Transcriptome analysis and physiological changes in the leaves of two Bromus inermis L. genotypes in response to salt stress

Abstract

Soil salinity is a major factor threatening the production of crops around the world. Smooth bromegrass (Bromus inermis L.) is a high-quality grass in northern and northwestern China. Currently, selecting and utilizing salt-tolerant genotypes is an important way to mitigate the detrimental effects of salinity on crop productivity. In our research, salt-tolerant and salt-sensitive varieties were selected from 57 accessions based on a comprehensive evaluation of 22 relevant indexes, and their salt-tolerance physiological and molecular mechanisms were further analyzed. Results showed significant differences in salt tolerance between 57 genotypes, with Q25 and Q46 considered to be the most salt-tolerant and salt-sensitive accessions, respectively, compared to other varieties. Under saline conditions, the salt-tolerant genotype Q25 not only maintained significantly higher photosynthetic performance, leaf relative water content (RWC), and proline content but also exhibited obviously lower relative conductivity and malondialdehyde (MDA) content than the salt-sensitive Q46 (p < 0.05). The transcriptome sequencing indicated 15,128 differentially expressed genes (DEGs) in Q46, of which 7,885 were upregulated and 7,243 downregulated, and 12,658 DEGs in Q25, of which 6,059 were upregulated and 6,599 downregulated. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that the salt response differences between Q25 and Q46 were attributed to the variable expression of genes associated with plant hormone signal transduction and MAPK signaling pathways. Furthermore, a large number of candidate genes, related to salt tolerance, were detected, which involved transcription factors (zinc finger proteins) and accumulation of compatible osmolytes (glutathione S-transferases and pyrroline-5-carboxylate reductases), etc. This study offers an important view of the physiological and molecular regulatory mechanisms of salt tolerance in two smooth bromegrass genotypes and lays the foundation for further identification of key genes linked to salt tolerance.

Keywords: Bromus inermis L.; accessions evaluation; physiological analysis; salt stress; transcriptome analysis.

Figure 1

The subordinate function values analysis for comprehensive evaluation of salt tolerance among 57 smooth bromegrass accessions. Subordinate function values: the value was calculated based on the switching function vector analysis. D value: the value of weighted membership function; the higher the D value, the stronger the salt tolerance of the genotype.

Figure 2

Effects of 300 mM NaCl on phenotype of smooth bromegrass seedlings under different salt stress times. ST, salt-tolerant genotype; SS, salt-sensitive genotype; CK, control treatment; T, salt treatment.

Figure 3

Effects of stress on physiological indexes of smooth bromegrass seedlings under different salt stress times. (A) MDA content. (B) Relative electrolyte leakage. (C) Relative water content. (D) Chlorophyll content (SPAD). (E) Proline content. (F) Soluble protein content. (G) Soluble sugar content. Significantly different means are shown with different letters, calculated using Tukey’s test (p < 0.05). ST, salt-tolerant genotype; SS, salt-sensitive genotype; CK, control treatment; T, salt treatment; MDA, malondialdehyde; SPAD, Soil Plant Analysis Development.

Figure 4

Comparison of DEG expression levels of different treatments and PCA with different treatments in smooth bromegrass. (A) Density map: the horizontal axis is the log10 (FPKM) value of the gene, and the vertical axis is the distribution density of the gene corresponding to the expression amount. (B) FPKM distribution: the middle horizontal line of the box is the median, the upper and lower edges of the box are 75%, and the upper and lower limits are 90%. The external shape is the kernel density estimation. (C) PCA. DEG, differentially expressed gene; PCA, principal component analysis.

Figure 5

The comprehensive description of the transcriptome analysis of the salt stress response of smooth bromegrass. (A) Bar charts of up- and downregulated genes in groups. (B) Venn diagram of upregulated and downregulated DEGs in the comparison group of T-0_vs_T-9 and S-0_vs_S-9. (C) Venn diagram of upregulated and downregulated DEGs in the comparison group of T-9_vs_S-9 and T-0_vs_S-0. (D) K-mean cluster analysis. DEGs, differentially expressed genes.

Figure 6

Different TFs of smooth bromegrass in response to salt stress. (A) T-0_vs_T-9. (B) S-0_vs_S-9. The chart shows the different kinds of transcription factors (TFs) on the vertical axis and the quantity of transcription factors on the horizontal axis.

Figure 7

GO pathway enrichment plot for gene expression during salt stress. (A) T-0_vs_T-9. (B) S-0_vs_S-9. (C) S-0_vs_T-0. (D) S-9_vs_T-9. The size and color of the circle indicate the number of transcripts and the significance value (p-value) of the rich factor, respectively. GO, Gene Ontology.

Figure 8

Scatterplot of KEGG pathway enrichment for DEGs under salt stress. (A) S-0_vs_S-9. (B) T-0_vs_T-9. (C) S-0_vs_T-0. (D) S-9_vs_T-9. The size and color of the circle indicate the number of transcripts and the significance value (p-value) of the rich factor, respectively. KEGG, Kyoto Encyclopedia of Genes and Genomes; DEG, differentially expressed gene.

Figure 9

Expression map of genes related to plant hormone signaling transduction. S, salt-sensitive genotype; T, salt-tolerant genotype.

Figure 10

Heat map of gene expression related to MAPK signal pathway—plant. S, salt-sensitive genotype; T, salt-tolerant genotype.

Figure 11

Heat map of DEG expression associated with photosynthetic, antioxidant, ROS, and ABC transport pathways under NaCl stress. S, salt-sensitive genotype; T, salt-tolerant genotype; DEG, differentially expressed gene; ROS, reactive oxygen species.

Figure 12

Heat map of the expression of the DEGs associated with interested pathways under salt stress. S, salt-sensitive genotype; T, salt-tolerant genotype; DEGs, differentially expressed genes.