Identification of the bHLH transcription factor family and functional characterization of CibHLH141 and CibHLH127 in response to iron deficiency stress of pecan (Carya illinoinensis).

The basic helix-loop-helix (bHLH) gene family plays vital roles across various plant species. However, the bHLH family has not been reported in pean (Carya illinoinensis). In this study, 164 CibHLHs were identified, exhibiting high evolutionary conservation as revealed by gene structure, conserved motifs, domains, and collinearity analyses. Promoter analysis revealed that many CibHLHs contain cis-acting elements related to abiotic stress responses. CibHLH141 and CibHLH127 were significantly upregulated in both leaves and roots of pecan under iron deficiency. The subcellular localization investigation confirmed that CibHLH141 and CibHLH127 are nuclear localization proteins. Transcriptional activity assays demonstrated that both possess transcriptional activity, with the active region located at the N-terminus. Transient overexpression in tobacco and stable overexpression in Arabidopsis confirmed their positive roles in enhancing tolerance to iron deficiency. Furthermore, in vivo and in vitro assays revealed stable interactions between CibHLH141, CibHLH127 and CibHLH119, with CibHLH119 also implicated in the iron regulatory network. Overall, these findings advance our understanding of the CibHLH gene family and lay a foundation for elucidating the molecular mechanisms of iron homeostasis in pecan. Moreover, this study offers theoretical support for breeding stress-resilient pecan cultivars, enhancing their ecological and economic value.