Genome-wide identification of bHLH transcription factors related to plant development and abiotic stress response in sand rice (Agriophyllum squarrosum (L.) Moq.)).

Sand rice (Agriophyllum squarrosum (L.) Moq.) is widely distributed across mobile and semi-mobile sand dunes in Central Asia and can thrive in extreme environments, including drought, salinity, high temperatures, and UV radiation. The basic helix-loop-helix (bHLH) transcription factors (TFs) play important roles in plant growth and development, as well as in responses to various environmental stresses. However, a comprehensive analysis of the bHLH family in sand rice has not yet been conducted. In this study, a total of 69 bHLH genes (AsbHLHs) were identified and classified into 18 subfamilies based on phylogenetic analysis. Their physicochemical properties, bHLH domains, conserved motifs, and gene structures were further examined. Most AsbHLHs within the same subfamily shared similar characteristics. cis-Regulatory elements (CREs) and Gene Ontology (GO) analyses suggested that AsbHLHs are involved in plant development, hormonal signaling, and stress responses. Transcriptomic analysis revealed tissue- and stress-responsive-specific expression profiles of AsbHLHs. qRT-PCR validation confirmed the expression of 69 AsbHLHs across different tissues and 17 genes under salt and drought stress, with AsbHLH7, 10, 20, 22, 32, 46, 53, 59, 64, 65, 68, and 69 proposed as potential regulators in stress response. Protein-protein interaction network predications showed that these proteins may potentially form complexes, which could participate in stress-related biological processes. Statistical analyses, including Mantel's test and redundancy analysis (RDA), revealed significant correlations between gene structural features and expression profiles. These findings suggest a potential framework for exploring the role of bHLH TFs in stress tolerance and adaptive mechanisms in sand rice.