Genome-Wide Analysis of Gene Expression Provides New Insights into Waterlogging Responses in Barley (Hordeum vulgare L.)

Abstract

Waterlogging is a major abiotic stress causing oxygen depletion and carbon dioxide accumulation in the rhizosphere. Barley is more susceptible to waterlogging stress than other cereals. To gain a better understanding, the genome-wide gene expression responses in roots of waterlogged barley seedlings of Yerong and Deder2 were analyzed by RNA-Sequencing. A total of 6736, 5482, and 4538 differentially expressed genes (DEGs) were identified in waterlogged roots of Yerong at 72 h and Deder2 at 72 and 120 h, respectively, compared with the non-waterlogged control. Gene Ontology (GO) enrichment analyses showed that the most significant changes in GO terms, resulted from these DEGs observed under waterlogging stress, were related to primary and secondary metabolism, regulation, and oxygen carrier activity. In addition, more than 297 transcription factors, including members of MYB, AP2/EREBP, NAC, WRKY, bHLH, bZIP, and G2-like families, were identified as waterlogging responsive. Tentative important contributors to waterlogging tolerance in Deder2 might be the highest up-regulated DEGs: Trichome birefringence, α/β-Hydrolases, Xylanase inhibitor, MATE efflux, serine carboxypeptidase, and SAUR-like auxin-responsive protein. The study provides insights into the molecular mechanisms underlying the response to waterlogging in barley, which will be of benefit for future studies of molecular responses to waterlogging and will greatly assist barley genetic research and breeding.

Keywords: RNA-Seq; barley; differentially expressed genes; transcription factors; waterlogging stress.

Figure 1

Cluster analysis of the genome-wide gene expression profiles of barley roots under waterlogging and control. Gene expression values (FPKM, fragments per kb transcript length per million reads of library size) were centralized by row using the Heatmap3 program. The genes were clustered by distances. The color scale in the above heatmap shows the expression level; blue indicates low transcript abundance while red indicates high abundance. YC0, Yerong 0 h control; YC72, Yerong 72 h control; YW72, Yerong 72 h of waterlogging; DC0, Deder2 0 h control; DC72, Deder2 72 h control; DC120, Deder2 120 h control; DW72, Deder2 72 h waterlogging; DW120, Deder2 120 h waterlogging.

Figure 2

Differentially expressed genes (DEGs) under waterlogging: (A) Volcano plot illustrating number of DEGs between flooded and control treatments of Yerong at 72 h (a), and Deder2 at 72 h (b) and 120 h (c). (B) Venn diagram illustrating unique and common DEGs in barley roots: (a) number of DEGs in Deder2 at 72 and 120 h of waterlogging stress; (b) number of unique and shared DEGs between Yerong at 72 h, and Deder2 at 72 and 120 h post waterlogging treatment. YW72, Yerong 72 h of waterlogging; DW72, Deder2 72 h waterlogging; DW120, Deder2 120 h waterlogging.

Figure 3

Gene Ontology (GO) enrichment analysis. The GO enrichment analyses were performed on waterlogging group against controls. The adjusted p-values of the enrichment significant were transformed by –log10 and clustered by GO terms. A darker color indicates greater significance. YW72YC0, Yerong 72 h waterlogging against 0 h control; YW72YC72, Yerong 72 h waterlogging against 72 h control; DW72DC0, Deder2 72 h waterlogging against 0 h control; DW72DC72, Deder2 72 h waterlogging against 72 h control; DW120DC0, Deder2 120 h waterlogging against 0 h control; DW120DC120, Deder2 120 h waterlogging against 120 h control; YC0DC0, Yerong 0 h control against Deder2 0 h control.

Figure 4

Summary of enriched Gene Ontology (GO) terms of down-regulated (left side) and up-regulated (right side) genes in waterlogged roots of Yerong at 72 h (YW72), as well as Deder2 under waterlogging stress at 72 h (DW72) and 120 h (DW120). Circle size represents number of genes in the GO term, and connections among circles represent overlapping gene sets of each GO term. Larger clusters are generally named based on contained GO terms. GO terms containing more than 500 genes were removed.

Figure 5

Transcription factors identified in differentially expressed genes in Yerong roots at 72 h (A) of waterlogging stress, and Deder2 roots at 72 (B) and 120 h (C). YW72, Yerong 72 h of waterlogging; DW72, Deder2 72 h waterlogging; DW120, Deder2 120 h waterlogging.

Figure 6

Schematic diagram of the main waterlogging stress responses in the roots of moderately-tolerant (72 h) and tolerant Deder2 (72 and 120 h) barley genotypes. NADP-GADH, NADP-dependent glyceraldehyde-3-phosphate dehydrogenase; PK, pyruvate kinase; ADH, alcohol dehydrogenase; LDH, lactate dehydrogenase; INV, acid β-fructofuranosidase; AS, asparagine synthetase; GS, glutamine synthetase; GOGAT, glutamate synthase; ACC, 1-aminocyclopropane-1-carboxylic acid synthase; ACO, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase; GST, glutathione S-transferase; MYB, myeloblastosis; AP2/EREBP, APETALA2/Ethylene-responsive Element Binding Proteins; NAC, this acronym is derived from three genes that were initially discovered to contain a particular domain (the NAC domain): NAM (for no apical meristem), ATAF1 and −2, and CUC2 (for cup-shaped cotyledon); WRKY, this TF binds a specific promoter sequence of the target gene, known as a W-box and the WRKY proteins contain one or two DNA binding domains of 60 amino acids containing the conserved heptapeptide WRKYGQK. Blue arrow indicates decreased expression, red arrow indicates increased expression, and black arrow indicates down-and up-regulation in Yerong roots at 72 h and Deder2 roots at 72 and 120 h of waterlogging treatment.