Transcriptome profiling at osmotic and ionic phases of salt stress response in bread wheat uncovers trait-specific candidate genes

Abstract

Background: Bread wheat is one of the most important crops for the human diet, but the increasing soil salinization is causing yield reductions worldwide. Improving salt stress tolerance in wheat requires the elucidation of the mechanistic basis of plant response to this abiotic stress factor. Although several studies have been performed to analyze wheat adaptation to salt stress, there are still some gaps to fully understand the molecular mechanisms from initial signal perception to the onset of responsive tolerance pathways. The main objective of this study is to exploit the dynamic salt stress transcriptome in underlying QTL regions to uncover candidate genes controlling salt stress tolerance in bread wheat. The massive analysis of 3'-ends sequencing protocol was used to analyze leave samples at osmotic and ionic phases. Afterward, stress-responsive genes overlapping QTL for salt stress-related traits in two mapping populations were identified.

Results: Among the over-represented salt-responsive gene categories, the early up-regulation of calcium-binding and cell wall synthesis genes found in the tolerant genotype are presumably strategies to cope with the salt-related osmotic stress. On the other hand, the down-regulation of photosynthesis-related and calcium-binding genes, and the increased oxidative stress response in the susceptible genotype are linked with the greater photosynthesis inhibition at the osmotic phase. The specific up-regulation of some ABC transporters and Na⁺/Ca²⁺ exchangers in the tolerant genotype at the ionic stage indicates their involvement in mechanisms of sodium exclusion and homeostasis. Moreover, genes related to protein synthesis and breakdown were identified at both stress phases. Based on the linkage disequilibrium blocks, salt-responsive genes within QTL intervals were identified as potential components operating in pathways leading to salt stress tolerance. Furthermore, this study conferred evidence of novel regions with transcription in bread wheat.

Conclusion: The dynamic transcriptome analysis allowed the comparison of osmotic and ionic phases of the salt stress response and gave insights into key molecular mechanisms involved in the salt stress adaptation of contrasting bread wheat genotypes. The leveraging of the highly contiguous chromosome-level reference genome sequence assembly facilitated the QTL dissection by targeting novel candidate genes for salt tolerance.

Keywords: Bread wheat; Comparative transcriptomics; Ionic stress; Osmotic stress; QTL dissection; Salt stress.

Fig. 1

Representation of the prolonged 3′-end of the gene TraesCS7D02G051200 with reads (in green) mapping beyond the gene model

Fig. 2

Photosynthesis rate curve of contrasting wheat genotypes studied during the osmotic phase adapted from Dadshani [27]. The shadows represent the standard deviation of the measurements, and the time points selected for the transcriptomic analysis are highlighted with blue arrows

Fig. 3

Venn diagrams of the salt-responsive genes in the contrasting genotypes studied. The total number of genes in each genotype and/or time point are shown above each diagram. (A) Diagram of the salt-responsive genes in Syn86 by time point; (B) diagram of the salt-responsive genes in Zentos by time point; (C) diagram of the salt-responsive genes in the two sampled days from the ionic stress phase; (D) diagram with the four genotypes. The blue number represents the genes shared by the tolerant genotypes, while the red number indicates the genes shared by the salt-susceptible

Fig. 4

Distribution of up- and down-regulated salt-responsive genes across stress time points. (A) Osmotic phase and (B) ionic phase time points

Fig. 5

GO terms over-represented during the salt stress response. (A) Up-regulated and (B) down-regulated categories identified in the four stress time points sampled during the osmotic phase; (C) up- and down-regulated categories observed in the two stress time points from the ionic phase. Bold ontologies are categories specific for each heatmap. The –log₁₀ transformation of the corrected p-values highlights the categories with greater significance that are therefore better over-represented. Transformed values > 3 are significant (corrected p-value < 0.001)

Fig. 6

Generalized fold change (GFOLD) values of the time course relative expression of selected gene ontologies in the contrasting genotypes at the osmotic phase. In each expression profile frame, the gray lines show the time course expression pattern of each gene, and the red or green lines are LOESS (locally estimated scatterplot smoothing) curves that represent the expression tendency of the clusters of genes

Fig. 7

Relative expression (GFOLD value) of transcripts from the transmembrane transport category with a role in ion homeostasis at 24 days after stress

Fig. 8

Overview of salt-responsive genes in QTL intervals in chromosome 2A. (A) Marker RAC875_c38018_278 detected by association mapping [28] and (B) marker BS00041707_51 detected by AB-QTL mapping [27]. Salt-responsive genes are marked with colors. The chromosome regions were retrieved from Ensembl Plants release 46 [41]

Fig. 9

Relative expression values calculated with the ∆∆ Ct method [47]. (A) TraesCS2D02G173600 expression with TaEf-1.2 as internal control. (B) TraesCS5D02G238700 expression with TaEf-1.1 as internal control. Different letters show significant differences in mean values from the two genotypes (p < 0.05). Mean relative expression values > 2.0 or < 1.0 (p < 0.05) indicated up-regulation (↑) or down-regulation (↓) of genes, respectively