Genome-wide analysis of the Tritipyrum bHLH gene family and the response of TtbHLH310 in salt-tolerance

Abstract

Background: The bHLH transcription factor is prevalent across the plant kingdom and is crucial for various abiotic stress responses in different plant species. Tritipyrum, an octoploid created from an intergeneric cross between Triticum aestivum (AABBDD) and Thinopyrum elongatum (EE), serves as a significant source of germplasm, facilitating the incorporation of desirable traits from Th. elongatum into T. aestivum. With the recent availability of the complete genome sequences of T. aestivum and Th. elongatum, it has become feasible to investigate the organization and expression patterns of bHLH genes within the Tritipyrum genome.

Results: In this study, a total of 398 bHLH genes (TtbHLH) were identified within the Tritipyrum genome. These genes were classified into twenty major groups based on evolutionary analysis, indicating that they share conserved motif compositions. The TtbHLH genes are distributed across 28 chromosomes and include 67 duplication events. Synteny analysis suggests a common ancestral lineage for the bHLH gene family. Transcriptome data and quantitative polymerase chain reaction (qPCR) expression profiling identified 29 TtbHLH genes with significantly elevated expression levels in response to various salt-stress conditions and recovery treatments. Notably, Tel1E01T336100 (TtbHLH310) demonstrated a pronounced sensitivity to salt stress and is phylogenetically related to the salt-tolerant gene AtbHLH6 in Arabidopsis thaliana. Additionally, Pearson correlation analysis revealed 485 genes that exhibited a strong positive correlation (R > 0.9) with TtbHLH310 expression, which was enriched in pathways related to metabolic activities, cellular processes, stimulus responses, and biological regulation. Further analysis through real-time PCR confirmed that TtbHLH310 is highly expressed in the roots, stems, and leaves under salt-stress conditions.

Conclusions: The findings indicate that TtbHLH310 may play a pivotal role in enhancing salt stress tolerance in plants. Its strong expression in response to salt stress highlights its potential as a valuable foreign gene for improving salt tolerance in wheat. These insights contribute to our understanding of the molecular mechanisms underpinning abiotic stress responses in Tritipyrum and may aid in the development of more resilient wheat varieties.

Keywords: Tritipyrum; TtbHLH310; Expression patterns; Genome-wide; Salt-tolerance; bHLH.

Fig. 1

Chromosomal and subgenomes distribution of bHLH genes in Tritipyrum

Fig. 2

Phylogenetic relationships and distance among the bHLH proteins from Tritipyrum and A. thaliana.A: Phylogenetic relationships among 438 bHLH proteins from Tritipyrum, A. thaliana and O. sativa; B: genetic distance among the same clades of bHLH genes. The box plot shows the median (black line), interquartile range (box), and maximum and minimum scores (whiskers) of each data set

Fig. 3

Distribution, duplication and synteny analysis of bHLH genes in Tritipyrum. Collinear correlations of bHLH in Tritipyrum genomes are displayed by Circos. Tritipyrum chromosomes are colored according to the inferred ancestral chromosomes following an established convention. In the center, the relative map position of 391 bHLH genes is shown on each of the 28 Tritipyrum chromosomes

Fig. 4

Synteny analyses between Tritipyrum and five representative plant species. Gray lines in the background indicate collinear blocks within Tritipyrum and other plant genomes, while red lines highlight syntenic bHLH gene pairs

Fig. 5

Expression patterns of TtbHLH genes under salt stresses and recovery treatments. A: Hierachical clustering of expression profiles of 225 TtbHLH genes were expressed in 11 samples including salt stress and recovery treatment. B and C: The BP (B) and MF (C) analysis of 225 expression genes

Fig. 6

A-C: Expression analysis of 29 bHLH genes in eleven samples by qPCR. Data were normalized to β-actin gene and vertical bars indicate standard deviation. D: The relationships between qPCR and transcriptional of 29 up-regulated expression genes. Values are the log2 ratio (salt stress or recovery treatment /CK treatment) for genes. The determine coefficient (r²) is indicated in the figure. All qPCR reactions were performed in three biological replicates

Fig. 7

Expression patterns and correlation of TtbHLH310.A: Relative expression levels of TtbHLH310 in roots under salt stress and recovery conditions; B: Relative expression levels of TtbHLH310 in roots, stems, and leaves under salt stress; C and D: The BP (C) and MF (D) analysis of 485 positively related to TtbHLH310 expression genes; E: four hundred eighty-five genes positively related (R > 0.9) with TtbHLH310 expression