Shengnn Ge,Debin Liu,Min Chu,Xinyu Liu,Yulei Wei,Xinyng Che,Lei Zhu,Lin He,*,Jingyu Xu,b,*

a Key Laboratory of Modern Agricultural Cultivation and Crop Germplasm Improvement of Heilongjiang Province/Key Laboratory of Low Carbon Green Agriculture in Northeast Plain,Ministry of Agriculture and Rural Affairs,China/Research Center of Saline and Alkali Land Improvement Engineering Technology in Heilongjiang Province/College of Agriculture,Heilongjiang Bayi Agricultural University,Daqing 163319,Heilongjiang,China

b National Coarse Cereal Engineering Research Center,Daqing 163319,Heilongjiang,China

Keywords:Sorghum(Sorghum bicolor L.Moench)Membrane lipid remodeling Lipidome Transcriptome Salt stress

ABSTRACT Salt stress limits plant growth and development.In this study,changes in membrane lipids were investigated in leaves of sorghum seedlings subjected to salt stress(150 mmol L-1 NaCl).Galactolipids(DGDG and MGDG)accounted for more than 65%of the total glycerolipids in sorghum leaves.The predominance of C36 molecular species in MGDG suggested that sorghum is an 18:3 plant.Under NaCl treatment,the content of major phospholipids(PC and PE)increased,accompanied by the activation of their metabolism pathways at the transcriptional level.In contrast,the proportion of MGDG and PG dropped drastically,leading to a decreased ratio of plastidic to non-plastidic lipids.An adjustment of glycerolipid pathway between the cytosolic and plastidic compartments was triggered by salt stress,as reflected by the increased conversion of PC to PA,providing precursors for galactolipid synthesis.The elevated DGDG resulted in increased DGDG/MGDG and bilayer/non-bilayer lipid ratios.The double-bond index of PC,PE,and DGDG increased markedly,evidently owing to the increased expression of FAD3 and FAD8.These findings will be helpful for understanding dynamic membrane lipid changes and adaptive lipid remodeling in sorghum response to salt stress.

1.Introduction

Soil salinization is one of the main environmental stresses that limit plant growth and production,and is an increasingly severe global problem[1].More than 800 million hectares of land worldwide are affected by salt stress,and this situation will continue to worsen[2].

Plant membrane lipids are classified into three main categories:sphingolipids,sterols,and glycerolipids[3].Glycerolipids are the most abundant and the main structural components of cell membranes[4].Membrane glycerolipids bind two fatty acids in the glycerol backbone at the first and second acyl(sn-1 and-2)positions,and condense with phosphorus(phospholipids)or sugar(glycolipid)molecules at the sn-3 site[5].In plant cells,the major phospholipids are phosphatidylcholine(PC)and phosphatidylethanolamine(PE),together with phosphatidylserine(PS),phosphatidylinositol(PI)and phosphatidic acid(PA),constituting the extra-plastidic membrane lipids[6].In plastids,monogalactosediacylglycerol (MGDG), digalactosyldiacylglycerol(DGDG),sulfoquinovosyldiacylglycerol(SQDG),and the only plastid-evolved phospholipid phosphatidylglycerol(PG),form the plastidic bilayer membrane[7].In the green tissues of plants,the dominant membrane lipids are MGDG and DGDG,which strongly influence plant chloroplast(and thylakoid)structure and photosynthetic characteristics[8].

In plant cells,the synthesis of glycerolipids can be completed via two distinct pathways compartmentalized in cytoplasmic or plastids.The pathway located in the cytoplasmic/endoplasmic reticulum(ER)is called the eukaryotic pathway;the other is implemented in plastids and is referred to as the prokaryotic pathway[9].In the eukaryotic pathway,galactolipids carry C18 fatty acids at the sn-2 position owing the specificity of the acyltransferase,whereas prokaryoticpathway-generated galactolipids carry only C16 fatty acids at the sn-2 position[9].These two pathways work differently among plant species.In Arabidopsis,the synthesis of galactolipids is jointly accomplished by the eukaryotic and prokaryotic pathways,and the major product MGDG contains similar proportions of C34 and C36 fatty acid molecular species,so that this plant is called a 16:3 plant[10].In some plant species,known as 18:3 plants[11],the synthesis of galactolipids depends almost entirely on the eukaryotic pathway,leading to a predominance of C36:6 MGDG molecules.

Despite the differential spatial distribution of these two pathways,they respond synergistically to various environmental factors[12-14].In the 16:3 plant Arabidopsis thaliana,the chloroplast pathway is activated under low-temperature stress,whereas high temperature promotes the eukaryotic pathway[15].In the 18:3 plants wheat and maize,channeling of glycerolipids from ER to chloroplast is observed under low temperature,as reflected by increased turnover of PC to PA for galactolipid synthesis[13,15].Alkaline stress influences the adjustment of membrane lipid pathways by activating extra-plastidic PC hydrolysis and plastidic galactolipid synthesis[14].

The altering of the distribution of acyl chains and the exchange of head groups between different membrane lipid classes is also known as lipid remodeling[5,6,15]and occurs frequently in response to external environmental stimuli.Membrane lipid remodeling has been shown in plants exposed to salinity,which affects the permeabilityof the cellmembrane[16].In wheat,PC andPE werenegatively correlated at high temperature,and the higher PC:PE ratio may indicate PE-to-PC conversion[17].Under cold stress,DGDG content increases and MGDG/DGDG ratio decreases,favoring an increase in cell membrane stability[18,19].Changes in membrane lipid composition in plants subjected to stress help maintain the stability and integrity of membrane structure[14,20,21].

Previous membrane-lipid studies have focused mainly on changes in total lipids of the plasma membrane,and information about the comprehensive lipid remodeling and salt-induced changes at the gene transcriptional level is scarce[5].Sorghum(Sorghum bicolor L.Moench)is an annual C4 cereal providing food,feed and fuel,and is a highly adaptable crop with high water-use efficiency and stress tolerance[22,23].The objective of the present study was to perform a combined lipidomic and transcriptomic analysis to identify dynamic membrane lipid metabolic changes and investigate the regulation of lipid remodeling in sorghum leaves under salt stress.Investigation of the biological response mechanisms of crops to salt stress will help to ensure crop productivity and make effective use of saline land[24].

2.Materials and methods

2.1.Plant growth,treatments,and sampling

Seeds of the sorghum cultivar Hei63027 were disinfected with 10% NaClO solution for 30 min,washed,and soaked in distilled water for 6-8 h.The soaked seeds were placed in germination boxes in an incubator set at 25 °C in the dark for germination.Sprouts with uniform shoot length were planted on perforated foam boards and incubated in 1/2 Hoagland nutrient solution(pH 5.5).The seedlings were placed in an artificial-climate chamber with the temperature set at 25±2°C,16 h/8 h(light/dark)photoperiodic cycle,and relative humidity about 65%.The 2-week-old seedlings were treated with 150 mmol L-1NaCl.The leaf tissues were sampled for RNA sequencing at 0,12,24,and 72 h after NaCl treatment,and three biological replicates were collected for each treatment.For lipidomic analysis,leaf tissues were sampled at 0,3,7,and 14 d after NaCl treatment with six biological replicates for each group of samples.

2.2.RNA-seq analysis and RT-PCR validation

RNA from the leaves of sorghum seedlings was extracted with TRIZOL reagent,and a cDNA library was constructed after quality inspection.After the database qualification,RNA-seq sequencing was performed and clean reads were further annotated with the KEGG(Kyoto Encyclopedia of Genes and Genomes),Nr(NCBI non-redundant protein sequences),Nt(NCBI non-redundant nucleotide sequences),COG(Clusters of Orthologous Groups of proteins),and Swiss-port(a manually annotated and reviewed protein sequence database)databases.Identificaion of differentially expressed genes(DEGs)employed a false-discovery rate＜0.01 and|log2fold change|≥1.The RNA-seq data have been submitted to the online SRA(sequential read file)database with accession number SRP333181.

To verify the reliability of the RNA-seq data, real-time quantitative RT-PCR amplification was performed for selected DEGs. The synthesis of cDNA and qRT-PCR was completed in 96-well plates with SYBR qRT-PCR Master Mix (Toyobo, Osaka, Japan) [25]. Sb18SRNA and SbACTIN were used as internal reference genes. The results of qRTPCR (Fig. S1) were quantified by the 2-ΔΔCTmethod [26].

2.3.Membrane lipid extraction and analysis

Extraction of polar lipids from sorghum seedlings was performed by a modification of Narayanan’s method[27].Three mL of isopropanol solution containing 0.01% BHT was added to a 50 mL glass tube with a screw cap and preheated to 75 °C,and fresh leaves of sorghum seedlings were quickly placed in the preheated glass tube and heated for 15 min.To the tube were added 1.5 mL of chloroform and 0.6 mL distilled water,followed by shaking at room temperature for 1 h.The extract was transferred to a new glass tube,and 4 mL of chloroform:methanol(2:1)containing 0.01% BHT was added.The mixture was vortexed and then shaken for 30 min.This extraction was repeated 3-4 times until the leaves turned white.Then 1 mL of 1 mol L-1KCl solution was added to the extraction mixture and the supernatant remaining after the vortexing,shaking,and centrifugation was discarded.The lipid extract was washed with 2 mL of distilled water and evaporated in a nitrogen blowing instrument,redissolved in 1 mL of chloroform solution,transferred to a 2-mL brown bottle with a Teflon-lined screw cap,and evaporated again under nitrogen stream.The nitrogen-blown brown vials were stored in a-20 °C refrigerator.Lipid content and composition were determined by electrospray ionization mass spectrometry(ESI-MS/MS)at Kansas Lipidomics Research Center(Manhattan,KS,USA).

2.4.Principal component analysis

Principal component analysis(PCA)was performed with Graph-Pad Prism 9.0(https://www.graphpad-prism.cn).Six biological replicates were used for lipidomic data analysis of lipid species:galactolipids(MGDG and DGDG),phospholipids(PC,PE,PA,PI,PS,and PG)and lysophospholipids(LPG,LPC,and LPA).

2.5.Statistical analysis

Statistical analysis was performed using GraphPad Prism,Microsoft Excel and SPSS Statistics,and the significance level was calculated by Student’s t-test.

3.Results

3.1.Changes of membrane lipids in sorghum leaves under salt stress

As revealed in Fig.1A,the molar percentages of galactolipids MGDG and DGDG were dominant in the leaves of sorghum seedlings.MGDG and DGDG together accounted for more than 65% of total glycerolipids,with MGDG accounting for 45% and DGDG for 20%.Among the six types of phospholipids,PG and PC were present in higher molar percentages,accounted for respectively 14%and 10%of total glycerolipids.The molar percentages of PI,PS,PA,and the three lysophospholipids were relatively low.Under salt stress,the content of MGDG decreased significantly with the extension of NaCl treatment,and dropped by 47.3% by the 14-d time point;whereas the molar percentage of DGDG increased under long-term NaCl treatment to 29%at 14 d.In the phospholipid classes,the molar percentage of plastidic phospholipid PG decreased by respectively 11.5%and 47.5% after 7 and 14 d treatment.The molar percentages of PC and PE increased drastically at the later stage of NaCl treatment,and rose more than two folds at the 14-d time point.A significant increase of LPC and LPE was observed on the 14-d time point.

PCA showed that under salt stress,the glycerolipid species in sorghum leaves were clearly separated by PC1 and PC2,which explained respectively 69.5% and 15.8% of total variance.MGDG,PG,PA,and LPG constituted the main components of PC1,while DGDG,PE,and PI constituted the main components of PC2(Fig.1B).

Spearman’s correlation coefficients(ρ)between the types of glycerolipids were calculated.As shown in Fig.1C,DGDG was negatively correlated with MGDG and PG,with correlations of-0.90 and-0.93,respectively.DGDG was positively correlated with most phospholipid species with correlations ranging from 0.84 to 0.89.MGDG was positively correlated with PG,and strongly negatively correlated with other phospholipids.PG was positively correlated with PA with a coefficient of 0.21,but showed a strong negative correlation with PC,PE,PI,and PS.Most extra-plastidic phospholipids and lysophospholipids were positively correlated,except that PA was negatively correlated with most other phospholipids.

The proportions of specific glycerolipids also underwent adjustment under salt stress.As shown in Fig.1D,the ratio of DGDG to MGDG increased significantly under salt stress,especially at the 14-d time point;whereas the ratio of PC:PE decreased slightly but not significantly.Bilayer lipids,including DGDG,PC,PG,and PI,are more beneficial to the stability of lipid bilayers than are nonbilayer lipids,including MGDG,PE,PA,and PS.The ratio of bilayer lipid:non-bilayer lipid was significantly increaseed at 14 d,suggesting an increase in membrane stability in response to salt stress.The ratio of plastidic membrane lipids(MGDG,DGDG,and PG)to nonplastidic lipids(phospholipids other than PG)declined under salt stress,particularly at the later stage of NaCl treatment,suggesting that salt stress caused damage to the photosynthetic membrane.

3.2.Changes in molecular species of phospholipids in sorghum leaves under salt stress

The fatty acid composition at sn-1&2 acyl positions of individual glycerolipids was determined by lipidomic analysis and marked as Cxx:x(total number of acyl carbon atoms:number of double bonds).As shown in Fig.2A,the six classes of phospholipids were composed of different molecular species dominated by C34 and C36 molecules,ranging from C34:1 to C36:6.Given that PG is the only phospholipid assembled in the plastids,the C34 molecular species was dominant in PG.For other non-plastidic phospholipids,the proportion of C34 and C36 molecules was similar(Fig.2A).Under salt stress,the molecular species of specific phospholipids underwent alterations.As shown in Fig.2B,in PG(plastidic phospholipid),the polyunsaturated molecular species C34:3 and C34:4 decreased significantly under long-term NaCl treatment,particularly at 14 d.In contrast,the C36:6 PA increased drastically at 14 d.The most noteworthy finding was the changes in molecular species of the major phospholipid PC.All C34 and C36 species in PC showed an upward trend in the later stage of salt stress treatment,except for C36:4,the only species that decreased significantly under salt stress(Fig.2B).

3.3.Molecular species analysis of galactolipids revealed that sorghum is an 18:3 plant species

As shown in Fig.3A,the molar percentage of C34:3,C36:6,C36:5,and C36:4 MGDG gradually decreased under salt stress and dropped significantly at the 14-d time point.However,the contents of C36:6,C36:5,and C36:3 DGDG gradually increased under salt stress(Fig.3A).

As revealed in Fig.3B,the proportion of C36 MGDG molecules accounted for most of the total MGDG molecules,with C36:6 MGDG accounting for 91.9% of total MGDGs.C36:6 DGDG accounted for 74.9% of total DGDGs,whereas C34:3 accounted for only 14.8%of total DGDGs(Fig.3B).According to the difference of lipid synthesis pathways[9,28],there is a class of plants called 18:3 plants,in which galactolipid synthesis relies on the ER pathway and produces C36-dominated MGDG and DGDG.In the present study,the predominance of C36 galactolipids implied dependence on ER pathway,suggesting that sorghum is an 18:3 plant.

C34:2 MGDG and C34:3 MGDG were positively correlated,with a correlation of 0.83,whereas C36:4 MGDG and C36:6 MGDG had a correlation of 0.89.C34:2 DGDG was positively correlated with C34:3 DGDG,and the correlation coefficient was 0.10;C36:4 DGDG was negatively correlated with C36:6 DGDG,and the coefficient was-0.17(Fig.3C).

3.4.Correlation and unsaturation index analysis of membrane lipids in sorghum leaves under salt stress

As shown in Fig.4,correlation analysis of phospholipids(PC,PE)and galactolipids(MGDG,DGDG)revealed that phospholipids(PC,PE)were negatively correlated with MGDG and positively correlated with DGDG,but that C36:4 and C34:2 PC were positively correlated with most MGDG and negatively correlated with most DGDG.The correlation between the molecular species of phospholipids PC and PE was calculated and it was found that there was an overall positive correlation among phospholipids,but C34:2 PC and C36:4 PC were negatively correlated with other molecular species of PC and PE,with C36:4 PC showing the largest negative correlation.There was no apparent conversion between PC and PE.There was an overall negative correlation between galactolipids,with stronger negative correlations among C36:2,C36:3,C36:5,and C36:6 DGDG and MGDG molecular species,suggesting a strong conversion relationship.These results are consistent with the previous ratio analysis of classified lipid species(Fig.1B).

Fatty acid unsaturation index is an indicator of the characteristics of plasma membrane,and strongly influences the adaptation of plants to abiotic stresses.In this study,the double-bond index(DBI)was calculated(Fig.5A),and the DBIs of galactolipids MGDG and DGDG were relatively large.Under salt stress,the DBI of DGDG increased with the prolongation of NaCl treatment,while the DBI of MGDG decreased significantly under salt treatment.In the phospholipids,the DBI of PC and PE increased significantly,especially at the 14-d time point,whereas the unsaturation of PG decreased gradually under salt stress.

Fig.1.Changes of membrane glycerolipids in sorghum leaves under salt stress.(A)Changes in glycerolipid content;values(mol%)are mean±standard error(n=6);the significance level was calculated by Student’s t-test;*indicates that the value is significantly different from the control(P＜0.05);**indicates that the value is extremely different from the control.(B)Principal component analysis of lipid groups.(C)Correlations among lipid species.(D)Ratio comparison among lipid species.The a,b,and c in figure D indicate the significance of the difference between contrasted groups.

3.5.Transcriptomic analysis of sorghum leaves under salt stress

Transcriptomic analysis was performed on RNA prepared from sorghum leaf samples collected at 0,12,24,and 72 h after 150 mmol L-1NaCl treatment.A total of 84.44 Gb clean data was obtained,and the clean reads were annotated with KEGG(Kyoto Encyclopedia of Genes and Genomes),Nr(NCBI non-redundant protein sequences),Nt(NCBI non-redundant nucleotide sequences),COG(Clusters of Orthologous Groups of proteins),and Swiss-port(a manually annotated and reviewed protein sequence database)databases.The accession ID of the RNA-seq data in the SRA database is SRP333181.DEGs were identified and a total of 384 co-expressed DEGs(|log2FC|≥1)were retrieved in the‘‘12 h vs.0 h”,‘‘24 h vs.0 h”,and‘‘72 h vs.0 h”comparison groups,of which 140 were up-regulated and 244 down-regulated(Fig.6A).In the‘‘24 h vs.12 h”,‘‘72 h vs.12 h”and‘‘72 h vs.24 h”comparison groups,there were 41 co-expressed genes,of which 30 were up-regulated and 11 down-regulated.There were 41 differentially co-expressed genes in the‘‘24 h vs.12 h”and‘‘72 h vs.24 h”comparison groups.There were 249 differentially co-expressed genes in‘‘72 h vs.12 h”and‘‘72 h vs.24 h”comparison groups,of which 177 were up-regulated and 72 were downregulated(Fig.6A).These genes may function in salt stress response.

The KEGG annotation analysis of DEGs is shown in Fig.6B,‘‘Metabolism”accounted for the main proportion(around 80%)in all three comparison groups.Partitioning analysis of‘‘Metabolism”showed that‘‘Lipid metabolism”accounted for a large proportion in all the metabolic processes.The‘‘Others”proportion was split into several parts,and‘‘Environmental information processing”accounted for a larger proportion in the‘‘24 h vs.0 h”comparison group;whereas in the‘‘72 h vs.0 h”comparison group,‘‘Genetic information processing”accounted for more than half of that part.

3.6.Differentially expressed genes associated with lipid metabolism under salt stress

Genes associated with lipid synthesis were identified in the sorghum transcriptome based on the annotations of the GO and KEGG databases and the Arabidopsis lipid gene database[29,30].As shown in Fig.7A,a total of 395 genes associated with lipid metabolism were identified in the‘‘12 h vs.0 h”comparison group,of which 177 were up-regulated and 218 down-regulated.Respectively 379(207 up-regulated and 172 down-regulated),and 387(198 up-regulated and 189 down-regulated)lipid-related genes were identified in the‘‘24 h vs.0 h”and‘‘72 h vs.0 h”comparison groups.The increase of up-regulated genes in the later stage of salt stress indicates that some lipid metabolism pathways had been activated.The up-regulated genes were enriched mainly in the‘‘Phospholipid signaling”,‘‘Fatty acid elongation & wax biosynthesis”pathways,and the number of up-regulated genes reached 30 in the‘‘24 h vs.0 h”and‘‘72 h vs.0 h”comparison groups.A higher proportion of up-regulated genes were also found in the‘‘Triacylglycerol synthesis”,‘‘Triacylglycerol & fatty acid degradation”,‘‘Oxilipin metabolism”,‘‘Eukaryotic phospholipid synthesis& editing”,and‘‘Eukaryotic galactolipid&sulfolipid synthesis”processes.

Fig.2.Changes in molecular species of phospholipids in sorghum leaves under salt stress.(A)Changes in molecular species of phospholipids;the significance level was calculated by Student’s t-test;‘‘0 d”is set as control;*indicates that the value is significantly different from the control(P＜0.05);**indicates that the value is extremely different from the control.(B)Differential changes of specific molecular species in PC and PG.The a,b,and c in the figure B indicate the significance of the difference between the contrasted groups.

Fig.3.Changes in galactolipids in sorghum leaves under salt stress.(A)Changes in molecular species of galactolipids.(B)The occupancy of galactolipid molecular species.(C)Correlations among galactolipid molecular species.Values(mol%)are mean value of replicates±standard error(n=6).‘‘0 d”is set as control;*indicates that the value is significantly different from the control(P＜0.05);**indicates that the value is extremely different from the control.

Fig.4.Correlation among molecular species of PC,PE,MGDG,and DGDG.The Spearman correlation was calculated basing on the molar percentage content of C34-C36 PC,PE,MGDG,and DGDG.Spearman’s correlations＜0 are negative and represented by green squares and those greater than 0 are positive and represented by red squares.

A further proportional analysis of specific lipid metabolism pathways also revealed that the‘‘Fatty acid elongation & wax biosynthesis”and‘‘Phospholipid signaling”pathways accounted for a large proportion of total genes associated with lipid synthesis(Fig.7B).These results together indicated that the‘‘Phospholipid signaling”and‘‘Fatty acid elongation & wax biosynthesis”pathways were activated under salt stress treatment.

The significant DEGs in major lipid metabolic pathways are presented in heat maps in Fig.7C.The expression of genes encoding the enzymes involved in the steps of de novo TAG biosynthesis,which are also the essential steps for PC biosynthesis,including GPAT,LPAAT,PAH,DGAT1,and DGAT3,were markedly upregulated under salt stress treatment.Genes involved in the de novo synthesis of PC were influenced by salt stress as well,including down-regulation of CEK(choline kinase),up-regulation of CCT(choline-phosphate cytidylyltransferase)and AAPT(aminoalcohol phosphotransferase).These findings indicated that the activated DAG-PC and de novo PC assembly steps increased PC synthesis,in agreement with the increase in PC content revealed by the lipidomic analysis.As the central intermediate in lipid metabolism,PC undergoes active degradation to provide DAG and PA as precursors for synthesis of other lipids.In the present study,7 of 8 PLD isoforms and 3 of 4 NPC were significantly up-regulated at most time points under salt stress.The expression of genes involved in the biosynthesis of galactolipids(MGDG and DGDG)was not obviously up-regulated under salt stress.The expression of 2 MGD isoforms was down-regulated,whereas the expression of 2 DGD isoforms was slightly up-regulated at different time points of salt treatment.The expression of DGD1 was up-regulated in the‘‘24 h vs.0 h”comparison group,while DGD2 was up-regulated in the‘‘12 h vs.0 h”comparison group,possibly reflecting an early response of DGD genes to salt stress.The genes encoding FAD3 and FAD8.2 were up-regulated,whereas the other FAD genes were mostly downregulated under salt stress.

3.7.Regulation of lipid metabolism in sorghum leaves under salt stress

Based on our findings from the transcriptome and lipidome data,a gene-metabolite regulation network in sorghum leaves under salt stress was constructed(Fig.8).The steps or pathways of transcriptional activation under salt stress are represented by red arrows.As shown in Fig.8,the activated steps or pathways were associated mainly with PC synthesis and turnover,galactolipid synthesis and degradation,and desaturation of fatty acids.

Fig.5.Changes in double-bond index(DBI)in sorghum leaves under salt stress.(A)Clustered DBI analysis of glycerolipids.(B)DBI of individual glycerolipids.DBI=(∑[N×mol%lipid])/100,N is the total number of double bonds in the two fatty acid chains of each glycerolipid molecule.The significance level was calculated by Student’s ttest;a,b,and c in the figure B indicate the significance of the difference between the contrasted groups.

In ER compartment,GPAT,LPAAT,PAH,and DGAT are the major catalytic enzymes involved in the biological assembly of TAG,and most of the genes encoding these enzymes were up-regulated under salt stress.The up-regulated expression of PAH and AAPT indicated that the PA-DAG-PC pathway was activated for PC production.The de novo PC synthesis pathway through CCT was also induced under salt stress.These changes together suggest increased PC production in response to salt stress at the transcriptional level,evidenced by elevated PC accumulation at the later stage of salt stress as revealed by lipidomic analysis.PC is a relatively abundant phospholipid and an intermediate in lipid metabolism.The ER-derived PC is converted by PLD to PA and finally by NPC to form DAG,which can be used for synthesis of MGDG and DGDG.Most genes encoding PLD and NPC were up-regulated under salt stress,but this up-regulation did not lead to a decrease in total PC level.However,the accumulation of one specific PC molecule,C36:4 PC,dropped greatly under salt stress,suggesting that it might be the favored substrate for the activated hydrolases PLD and NPC.The increased accumulation of polyunsaturated PCs,C34:3 and C36:6 PC,under salt stress could be attributed to the induced expression of the desaturase FAD3.

In the chloroplast compartment,although the synthesis of galactolipids seemed to be inhibited at both the transcriptional and metabolic levels under salt stress,the steps governing the synthesis and desaturation of DGDG were induced as illustrated by the up-regulated DGD2 and FAD8 genes,which led to the increased DGDG accumulation.Up-regulation of DGL genes in the chloroplast might result in galactolipid degradation,converting MGDG and DGDG into free fatty acids(FFA).The down-regulation of genes involved in PG synthesis pathway led to lowered PG production under salt stress(Fig.8).The impaired accumulation of plastidic lipids in sorghum leaves may give rise to dysfunction of the photosynthetic membrane of the chloroplast.

4.Discussion

The cell membrane is a barrier between plants and external stimuli,owing to its specific structure,typical permeability,and other protective properties[31].Glycerolipids are essential components of the plasma membrane,and large metabolic changes and lipid remodeling occur when plants are subjected to environmental stress[13,25,32,33].

Phospholipids are the main building blocks of biological membranes,with PC and PE the major membrane phospholipids in plant cells[34].In the present study,the total molar percentage of phospholipids was elevated in sorghum leaves under salt stress,with the molar percentage content of PC and PE increased about twofold at 14 d relative to 0 d.The PC and PE synthesis pathways were activated at the transcriptional level by salt treatment,as reflected by the up-regulation of genes involved in those pathways.In a previous study[35],PC content in chloroplast membranes of leaves and bundle sheaths of maize increased under salt stress.

Fig.6.Differentially expressed gene(DEG)distribution and Kyoto Encyclopedia of Genes and Genomes(KEGG)classification in the transcriptome of sorghum leaves under salt stress.(A)Venn diagram showing DEGs;colored shapes indicate the expression value.Pink indicates high level,and green indicates low level.(B)KEGG annotation of genes associated with lipid metabolism.

Membrane galactolipids(MGDG and DGDG)are dominant lipids in photosynthetic membranes of plant leaves and are stronlgy affected by abiotic stresses[36].MGDG is the most abundant galactolipid in the thylakoid membrane and influences chloroplast structure and photosynthetic performance[37].In the present study,the galactolipids in sorghum leaves decreased by 26.6% under salt stress.Although the content of DGDG increased,MGDG decreased by 47.3% at 14 d compared with 0 d.Marked phenotypic changes of sorghum seedlings under salt stress were also observed.The plant growth was severely restricted,and the leaves of sorghum seedlings gradually turned yellow and wilted with increasing duration of salt stress,indicating that the photosynthetic system of the plant was impaired(Fig.S2).The reduction of glycolipids in the leaves of rice and cucumber seedlings results in a decrease in chlorophyll content in the leaves[37,38].Reduction in MGDG seems to be a general response of plants to osmotic stress caused by drought,salt or freezing damage[39].Drought caused a decrease in MGDG and an increase in DGDG in the leaves of two maize varieties[40].Synthesis of DGDG was triggered by the up-regulation of DGD2 under salt-alkali stress[14].The transcriptome analysis in the present study revealed the presence of down-regulated MGD and up-regulated DGD isoforms,possibly accounting for the differing changes in MGDG and DGDG.

Fig.7.Functional classification of DEGs associated with lipid metabolism in sorghum leaves under salt stress.(A)Functional categorization of lipid-related DEGs involved in various lipid metabolic pathways.In each category,purple columns represent up-regulated genes and green columns down-regulated genes.The number of genes in each category is shown on the x axis.(B)The proportional distribution of lipid-related DEGs.(C)The significant salt response lipid-related DEGs.Heat maps were constructed to illustrate the differential expression profiles of important lipid-associated DEGs.Numbers in each color block represent log2(fold-change)of the corresponding genes,and negative numbers represent down-regulated DEGs.Color scale is provided.

In plant cells,phospholipids PC,PE,PI,and PS constitute nonplastidic membrane lipids,whereas the glycolipids MGDG,DGDG,SQDG,and PG constitute the plastidic membrane lipids[6,7].In the green tissues of plants,plastidic lipids are predominant and associated closely with plant thylakoid membrane structure and photosynthetic characteristics[8].In the present study,the ratio of plastidic to non-plastidic membrane lipids dropped to a very low level at 14 d salt treatment.It could be speculated that the sharp reduction in the level of galactolipids and the lowered ratio of plastidic to non-plastidic lipids lead to damage to photosynthetic membranes,and result in dysfunction of the chloroplast and photosynthesis in sorghum leaves under salt stress(Fig.9).

Membrane lipids are usually classified as bilayer or non-bilayer lipids[41].Whereas bilayer lipids ensure membrane stability,nonbilayer lipids function in mediating protein interactions and increasing the plasticity of lipid bilayer structures[42].The bilayer membrane lipids include PC,PG,PI,DGDG,and SQDG;while nonbilayer membrane lipids include PA,PE,PS,and MGDG[6].In the present study,the ratio of bilayer to non-bilayer lipid in the leaves of sorghum seedlings increased by 65.2% under salt stress.The major bilayer lipid PC increased significantly,whereas the ratio of PC/PE remains basically unchanged.In contrast,the ratio of DGDG/MGDG increased drastically at 14 d(145%),suggesting the contribution of bilayer lipid DGDG to the maintenance of chloroplast membrane stability under salt stress(Fig.9).There is evidence[43]that the DGDG/MGDG ratio is necessary for the stability of photosynthetic membranes.In maize,varieties with delayed leaf senescence under drought conditions showed higher DGDG content and DGDG/MGDG ratio[40].

In the present study,the C36:6 species in MGDG and DGDG in the leaves of sorghum seedlings accounted for the largest proportion,indicating that sorghum is an 18:3 plant.In 18:3 plants,the main synthetic precursors of MGDG and DGDG come from the degradation of eukaryotic-pathway-evolved PC,and the flux of PC hydrolyzates tends to be increased under various stress conditions[15,44].Under salt stress,although the total amount of PC did not show a large decrease,C36:4 PC was the only molecule showing a large reduction among all molecular species,suggesting that C36:4 was the most favorable precursor for glycolipid synthesis.In agreement with this finding,previous studies[13,15]have shown that C36:4 PC is the specific molecular species contributing to lipid flux shared between the cytoplasm and plastid pathways when plants encounter temperature stress.Our findings indicated that under salt stress,C36:4 PC was also the major contributor to plastidic lipid synthesis.Phospholipase D(PLD)and non-specific phospholipase C(NPC)catalyze the degradation of PC into PA and DAG,respectively,and participate in lipid remodeling[45].In the transcriptome data,genes involved in the PLD and PLC/DGK pathways were up-regulated under salt stress,implying activated PC degradation to ensure the flow of lipid intermediates from cytoplasm to chloroplast for photosynthetic membrane lipid assembly.

Fig.8.The gene-metabolite network of lipid metabolism in sorghum leaves under salt stress.The relative changes of lipid molecular species[(3 d or 7 d or 14 d mol%-0 d mol%)/0 d mol%]and the relative expression levels of related genes(72 h vs.0 h)are marked as heat-map icons.Changes in lipid content are represented by diamond-shaped heat map icons,and transcription levels are represented by square heat-map icons.The steps or pathways of transcriptional activation under salt stress are represented by red arrows.

Fatty acid unsaturation index is a major indicator of the characteristics of the plasma membrane,and is also closely associated with adaptation of plants to abiotic stresses[46,47].In the present study,the DBI of the bilayer membrane lipids(DGDG,PC,PI)underwent significant elevation.The DBI of DGDG increased by 30.5%,while DBI of PC and PI increased by 144.0% and 171.8%,respectively.Thus,it could be speculated that the fluidity of the bilayer membrane increased under salt stress.In Phanerochaete rose,the DBI of all glycerophospholipids increased after cold treatment[48].In Fabaceae,200 mmol L-1NaCl treatment led to increased unsaturation of MGDG,DGDG,and PG,which might be beneficial to salt stress adaption and protection of the integrity of the chloroplast membrane[37].Fatty acid unsaturation is determined by the activity of fatty acid desaturase(FAD),which introduces double bonds at specific acyl chain positions[32,49].In the ER compartment,FAD2 converts oleic acid(18:1)to linoleic acid(18:2),which is further desaturated by FAD3 to linolenic acid(18:3)[50,51];in plastids,FAD7 and FAD8 are responsible for catalyzing the 18:2 to 18:3 conversion[52].In our transcriptome data,the genes encoding FAD3 and FAD8.2 were up-regulated,while other FAD genes were mostly down-regulated under salt stress,indicating that the 18:2 to 18:3 conversion was promoted under salt stress.In transgenic tobacco plants,overexpression ofω-3 desaturase(FAD3 or FAD8)increased tolerance to salt and drought stress[53].Knockout of FAD8 led to reduced membrane fluidity in rice under cold stress[54].

Although TAG is not usually produced at high levels in vegetative tissues of plants,its accumulation in these tissues functions in plant response to abiotic stresses[55].TAG accounts for＜1% of total lipid content in Arabidopsis leaf tissue,but it is related to the short-term supply of glycerolipid intermediates during membrane lipid remodeling[15,56].TAG regulates the conversion of excessive DAG to TAG through the catalysis of DGAT and DGK,so that plants can resist freezing stress[57,58].In the present study,the expression of genes encoding the enzymes involved in the steps of de novo TAG biosynthesis,including DGAT1 and DGAT3,was up-regulated under salt stress treatment.Although we did not investigate changes in TAG in this study,it can be inferred from the gene transcription that TAG is essential in membrane-lipid metabolic response to salt stress.

Fig.9.Schematic diagram of membrane lipid changes and adaptive remodeling in response to salt stress in sorghum leaves.A decrease in the proportion of plastidic lipids in sorghum leaves may severely damage the stability of photosynthetic membranes and the function of the chloroplast.Synergistically,several adaptive membrane lipid adjustments also occur,including increased conversion of C36:4 PC into C36:6 MGDG and C36:6 DGDG,increased ratio of bilayer lipid/non-bilayer lipid,and an increased double-bond index(DBI)in the major phospholipids and DGDG,which may increase membrane stability and fluidity in response to salt stress.

Membrane lipid remolding is a necessary modulation for plant to cope with abiotic stresses.In the present study,active metabolic changes in membrane lipids were observed in sorghum seedlings under salt stress.As summarized in Fig.9,salt stress led to a significant decrease in the proportion of plastidic lipids in sorghum leaves,particularly MGDG and PG,which may greatly damage the stability of photosynthetic membrane and the function of chloroplast.Synergistically,several adaptive membrane lipid adjustments also occur for sorghum leaves to adapt to external stimuli.First,the adjustment of the glycerolipid pathway between the cytosolic and plastidic compartment resulted in an intensive conversion of C36:4 PC into C36:6 MGDG and then into C36:6 DGDG to complement the chloroplast lipids and ensure the integrity of the photosynthetic membrane.Second,the ratio of bilayer lipid to non-bilayer lipid increased,especially the ratio of DGDG/MGDG,which is conducive to stabilizing the photosynthetic membrane.Thirdly,the elevation of the DBI in the major phospholipids and DGDG implied increased membrane fluidity in response to salt stress.These findings are helpful for understanding the dynamic regulation of membrane lipids in plants under salt stress and for identifying some adaptive roles of membrane-lipid remodeling in plant adaptation to salt stress.

CRediT authorship contribution statement

Shengnan Ge:Validation,Visualization,Writing-original draft.Debin Liu and Min Chu:Conceptualization,Writing-review &editing.Xinyu Liu and Yulei Wei:Data curation.Xinyang Che and Lei Zhu:Formal analysis.Lin He and Jingyu Xu:Conceptualization,Funding acquisition,Project administration,Writingreview & editing.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

We gratefully acknowledge Dr.Ruth Welti and Mary Roth(Kansas Lipidomics Research Center)for their help with lipidomic analysis.This work was supported by the Natural Science Foundation of Heilongjiang Province(ZD2020C007,QC2017024),Heilongjiang Bayi Agricultural University Support Program for San Heng San Zong(ZDZX202101),and Special Funds from the Central Finance to Support the Development of Local Universities(TO SXL).

Appendix A.Supplementary data

Supplementary data for this article can be found online at https://doi.org/10.1016/j.cj.2022.03.006.