in TrSAMS-transgenic Arabidopsis thaliana, which could be more beneficial to maintain membrane integrity and functionality under heat stress. In addition, TrSAMS-transgenic plants also showed significantly higher levels of lysophospholipids (LPA, LPI, LPC, LPE, and LPG) than WT under heat stress. Research is increasingly focusing on investigating possible functions of these lysophospholipids in plants. For example, it has been demonstrated that the LPA, LPE, and LPC regulate signal transmission, wound response, pathogen infection, or vacuolar H+ pool in different plants [57,58,59,60]. However, their regulatory roles in thermotolerance are still unclear and deserve to be further investigated in plant species.

Changes in lipid composition and ratio affect the formation of lipid bilayers when plants respond to high temperature. One of the important thermal adaptabilities in plant species is a possible prevention of phase transition from bilayer membranes to non-bilayer ones [20]. Heat stress causes thylakoid membrane peroxidation resulting in the disruption of chloroplast function and a subsequent decline in photosynthesis [54]. DGDG and MGDG are two abundant thylakoid lipids in plants. Although both of them play vital roles in thylakoid membrane architecture, an increase in DGDG:MGDG ratio exhibits higher contributions to the improvement of thylakoid membrane stability under abiotic stress, because DGDG is known as a bilayer-forming lipid, but the MGDG exhibits a spontaneous tendency to form non-bilayer lipid phase [61,62]. It has been proved that decreased DGDG and MGDG contents led to declines in Chl content and photochemical efficiency [63]. Higher DGDG:MGDG ratio contributed to better stability of thylakoid membrane for photosynthesis in Arabidopsis thaliana subjected to heat stress [64]. Enhanced heat sensitivity in Arabidopsis thaliana mutation with impaired biosynthesis of DGDG has also been found in a previous study [64]. TrSAMS-transgenic Arabidopsis thaliana not only had significantly higher DGDG content, but also attempted to maintain higher DGDG:MGDG ratio than WT undergoing a long period of heat stress (10 d). This could be propitious to prevent stress-induced non-bilayer phase formation, helping to retain membrane stability and integrity under heat stress.

Unlike Phls and Glls, the function of Spls in relation to plant adaptability to abiotic stress is not well documented. Most of Spls such as Cer, Hex1Cer, and CerG2GNAc1 perform as structural components of cell membranes, and some of them also act as lipid signaling molecules such as Cer for regulating cellular metabolism and renewal [21,65]. It has been elucidated that the change in Cer content affected endogenous phytohormone levels and programmed cell death (PCD) in plants [66]. Diethyl aminoethyl hexanoate (DA-6) induced the improvement in drought tolerance associated with enhanced accumulation of total Spls, Cer, Hex1Cer, and CerG2GNAc1 in leaves of white clover [67]. As compared to WT, a significant increase in endogenous Spd content was accompanied by higher Cer, Hex1, and CerG2GNAc1 levels in TrSAMS-transgenic Arabidopsis thaliana during heat stress. However, how do these Glls as structural materials affect cell membrane stability and fluidity under heat stress? What are the main downstream defensive pathways regulated by these Glls contributing to thermotolerance in plants? These questions need to be verified by further study in plant species.

4. Materials and Methods

4.1. Plant Material and Treatments

For the cultivation and treatment of white clover (cv. Ladino), seeds germinated in quartz sands and distilled water for 8 days in a growth chamber, which provided normal growth condition (23/19 °C (day/night), 14 h photoperiod, 65% relative humidity, and 700 µmol·m−2·s−1 PAR). Seedlings were then cultivated in the Hoagland’s solution [68] for 12 days and then removed carefully from quartz sands and suspended through small holes in Styrofoam boards for 7 days of hydroponic cultivation in the Hoagland’s solution. For the Spd pretreatment, 27-days-old seedlings were cultivated in the Hoagland’s solution containing Spd (70 μmol/L) for 3 days. Non-pretreated seedlings were cultivated in Hoagland’s solution without the Spd for 3 days. Spd-pretreated and untreated seedlings were then placed in normal growth chambers (mentioned above) or high temperature growth chambers (38/33 °C (day/night), 14 h photoperiod, 65% relative humidity, and 700 µmol·m−2·s−1 PAR) for 20 days. All solutions were refreshed every day. Two transgenic lines (OE4 and OE5) overexpressing a TrSAMS cloned from the white clover (cv. Ladino) were selected as experimental materials [69], since these two lines exhibited significantly higher endogenous Spd content than wild type under normal condition and heat stress (Figure 3B). For the cultivation and treatment of Arabidopsis thaliana, wild type (Col-0) and transgenic lines were grown in pots (9.5 cm length, 9.5 cm width, and 9 cm height) with peat soil and irrigated weekly with half-strength Hoagland’s solution for 20 days in a growth chamber (21/18 °C (day/ night), 70% relative humidity, 16 h photoperiod, and 350 µmol·m−2·s−1 PAR). Plants were then moved into the high temperature chambers (38/33 °C (day/ night), 70% relative humidity, 16 h photoperiod, and 350 µmol·m−2·s−1 PAR) for 10 days or kept in the normal growth chambers for 10 days. All plants were completely arranged in growth chambers. Leaf samples were collected for six independent biologic replications per treatment and twenty plants per replication were used.

4.2. Growth and Physiological Measurements

Chl content was detected based on the method of Barnes et al. [70]. Fresh leaves (0.15 g) were cut from plants and soaked immediately in 15 mL of dimethylsulfoxide solution at room temperature for 48 h. The absorbance of extracts was determined using a spectrophotometer (Spectronic Instruments) at 663 nm and 645 nm. EL was detected using a conductivity meter (Model 32, Yellow Springs Instrument Company). The surface of fresh leaves (0.15 g) was cleaned carefully using deionized water and then immersed in 35 mL of deionized water for 24 h. The initial conductivity (Cinitial) of soaks was detected. Leaves were then autoclaved at 120 °C for 15 min to detect the maximum conductance (Cmax) of soaks. The EL was calculated as the percentage of Cinitial/Cmax [71]. For the determination of MDA content, fresh leaves (0.15 g) were ground with 2 mL of 50 mM cold PBS (pH 7.8) and the mixture was centrifuged at 10,000× g for 30 min. The supernatant (0.5 mL) was mixed with the reaction solution (1.0 mL, 20% w/v trichloroacetic acid and 0.5% w/v thiobarbituric acid) and then incubated at 95 °C for 15 min. After being centrifuged at 8000× g for 10 min, the absorbance of reaction solution was measured at 532 and 600 nm [72]. For PIABS and PSII Fv/Fm, a single layer of leaves was clipped for 30 min dark adaptation and readings of Fv/Fm and PIABS were recorded with the Chl fluorescence meter (Pocket PEA). SL, RL, LL, and LW were detected using 10 plants of each replicate and a total of 40 plants were used for each treatment. Fresh leaves were harvested, and FW of above-ground biomass was detected immediately. These leaves dried in an oven at 105 °C for 20 min and then at 70 °C for 3 days to get the DW of above-ground biomass.

4.3. Measurements of Endogenous Spermidine

Fresh leaves (0.2 g) were ground with 1.5 mL cold perchloric acid (5%, v:v) on the ice. After being incubated at 4 °C for 1 h, the homogenate was centrifuged at 10,000× g for 30 min (4 °C). Supernatant (0.5 mL) was mixed with 2 M NaOH (2 mL) and benzoyl chlorides (10 mL), and then the mixture was incubated at 37 °C for 30 min. Saturated NaCl solution (2 mL) and cold diethylether (2 mL) were added into the mixture in proper order and mixed by shaking. The supernatant ether phase (1 mL) was evaporated to dryness and re-dissolved in 1 mL of methanol for determination of Spd through high performance liquid chromatography (HPLC, Agilent-1200, Agilent Technologies, Santa Clara, CA, USA). Benzoyl Spd extract (20 µL) was loaded onto a reverse-phase Tigerkin®C18 column (150 mm × 4.6 mm, and 5 µm particle size). Mobile phase was methanol–H2O (64:36, v:v) and column temperature was maintained at 25 °C. The Spd peak was monitored with a UV detector at 254 nm with a flow rate of 1 mL/min [73].

4.4. Metabolomics Analysis

For metabolites extraction, leaf tissues (100 mg) were ground with 500 µL of prechilled solution containing 80% methanol and 0.1% formic acid. After being incubated on the ice for 5 min, the homogenate was centrifuged at 12,000× g for 10 min (4 °C). The supernatant was diluted by hyperpure water to final concentration containing 53% methanol and then transferred to a fresh tube for the centrifugation (15,000 g, 4 °C, and 10 min). Finally, the supernatant was injected into the LC-MS/MS system, which is a Vanquish UHPLC system (Thermo Fisher, Waltham, MA, USA) coupled with an Orbitrap Q Exactive series mass spectrometer (Thermo Fisher, Waltham, MA, USA). A Hyperil Gold column (C18, 100 mm × 2.1 mm, and 1.9 µm particle size) was used (a flow rate of 0.2 mL/min, 0.1% formic acid in water, and methanol). Mass spectrometers of Q Exactive series were operated in positive/negative polarity mode with spray voltage of 3.2 kV, capillary temperature of 320 °C, sheath gas flow rate of 35 arb, and aux gas flowrate of 10 arb. Raw data were processed using the Compound Discoverer 3.1 (Thermo Fisher, Waltham, MA, USA) to perform peak alignment, peak picking, and quantitation for each metabolite. Normalized data were used to predict the molecular formula based on additive ions, molecular ion peaks, and fragment ions. Peaks were matched with the mzCloud (https://www.mzcloud.org/), mzVault, and MassList database to obtain accurate qualitative and relative quantitative results. Metabolic pathways of identified metabolites were annotated using the KEGG database (http://www.genome.jp/kegg/) and Lipidmaps database (http://www.lipidmaps.org/). All metabolites with p-value < 0.05 and fold change ≥ 2.0 (significant increase) or ≤ 0.5 (significant decrease) were identified as DAMs.

4.5. Lipidomics Analysis

For lipids extraction, fresh leaves (0.15 g) were lyophilized in a Freeze Dryer for 3 days until consistent weight. Lyophilized leaves were ground into fine powders on the ice. Fine powders (20 mg) were mixed with 300 µL of chloroform and methanol (v:v, 2:1) and then shaken for 30 min. The mixture was centrifuged for 20 min at 12,000× g and the supernatant was removed into a new centrifuge tube. The isopropanol (300 µL) was added into the residues and then shaken for 30 min again. After being centrifuged at 12,000× g for 20 min, the supernatant was taken out and mixed with the previous supernatant uniformly. The mixed supernatant (200 µL) was used for the analysis of lipids by using a Vanquish UHPLC/Q Exactive Plus (Thermo Fisher, Waltham, MA, USA). Column (ACQUITY UPLC BEH C18, 100 × 2.1 mm, and 1.7 μm) temperature was maintained at 55 °C. For the mobile phase A, acetonitrile/water (60:40) with 10 mM ammonium formate and 0.1% formic acid were used. Isopropanol/acetonitrile (90:10) with 10 mM ammonium formate and 0.1% formic acid were used as the mobile phase B. Gradient or flow rate was set to 95/5~0/100 in 17 min or 0.4 mL/min, respectively. Data were acquired by software Xcalibur and identified by Lipidsearch 4.2 (Thermo Fisher, Waltham, MA, USA). Unsaturation index of total lipids, Phls, Glls, or Spls was calculated according to the formula (n × mol% lipid)/100 (n indicates the number of double bonds in the lipid molecular species and mol% indicates the composition percentage of individual lipid molecular species) [74].

4.6. Gene Expression Analysis

Transcript levels of genes involved in PI and PA metabolism including AtPI4K-alpha, AtPIP5K1, AtPLC5, AtDGK2, and AtPLD-delta were detected using real-time quantitative polymerase chain reaction (qRT-PCR). Total RNAs were extracted from fresh leaves of wild type and transgenic plants using RNeasy Mini Kit (Tiangen, Beijing, China) and then reverse-transcribed to cDNAs using a Revert Aid First Stand cDNA Synthesis Kit (Fermentas, Beijing, China). Primers were recorded in Supplementary Table S1 and AtACT2 was used as the reference gene [75,76,77,78,79]. Operation steps of qRT-PCR included 5 min at 94 °C, denaturation at 95 °C for 30 s (40 repeats), annealing at 58–61 °C for 30 s, and extension at 72 °C for 30 s (Supplementary Table S1). The formula 2−ΔΔCT in the study of Livak and Schmittgen was used to calculate transcript levels of genes [80].

4.7. Statistical Analysis

PCA and variances of growth, physiological, and lipidomics parameters were analyzed using a statistical program (SPSS v20.0, IBM, Amunk, NY, USA). Differences among treatment means were tested using Fisher’s protected least significance (LSD) test at a 0.05 probability level. HSI was used to evaluate relative change of lipids under heat stress condition (3 h or 10 d) as compared to normal condition (0 h) based on the formula: HSI = (value of parameter under heat stress)/(value of parameter under normal condition) × 100 [81].

5. Conclusions

In conclusion, significant increase in endogenous Spd content through exogenous application of Spd or TrSAMS overexpression could effectively alleviate heat-induced growth retardation, oxidative damage to lipids, and declines in photochemical efficiency and cell membrane stability. The analysis of metabolomics showed that the main metabolic pathways were regulated by Spd in white clover including the amino acids and vitamins metabolism, biosynthesis of secondary metabolites, and lipids metabolism under high temperature condition. TrSAMS-transgenic Arabidopsis thaliana up-regulated PIP2 and PA signaling pathways and other Phls (PS, PG, PC, and CL) levels associated with better maintenance of membrane integrity and functionality in response to heat stress. In addition, TrSAMS-transgenic Arabidopsis thaliana also could maintain higher DGDG level and DGDG:MGDG ratio, which was beneficial to retain bilayer phase contributing to thylakoid membrane stability and integrity undergoing a long period of heat stress.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms232012247/s1.

Click here for additional data file. (465.1KB, zip)

Author Contributions

Z.L., Y.P. and X.Z. designed experiments and improved the manuscript; Y.Z. (Yue Zhao), Z.L., L.L. and B.C. performed experiments; Z.L., B.C. and Y.Z. (Yue Zhao) wrote the manuscript; Y.Z. (Yan Zhang), G.F., L.H., Y.P. and X.Z. improved the manuscript. Y.P. and X.Z. provided materials and editorial advice to improve the manuscript. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Conflicts of Interest

Authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Funding Statement

This research was supported by the earmarked fund for CARS (CARS-34) and China Postdoctoral Science Foundation (2021T140058).

Footnotes

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

References

  • 1.Kotak S., Larkindale J., Lee U., von Koskull-Döring P., Vierling E., Scharf K.D. Complexity of the heat stress response in plants. Curr. Opin. Plant Biol. 2007;10:310–316. doi: 10.1016/j.pbi.2007.04.011. [DOI] [PubMed] [Google Scholar]
  • 2.Gourdji S.M., Sibley A.M., Lobell D.B. Global crop exposure to critical high temperatures in the reproductive period: Historical trends and future projections. Environ. Res. Lett. 2013;8:024041. doi: 10.1088/1748-9326/8/2/024041. [DOI] [Google Scholar]
  • 3.Bokszczanin K. Perspectives on deciphering mechanisms underlying plant heat stress response and thermotolerance. Front. Plant. 2013;4:315. doi: 10.3389/fpls.2013.00315. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Takashi H., Katsutomo T., Ken-Ichi S., Yasuyuki Y. Molecular cloning of plant spermidine synthases. Plant Cell Physiol. 1998;39:73–79. doi: 10.1093/oxfordjournals.pcp.a029291. [DOI] [PubMed] [Google Scholar]
  • 5.de Sousa Araújo S., dos Santos A.L.W., Duque A.S. Engineering polyamine metabolic pathways for abiotic stress tolerance in plants. In: Hossain M.A., Kumar V., Burritt D.J., Fujita M., Mäkelä P.S.A., editors. Osmoprotectant-Mediated Abiotic Stress Tolerance in Plants: Recent Advances and Future Perspectives. Springer International Publishing; Cham, Switzerland: 2019. pp. 287–318. [DOI] [Google Scholar]
  • 6.Zhang Y., Li Z., Li Y.P., Zhang X.Q., Ma X., Huang L.K., Yan Y.H., Peng Y. Chitosan and spermine enhance drought resistance in white clover, associated with changes in endogenous phytohormones and polyamines, and antioxidant metabolism. Funct. Plant Biol. 2018;45:1205–1222. doi: 10.1071/FP18012. [DOI] [PubMed] [Google Scholar]
  • 7.Jahan M.S., Shu S., Wang Y., Chen Z., He M., Tao M., Sun J., Guo S. Melatonin alleviates heat-induced damage of tomato seedlings by balancing redox homeostasis and modulating polyamine and nitric oxide biosynthesis. BMC Plant Biol. 2019;19:414. doi: 10.1186/s12870-019-1992-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 8.Li S., Jin H., Zhang Q. The Effect of exogenous spermidine concentration on polyamine metabolism and salt tolerance in Zoysiagrass (Zoysia japonica Steud) subjected to short-term salinity stress. Front. Plant Sci. 2016;7:1221. doi: 10.3389/fpls.2016.01221. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Tang S., Zhang H., Li L., Liu X., Chen L., Chen W., Ding Y. Exogenous spermidine enhances the photosynthetic and antioxidant capacity of rice under heat stress during early grain-filling period. Funct. Plant Biol. 2018;45:911–921. doi: 10.1071/FP17149. [DOI] [PubMed] [Google Scholar]
  • 10.Fu Y., Gu Q., Dong Q., Zhang Z., Lin C., Hu W., Pan R., Guan Y., Hu J. Spermidine enhances heat tolerance of rice seeds by modulating endogenous starch and polyamine metabolism. Molecules. 2019;24:1395. doi: 10.3390/molecules24071395. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Cheng L., Zou Y., Ding S., Zhang J., Yu X., Cao J., Gang L. Polyamine accumulation in transgenic tomato enhances the tolerance to high temperature stress. J. Integr. Plant Biol. 2010;51:489–499. doi: 10.1111/j.1744-7909.2009.00816.x. [DOI] [PubMed] [Google Scholar]
  • 12.Cheng L., Sun R.R., Wang F.Y., Peng Z., Kong F.L., Wu J., Cao J.S., Lu G. Spermidine affects the transcriptome responses to high temperature stress in ripening tomato fruit. J. Zhejiang Univ. SCIENCE B. 2012;13:283–297. doi: 10.1631/jzus.B1100060. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Liang L.L., Cao Y.Q., Wang D., Peng Y., Zhang Y., Li Z. Spermine alleviates heat-induced senescence in creeping bentgrass by regulating water and oxidative balance, photosynthesis, and heat shock proteins. Biol. Plant. 2021;65:184–192. doi: 10.32615/bp.2021.008. [DOI] [Google Scholar]
  • 14.Luo L., Li Z., Tang M.Y., Cheng B.Z., Zeng W.H., Peng Y., Nie G., Zhang X.Q. Metabolic regulation of polyamines and γ-aminobutyric acid in relation to spermidine-induced heat tolerance in white clover. Plant Biol. 2020;22:794–804. doi: 10.1111/plb.13139. [DOI] [PubMed] [Google Scholar]
  • 15.Ali B., Qian P., Jin R., Ali S., Khan M., Aziz R., Tian T., Zhou W. Physiological and ultra-structural changes in Brassica napus seedlings induced by cadmium stress. Biol. Plant. 2014;58:131–138. doi: 10.1007/s10535-013-0358-5. [DOI] [Google Scholar]
  • 16.Ali B., Gill R.A., Yang S., Gill M.B., Ali S., Rafiq M.T., Zhou W. Hydrogen sulfide alleviates cadmium-induced morpho-physiological and ultrastructural changes in Brassica napus. Ecotoxicol. Environ. Saf. 2014;110:197–207. doi: 10.1016/j.ecoenv.2014.08.027. [DOI] [PubMed] [Google Scholar]
  • 17.Qi H., Kang D., Zeng W., Jawad Hassan M., Peng Y., Zhang X., Zhang Y., Feng G., Li Z. Alterations of endogenous hormones, antioxidant metabolism, and aquaporin gene expression in relation to γ-aminobutyric acid-regulated thermotolerance in white clover. Antioxidants. 2021;10:1099. doi: 10.3390/antiox10071099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 18.Ali B., Gill R.A., Yang S., Gill M.B., Farooq M.A., Liu D., Daud M.K., Ali S., Zhou W. Regulation of cadmium-induced proteomic and metabolic changes by 5-aminolevulinic acid in leaves of Brassica napus L. PLoS ONE. 2015;10:e0123328. doi: 10.1371/journal.pone.0123328. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Watson A.D. Thematic review series: Systems biology approaches to metabolic and cardiovascular disorders. Lipidomics: A global approach to lipid analysis in biological systems. J. Lipid Res. 2006;47:2101–2111. doi: 10.1194/jlr.R600022-JLR200. [DOI] [PubMed] [Google Scholar]
  • 20.Liu X., Ma D., Zhang Z., Wang S., Du S., Deng X., Yin L. Plant lipid remodeling in response to abiotic stresses. Environ. Exp. Bot. 2019;165:174–184. doi: 10.1016/j.envexpbot.2019.06.005. [DOI] [Google Scholar]
  • 21.Orešič M., Hänninen V.A., Vidal-Puig A. Lipidomics: A new window to biomedical frontiers. Trends Biotechnol. 2008;26:647–652. doi: 10.1016/j.tibtech.2008.09.001. [DOI] [PubMed] [Google Scholar]
  • 22.Hsu F.F. Mass spectrometry-based shotgun lipidomics—A critical review from the technical point of view. Anal. Bioanal. Chem. 2018;410:6387–6409. doi: 10.1007/s00216-018-1252-y. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Narayanan S., Zoong Lwe Z., Gandhi N., Welti R., Fallen B., Smith J., Rustgi S. Comparative lipidomic analysis reveals heat stress responses of two soybean genotypes differing in temperature sensitivity. Plants. 2020;9:457. doi: 10.3390/plants9040457. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Higashi Y., Okazaki Y., Myouga F., Shinozaki K., Saito K. Landscape of the lipidome and transcriptome under heat stress in Arabidopsis thaliana. Sci. Rep. 2015;5:10533. doi: 10.1038/srep10533. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Dureja-Munjal I., Acharya M.K., Guha-Mukherjee S. Effect of hormones and spermidine on the turnover of inositolphospholipids in Brassica seedlings. Phytochemistry. 1992;31:1161–1163. doi: 10.1016/0031-9422(92)80252-A. [DOI] [Google Scholar]
  • 26.Radhakrishnan R., Lee I.J. Regulation of salicylic acid, jasmonic acid and fatty acids in cucumber (Cucumis sativus L.) by spermidine promotes plant growth against salt stress. Acta Physiol. Plant. 2013;35:3315–3322. doi: 10.1007/s11738-013-1364-0. [DOI] [Google Scholar]
  • 27.Jagadish S.V.K., Way D.A., Sharkey T.D. Plant heat stress: Concepts directing future research. Plant Cell Environ. 2021;44:1992–2005. doi: 10.1111/pce.14050. [DOI] [PubMed] [Google Scholar]
  • 28.Mostofa M.G., Yoshida N., Fujita M. Spermidine pretreatment enhances heat tolerance in rice seedlings through modulating antioxidative and glyoxalase systems. Plant Growth Regul. 2014;73:31–44. doi: 10.1007/s10725-013-9865-9. [DOI] [Google Scholar]
  • 29.Hildebrandt T.M. Synthesis versus degradation: Directions of amino acid metabolism during Arabidopsis abiotic stress response. Plant Mol. Biol. 2018;98:121–135. doi: 10.1007/s11103-018-0767-0. [DOI] [PubMed] [Google Scholar]
  • 30.Li Z., Yu J., Peng Y., Huang B. Metabolic pathways regulated by γ-aminobutyric acid (GABA) contributing to heat tolerance in creeping bentgrass (Agrostis stolonifera) Sci. Rep. 2016;6:30338. doi: 10.1038/srep30338. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 31.Takeuchi S., Kono Y., Kawarada A., Ota Y., Nakayama M. Nicotinamide as a plant growth regulator isolated from rice hulls. Agric. Biol. Chem. 1975;39:859–861. doi: 10.1080/00021369.1975.10861684. [DOI] [Google Scholar]
  • 32.Hassanein R.A., Bassiouny F., Barakat D.M., Khalil R. Physiological effects of nicotinamide and ascorbic acid on Zea mays plant grown under salinity stress. I-Changes in growth, some relevant metabolic activities and oxidative defense systems. Res. J. Agric. Biol. Sci. 2009;31:72–80. [Google Scholar]
  • 33.Abdelhamid M.T., Sadak M.S., Schmidhalter U.R.S., El-Saady A.K.M. Interactive effects of salinity stress and nicotinamide on physiological and biochemical parameters of faba bean plant. Acta Biológica Colomb. 2013;18:499–509. [Google Scholar]
  • 34.Colinas M., Eisenhut M., Tohge T., Pesquera M., Fernie A., Weber A., Fitzpatrick T. Balancing of B6 vitamers is essential for plant development and metabolism in Arabidopsis. Plant Cell. 2016;28:439–453. doi: 10.1105/tpc.15.01033. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Vanderschuren H., Boycheva S., Li K.T., Szydlowski N., Gruissem W., Fitzpatrick T.B. Strategies for vitamin B6 biofortification of plants: A dual role as a micronutrient and a stress protectant. Front. Plant Sci. 2013;4:143. doi: 10.3389/fpls.2013.00143. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Larkindale J., Knight M.R. Protection against heat stress-induced oxidative damage in Arabidopsis involves calcium, abscisic acid, ethylene, and salicylic acid. Plant Physiol. 2002;128:682–695. doi: 10.1104/pp.010320. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 37.Ding W., Song L., Wang X., Bi Y. Effect of abscisic acid on heat stress tolerance in the calli from two ecotypes of Phragmites communis. Biol. Plant. 2010;54:607–613. doi: 10.1007/s10535-010-0110-3. [DOI] [Google Scholar]
  • 38.Thussagunpanit J., Jutamanee K., Kaveeta L., Chai-arree W., Pankean P., Suksamrarn A. Effects of a brassinosteroid and an ecdysone analogue on pollen germination of rice under heat stress. J. Pestic. Sci. 2013;38:105–111. doi: 10.1584/jpestics.D13-029. [DOI] [Google Scholar]
  • 39.Qian Y., Zhu C., Xia K., Gan L. Effects of methyl dihydrojasmonate on seedling growth and drought resistance in tall fescue. J. Nanjing Agric. Univ. 2009;32:47–51. [Google Scholar]
  • 40.He M., Ding N.Z. Plant unsaturated fatty acids: Multiple roles in stress response. Front. Plant Sci. 2020;11:562785. doi: 10.3389/fpls.2020.562785. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 41.Hu T., Sun X.Y., Zhao Z.J., Amombo E., Fu J.M. High temperature damage to fatty acids and carbohydrate metabolism in tall fescue by coupling deep transcriptome and metabolome analysis. Ecotoxicol. Environ. Saf. 2020;203:110943. doi: 10.1016/j.ecoenv.2020.110943. [DOI] [PubMed] [Google Scholar]
  • 42.Higashi Y., Saito K. Lipidomic studies of membrane glycerolipids in plant leaves under heat stress. Prog. Lipid Res. 2019;75:100990. doi: 10.1016/j.plipres.2019.100990. [DOI] [PubMed] [Google Scholar]
  • 43.Abdelrahman M., Ishii T., El-Sayed M., Tran L.S.P. Heat sensing and lipid reprograming as a signaling switch for heat stress responses in wheat. Plant Cell Physiol. 2020;61:1399–1407. doi: 10.1093/pcp/pcaa072. [DOI] [PubMed] [Google Scholar]
  • 44.Larkindale J., Huang B. Changes of lipid composition and saturation level in leaves and roots for heat-stressed and heat-acclimated creeping bentgrass (Agrostis stolonifera) Environ. Exp. Bot. 2004;51:57–67. doi: 10.1016/S0098-8472(03)00060-1. [DOI] [Google Scholar]
  • 45.Routaboul J.M., Skidmore C., Wallis J.G., Browse J. Arabidopsis mutants reveal that short- and long-term thermotolerance have different requirements for trienoic fatty acids. J. Exp. Bot. 2011;63:1435–1443. doi: 10.1093/jxb/err381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 46.Navarro-Reig M., Tauler R., Iriondo-Frias G., Jaumot J. Untargeted lipidomic evaluation of hydric and heat stresses on rice growth. J. Chromatogr. B Anal. Technol. Biomed. Life Sci. 2019;1:148–156. doi: 10.1016/j.jchromb.2018.11.018. [DOI] [PubMed] [Google Scholar]
  • 47.Heilmann I. Phosphoinositide signaling in plant development. Development. 2016;143:2044–2055. doi: 10.1242/dev.136432. [DOI] [PubMed] [Google Scholar]
  • 48.Hou Q., Ufer G., Bartels D. Lipid signalling in plant responses to abiotic stress. Plant Cell Environ. 2016;39:1029–1048. doi: 10.1111/pce.12666. [DOI] [PubMed] [Google Scholar]
  • 49.Wang X., Huang B. Lipid- and calcium-signaling regulation of HsfA2c-mediated heat tolerance in tall fescue. Environ. Exp. Bot. 2017;136:59–67. doi: 10.1016/j.envexpbot.2017.01.008. [DOI] [Google Scholar]
  • 50.Zhang X., Xu Y., Huang B. Lipidomic reprogramming associated with drought stress priming-enhanced heat tolerance in tall fescue (Festuca arundinacea) Plant Cell Environ. 2019;42:947–958. doi: 10.1111/pce.13405. [DOI] [PubMed] [Google Scholar]
  • 51.Liu Y.N., Lu X.X., Ren A., Shi L., Zhu J., Jiang A.L., Yu H.S., Zhao M.W. Conversion of phosphatidylinositol (PI) to PI4-phosphate (PI4P) and then to PI(4, 5)P2 is essential for the cytosolic Ca2+ concentration under heat stress in Ganoderma lucidum. Environ. Microbiol. 2018;20:2456–2468. doi: 10.1111/1462-2920.14254. [DOI] [PubMed] [Google Scholar]
  • 52.Vincent P., Maneta-Peyret L., Cassagne C., Moreau P. Phosphatidylserine delivery to endoplasmic reticulum-derived vesicles of plant cells depends on two biosynthetic pathways. FEBS Lett. 2001;498:32–36. doi: 10.1016/S0014-5793(01)02475-9. [DOI] [PubMed] [Google Scholar]
  • 53.Vance J.E., Steenbergen R. Metabolism and functions of phosphatidylserine. Prog. Lipid Res. 2005;44:207–234. doi: 10.1016/j.plipres.2005.05.001. [DOI] [PubMed] [Google Scholar]
  • 54.Wang G., Li F., Zhang J., Zhao M., Hui Z., Wang W. Overaccumulation of glycine betaine enhances tolerance of the photosynthetic apparatus to drought and heat stress in wheat. Photosynthetica. 2010;48:30–41. doi: 10.1007/s11099-010-0006-7. [DOI] [Google Scholar]
  • 55.Pineau B., Bourge M., Marion J., Mauve C., Gilard F., Maneta-Peyret L., Moreau P., Satiat-Jeunemaître B., Brown S.C., De Paepe R. The importance of cardiolipin synthase for mitochondrial ultrastructure, respiratory function, plant development, and stress responses in Arabidopsis. Plant Cell. 2013;25:4195–4208. doi: 10.1105/tpc.113.118018. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 56.Pan R., Jones A.D., Hu J. Cardiolipin-mediated mitochondrial dynamics and stress response in Arabidopsis. Plant Cell. 2014;26:391–409. doi: 10.1105/tpc.113.121095. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 57.Lee S., Suh S., Kim S., Crain R.C., Kwak J.M., Nam H.G., Lee Y. Systemic elevation of phosphatidic acid and lysophospholipid levels in wounded plants. Plant J. 1997;12:547–556. doi: 10.1046/j.1365-313X.1997.00547.x. [DOI] [Google Scholar]
  • 58.Wi S.J., yeon Seo S., Cho K., Nam M.H., Park K.Y. Lysophosphatidylcholine enhances susceptibility in signaling pathway against pathogen infection through biphasic production of reactive oxygen species and ethylene in tobacco plants. Phytochemistry. 2014;104:48–59. doi: 10.1016/j.phytochem.2014.04.009. [DOI] [PubMed] [Google Scholar]
  • 59.Viehweger K., Dordschbal B., Roos W. Elicitor-activated phospholipase A2 generates lysophosphatidylcholines that mobilize the vacuolar H+ pool for pH signaling via the activation of Na+-dependent proton fluxes. Plant Cell. 2002;14:1509–1525. doi: 10.1105/tpc.002329. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 60.Lee B.H., Choi S.H., Kim H.J., Jung S.W., Kim H.K., Nah S.Y. Plant lysophosphatidic acids: A rich source for bioactive lysophosphatidic acids and their pharmacological applications. Biol. Pharm. Bull. 2016;39:156–162. doi: 10.1248/bpb.b15-00575. [DOI] [PubMed] [Google Scholar]
  • 61.Chen D., Wang S., Qi L., Yin L., Deng X. Galactolipid remodeling is involved in drought-induced leaf senescence in maize. Environ. Exp. Bot. 2018;150:57–68. doi: 10.1016/j.envexpbot.2018.02.017. [DOI] [Google Scholar]
  • 62.Narayanan S., Tamura P.J., Roth M.R., Prasad P.V., Welti R. Wheat leaf lipids during heat stress: I. High day and night temperatures result in major lipid alterations. Plant Cell Environ. 2016;39:787–803. doi: 10.1111/pce.12649. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 63.Dörmann P., Benning C. Galactolipids rule in seed plants. Trends Plant Sci. 2002;7:112–118. doi: 10.1016/S1360-1385(01)02216-6. [DOI] [PubMed] [Google Scholar]
  • 64.Chen J., Burke J.J., Xin Z., Xu C., Velten J. Characterization of the Arabidopsis thermosensitive mutant atts02 reveals an important role for galactolipids in thermotolerance. Plant Cell Environ. 2006;29:1437–1448. doi: 10.1111/j.1365-3040.2006.01527.x. [DOI] [PubMed] [Google Scholar]
  • 65.Michaelson L.V., Napier J.A., Molino D., Faure J.D. Plant sphingolipids: Their importance in cellular organization and adaption. Biochim. Et Biophys. Acta (BBA)-Mol. Cell Biol. Lipids. 2016;1861:1329–1335. doi: 10.1016/j.bbalip.2016.04.003. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 66.Berkey R., Bendigeri D., Xiao S. Sphingolipids and plant defense/disease: The “death” connection and beyond. Front. Plant Sci. 2012;3:68. doi: 10.3389/fpls.2012.00068. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 67.Hassan M.J., Qi H., Cheng B., Hussain S., Peng Y., Liu W., Feng G., Zhao J., Li Z. Enhanced adaptability to limited water supply regulated by diethyl aminoethyl hexanoate (DA-6) associated with lipidomic reprogramming in two white clover genotypes. Front. Plant Sci. 2022;13:879331. doi: 10.3389/fpls.2022.879331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 68.Hogland C., Arnon D. The solution-culture method for growing plants without soil. Calif. Agric. Exp. Circ. 1950;247:16–21. [Google Scholar]
  • 69.Li Z., Hou J., Zhang Y., Zeng W., Cheng B., Hassan M., Zhang Y., Pu Q., Peng Y. Spermine regulates water balance associated with Ca2+-dependent aquaporin (TrTIP2-1, TrTIP2-2 and TrPIP2-7) expression in plants under water stress. Plant Cell Physiol. 2020;61:1576–1589. doi: 10.1093/pcp/pcaa080. [DOI] [PubMed] [Google Scholar]
  • 70.Barnes J.D., Balaguer L., Manrique E., Elvira S., Davison A.W. A reappraisal of the use of DMSO for the extraction and determination of chlorophylls a and b in lichens and higher plants. Environ. Exp. Bot. 1992;32:85–100. doi: 10.1016/0098-8472(92)90034-Y. [DOI] [Google Scholar]
  • 71.Blum A., Ebercon A. Cell membrane stability as a measure of drought and heat tolerance in wheat. Crop Sci. 1981;21:43–47. doi: 10.2135/cropsci1981.0011183X002100010013x. [DOI] [Google Scholar]
  • 72.Dhindsa R., Plumb-Dhindsa P., Thorpe T. Leaf senescence: Correlated with increased levels of membrane permeability and lipid peroxidation, and decreased levels of superoxide dismutase and catalase. J. Exp. Bot. 1981;32:93–101. doi: 10.1093/jxb/32.1.93. [DOI] [Google Scholar]
  • 73.Duan J., Li J., Guo S., Kang Y. Exogenous spermidine affects polyamine metabolism in salinity-stressed Cucumis sativus roots and enhances short-term salinity tolerance. J. Plant Physiol. 2008;165:1620–1635. doi: 10.1016/j.jplph.2007.11.006. [DOI] [PubMed] [Google Scholar]
  • 74.Su K., Bremer D.J., Jeannotte R., Welti R., Yang C. Membrane lipid composition and heat tolerance in cool-season turfgrasses, including a hybrid bluegrass. J. Am. Soc. Hortic. Sci. 2009;134:511–520. doi: 10.21273/JASHS.134.5.511. [DOI] [Google Scholar]
  • 75.Gutierrez L., Mauriat M., Guénin S., Pelloux J., Lefebvre J.F., Louvet R., Rusterucci C., Moritz T., Guerineau F., Bellini C., et al. The lack of a systematic validation of reference genes: A serious pitfall undervalued in reverse transcription-polymerase chain reaction (RT-PCR) analysis in plants. Plant Biotechnol. J. 2008;6:609–618. doi: 10.1111/j.1467-7652.2008.00346.x. [DOI] [PubMed] [Google Scholar]
  • 76.Gómez-Merino F.C., Arana-Ceballos F.A., Trejo-Téllez L.I., Skirycz A., Brearley C.A., Dörmann P., Mueller-Roeber B. Arabidopsis AtDGK7, the smallest member of plant diacylglycerol kinases (DGKs), displays unique biochemical features and saturates at low substrate concentration: The DGK inhibitor R59022 differentially affects AtDGK2 and AtDGK7 activity in vitro and alters plant growth and development. J. Biol. Chem. 2005;280:34888–34899. doi: 10.1074/jbc.M506859200. [DOI] [PubMed] [Google Scholar]
  • 77.Vaz Dias F., Serrazina S., Vitorino M., Marchese D., Heilmann I., Godinho M., Rodrigues M., Malhó R. A role for diacylglycerol kinase 4 in signalling crosstalk during Arabidopsis pollen tube growth. New Phytol. 2019;222:1434–1446. doi: 10.1111/nph.15674. [DOI] [PubMed] [Google Scholar]
  • 78.Wada Y., Kusano H., Tsuge T., Aoyama T. Phosphatidylinositol phosphate 5-kinase genes respond to phosphate deficiency for root hair elongation in Arabidopsis thaliana. Plant J. 2015;81:426–437. doi: 10.1111/tpj.12741. [DOI] [PubMed] [Google Scholar]
  • 79.Hunt L., Otterhag L., Lee J., Lasheen T., Hunt J., Seki M., Shinozaki K., Sommarin M., Gilmour D., Pical C. Gene-specific expression and calcium activation of Arabidopsis thaliana phospholipase C isoforms. New Phytol. 2004;162:643–654. doi: 10.1111/j.1469-8137.2004.01069.x. [DOI] [PubMed] [Google Scholar]
  • 80.Livak K.J., Schmittgen T.D. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods. 2001;25:402–408. doi: 10.1006/meth.2001.1262. [DOI] [PubMed] [Google Scholar]
  • 81.Bouslama M., Schapaugh W.T., Jr. Stress tolerance in soybeans. I. Evaluation of three screening techniques for heat and drought tolerance. Crop Sci. 1984;24:933–937. doi: 10.2135/cropsci1984.0011183X002400050026x. [DOI] [Google Scholar]

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