Molecular Mechanisms of Salt Tolerance in Naked Barley at Seedling Stage Based on Integrated Transcriptomic and Metabolomic Analysis

doi:10.7668/hbnxb.20195964

Abstract

Abstract:

Salt stress causes a significant threat to crop yield and quality.As one of the pioneer crop species in salt tolerance research,barley holds critical significance;the exploration of its salt tolerance mechanisms is capable of providing a theoretical foundation for crop salt-tolerance breeding programs.Two naked barley landraces,namely B87 with salt-sensitivity and B94 with salt-tolerance,were employed as experimental materials.At the three-leaf stage,their seedlings were exposed to a 200 mmol/L NaCl treatment for 7 days.Subsequent to the treatment,the above-ground tissues were collected for transcriptomic and metabolomic sequencing.By means of integrated multi-omics analysis,this study was designed to elucidate the molecular mechanisms governing salt tolerance in naked barley.The results demonstrated that 2 240 differentially expressed genes (DEGs) and 198 differentially abundant metabolites (DAMs) were identified in B87 via transcriptomic and metabolomic profiling,whereas 923 DEGs and 232 DAMs were detected in B94.Venn diagram analysis further revealed that the salt-tolerant naked barley B94 contained 480 specific DEGs and 129 specific DAMs.Furthermore, GO and KEGG analyses were separately performed on the DEGs and DAMs. And the DEGs of B94 were significantly enriched in 11 unique pathways, while its DAMs were only significantly enriched in 1 unique pathway. In addition, correlation analysis between the transcriptome and metabolome was conducted, and it was found that the changes in genes and metabolites exhibited both consistency and inconsistency. These research efforts not only enhance the current understanding of the molecular mechanisms underlying salt tolerance in naked barley,but also provide valuable insights and candidate targets for the development of salt-tolerant naked barley cultivars in future breeding.

Key words: Naked barley, Salt tolerance, Transcriptome, Metabolome, qPCR

CLC Number:

S512.3大麦

LIU Shisen, YANG Yicheng, FENG Shiji, GUO Zhenzhu, ZHANG Shuwei, GUO Guimei, WANG Yu, ZHOU Longhua, LIU Chenghong, CHEN Zhiwei. Molecular Mechanisms of Salt Tolerance in Naked Barley at Seedling Stage Based on Integrated Transcriptomic and Metabolomic Analysis[J]. Acta Agriculturae Boreali-Sinica, 2025, 40(6): 40-49. doi: 10.7668/hbnxb.20195964.

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Figures/Tables 9

Tab.1 Quality of RNA-seq data of shoots of naked barley seedlings grown under control (CK) and salt treatment (ST)

样本 Sample	原始碱基数/ Gb Raw bases	有效碱基数/ Gb Clean bases	Q30/ %	GC含量/ % GC content
CK87_1	7.03	6.94	95.50	56.87
CK87_2	7.15	7.04	95.55	56.71
CK87_3	7.09	7.02	95.30	56.96
CK94_1	7.06	6.96	95.36	57.21
CK94_2	6.99	6.91	95.49	56.99
CK94_3	7.07	7.01	95.71	56.50
ST87_1	7.16	7.04	95.46	55.80
ST87_2	6.97	6.89	95.99	57.02
ST87_3	6.96	6.88	95.76	56.23
ST94_1	7.01	6.92	95.71	56.53
ST94_2	6.96	6.89	95.46	56.20
ST94_3	7.00	6.94	95.66	57.17

Tab.2 Primers used for qPCR analysis

基因名称 Gene name	引物序列(5'—3') Primer sequences(5'—3')	扩增长度/bp Amplicon	扩增效率 PCR efficiency	引物来源 Primer origin
HORVU2Hr1G029900	F:CAACCGTAGGTACTCCTGCC	119	1.867 2	本研究
	R:AGCACCGACAAGCATCACAT
HORVU7Hr1G090410	F:CGCGACGACATCTCCATCTT	269	1.810 3
	R:TTCCCGGACTTGTCAGAACG
HORVU6Hr1G000510	F:GCTGCACGGATTATGCACTG	241	1.812 1
	R:CTCGGGGGTTTCGAACTTGA
HORVU5Hr1G010130	F:TGCAGATCGCTCTCTTCGTC	105	1.860 4
	R:AATCAGGGCGGTTAGTGTCG
HORVU2Hr1G110230	F:GATGGTAGTGTCGCTCCTCG	117	1.873 6
	R:GACCTCCGGAGCGATGTATG
HORVU3Hr1G029350	F:AGGATTCTCCAGTTTGCTGCT	199	1.803 7
	R:ACACGCAACACCTTCTCCTT
HORVU1Hr1G093780	F:TCTGCTCGGGCCTCAAGTT	133	1.833 4
	R:TCGATGTGCTCCGTGTTGTA
U61	F:GAGGAAACGAAACCTGTGC	65	1.828 7	[17]
	R:ACTTCTTAGAGGGTTGTGTTAC
snoR14	F:GATGTTTATGTATGATAGTCTGTC	67	1.811 8
	R:GTCGGGATGTATGCGTGTC

Fig.1 Analysis of differentially expressed genes (DEGs) in shoots of B87 and B94 seedlings under salt stress A.Cluster analysis of DEGs in B87;B.Cluster analysis of DEGs in B94;C.The histogram of DEGs in B87 and B94,respectively;D.Venn diagram of DEGs between B87 and B94.

Fig.2 GO enrichment analysis of differentially expressed genes (DEGs) in B87 and B94 A.Enriched GO terms of up-regulated genes in B87:1.Defense response,2.rRNA processing,3.Response to biotic stimulus,4.Drug transmembrane transport,5.Oxylipin biosynthetic process,6.Response to water,7.RNA catabolic process,8.snRNA pseudouridine synthesis,9.Positive regulation of seed germination,10.Maturation of SSU-rRNA,11.Protein complex oligomerization,12.Dhurrin biosynthetic process,13.rRNA pseudouridine synthesis,14.L-asparagine biosynthetic process,15.mRNA pseudouridine synthesis,16.Arginine catabolic process,17.Chloroplast membrane,18.Small-subunit processome,19.Box H/ACA snoRNP complex,20.Small nucleolar ribonucleoprotein complex,21.Iron ion binding,22.Toxin activity,23.snoRNA binding,24.Endoribonuclease activity,25.Linoleate 13S-lipoxygenase activity,26.Ribonuclease T2 activity,27.Chitinase activity,28.rRNA N-glycosylase activity,29.Asparagine synthase (glutamine-hydrolyzing) activity,30.Cadmium ion transmembrane transporter activity;B.Enriched GO terms of up-regulated genes in B94:1.Defense response,2.Response to cadmium ion,3.Defense response to fungus,4.Carbohydrate metabolic process,5.Response to biotic stimulus,6.Lipid transport,7.Response to wounding,8.Oxylipin biosynthetic process,9.Regulation of transcription by RNA polymerase Ⅱ,10.Response to arsenic-containing substance,11.Response to nitric oxide,12.Response to water,13.Regulation of ethylene-activated signaling pathway,14.Response to iron ion starvation,15.Killing of cells of other organism,16.Anthocyanin-containing compound biosynthetic process,17.Positive regulation of seed germination,18.L-pipecolic acid biosynthetic process,19.Extracellular region,20.Toxin activity,21.Monooxygenase activity,22.Lipid binding,23.Glucan endo-1,3-beta-D-glucosidase activity,24.Scopolin beta-glucosidase activity,25.Beta-glucosidase activity,26.Serine-type endopeptidase inhibitor activity,27.Linoleate 13S-lipoxygenase activity,28.Protein C-terminus binding,29.Ribonuclease T2 activity,30.Sucrose synthase activity;C.Enriched GO terms of down-regulated genes in B87:1.Response to oxidative stress,2.Hydrogen peroxide catabolic process,3.Photosynthesis,light harvesting,4.Cell wall biogenesis,5.Xyloglucan metabolic process,6.Microtubule-based process,7.Zinc ion transmembrane transport,8.Cutin biosynthetic process,9.Plasmodesmata-mediated intercellular transport,10.Oligopeptide transport,11.Arabinan catabolic process,12.Plasma membrane,13.Extracellular region,14.Cell wall,15.Plasmodesma,16.Apoplast,17.Plant-type cell wall,18.Anchored component of membrane,19.Anchored component of plasma membrane,20.DNA-binding transcription factor activity,21.Peroxidase activity,22.Manganese ion binding,23.Nutrient reservoir activity,24.Xyloglucan:xyloglucosyl transferase activity,25.Structural constituent of cytoskeleton,26.Hydrolase activity,hydrolyzing O-glycosyl compounds,27.Protochlorophyllide reductase activity,28.Zinc ion transmembrane transporter activity,29.Nitrate transmembrane transporter activity,30.Alpha-L-arabinofuranosidase activity;D.Enriched GO terms of down-regulated genes in B94:1.Cell wall organization,2.Nucleosome assembly,3.Hydrogen peroxide catabolic process,4.Lipid catabolic process,5.Lignin catabolic process,6.Fruit ripening,7.Ethylene biosynthetic process,8.Oligopeptide transport,9.Extracellular region,10.Nucleosome,11.Cell wall,12.Apoplast,13.Plant-type cell wall,14.DNA binding,15.Protein heterodimerization activity,16.Heme binding,17.Peroxidase activity,18.Copper ion binding,19.Oxidoreductase activity,oxidizing metal ions,20.Hydroquinone:oxygen oxidoreductase activity,21.3-oxo-arachidoyl-CoA synthase activity,22.3-oxo-cerotoyl-CoA synthase activity,23.3-oxo-lignoceronyl-CoA synthase activity,24.Very-long-chain 3-ketoacyl-CoA synthase activity,25.Structural constituent of cell wall,26.Glucan exo-1,3-beta-glucosidase activity,27.Sucrose alpha-glucosidase activity,28.Nitrate transmembrane transporter activity,29.Sucrose 1F-fructosyltransferase activity,30.Asparaginase activity.

Fig.3 KEGG enrichment analysis of differentially expressed genes (DEGs) in B87 and B94 A.Enriched KEGG pathways of up-regulated gene in B87:1.Ribosome biogenesis in eukaryotes,2.Linoleic acid metabolism,3.Alanine,aspartate and glutamate metabolism,4.Cyanoamino acid metabolism,5.Glucosinolate biosynthesis,6.Isoquinoline alkaloid biosynthesis,7.Arginine and proline metabolism,8.ABC transporters,9.Taurine and hypotaurine metabolism,10.beta-Alanine metabolism,11.Tyrosine metabolism,12.Butanoate metabolism,13.MAPK signaling pathway-plant,14.Phenylalanine metabolism,15.Cysteine and methionine metabolism,16.RNA degradation,17.Glycine,serine and threonine metabolism,18.Tropane,piperidine and pyridine alkaloid biosynthesis,19.Glutathione metabolism,20.Starch and sucrose metabolism,21.Indole alkaloid biosynthesis,22.Flavone and flavonol biosynthesis;B.Enriched KEGG pathways of up-regulated genes in B94:1.Linoleic acid metabolism,2.Cyanoamino acid metabolism,3.MAPK signaling pathway-plant,4.Plant hormone signal transduction,5.Cutin,suberine and wax biosynthesis,6.Flavone and flavonol biosynthesis,7.Glucosinolate biosynthesis,8.Starch and sucrose metabolism,9.Other glycan degradation,10.Alanine,aspartate and glutamate metabolism,11.Butanoate metabolism;C.Enriched KEGG pathways of down-regulated genes in B87:1.Phenylpropanoid biosynthesis,2.Photosynthesis-antenna proteins,3.Plant-pathogen interaction,4.Porphyrin and chlorophyll metabolism,5.Phagosome,6.Diterpenoid biosynthesis,7.MAPK signaling pathway-plant,8.Glycosaminoglycan degradation,9.Cutin,suberine and wax biosynthesis,10.Nitrogen metabolism,11.Indole alkaloid biosynthesis,12.Amino sugar and nucleotide sugar metabolism,13.Glycerophospholipid metabolism;D.Enriched KEGG pathways of down-regulated genes in B94:1.Fatty acid elongation,2.Phenylpropanoid biosynthesis,3.Nitrogen metabolism,4.Plant-pathogen interaction,5.Diterpenoid biosynthesis,6.Sesquiterpenoid and triterpenoid biosynthesis,7.Cysteine and methionine metabolism,8.Indole alkaloid biosynthesis,9.Betalain biosynthesis,10.Alanine,aspartate and glutamate metabolism,11.Plant hormone signal transduction,12.Cyanoamino acid metabolism,13.Biosynthesis of unsaturated fatty acids,14.MAPK signaling pathway-plant,15.Brassinosteroid biosynthesis.

Fig.4 Analysis of differentially abundant metabolites (DAMs) in shoots of B87 and B94 seedlings under salt stress A.Principal component analysis (PCA) of metabolites in all samples;B.The histogram of DAMs;C.Venn diagram of up-regulated abundant metabolites between B87 and B94;D.Venn diagram of down-regulated abundant metabolites between B87 and B94.

Fig.5 KEGG enrichment analysis of differentially abundant metabolites (DAMs) in B87 and B94 A.Enriched pathways of up-regulated metabolites in B87:1.Tryptophan metabolism,2.D-Amino acid metabolism,3.Glycine,serine and threonine metabolism,4.Tyrosine metabolism;B.Enriched pathways of up-regulated metabolites in B94:1.Alpha-Linolenic acid metabolism,2.Glycine,serine and threonine metabolism,3.Tryptophan metabolism;C.Enriched pathways of down-regulated metabolites in B94:1.Carbon fixation in photosynthetic organisms,2.Alanine,aspartate and glutamate metabolism,3.Cyanoamino acid metabolism,4.Galactose metabolism,5.Aminoacyl-tRNA biosynthesis,6.Glycerophospholipid metabolism,7.D-Amino acid metabolism,8.One carbon pool by folate;D.Enriched pathways of down-regulated metabolites in B94:1.Alanine,aspartate and glutamate metabolism.

Fig.6 Cytoscape network of DEGs and DAMs A.B87;B.B94.Circles represent genes,squares represent metabolites,and red and green lines indicate positive and negative correlations,respectively.

Fig.7 Validation by qPCR Different letters above the rectangular box of each histogram represent that the expression level of the corresponding gene is significantly different between the control(CK) and the salt treatment (ST) (P<0.05).The heatmap in the lower right corner shows their situations in transcriptome analysis.

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