ZnO nanoparticles alleviate salinity stress through transcriptional and antioxidant regulation in chickpea

This study evaluated the potential ameliorative effects of zinc oxide nanoparticles (ZnO NPs) on chickpea under salinity stress and examined the possible involvement of selected members of the bHLH transcription factor family in this response. A genome-wide analysis identified 48 CabHLH genes in the Cicer arietinum genome, and their structural features, chromosomal distribution, duplication patterns, phylogenetic relationships, conserved motifs, promoter cis-elements, and predicted interaction profiles were characterized using bioinformatics approaches. The expression responses of selected CabHLH genes were then assessed by RT-qPCR in root and shoot tissues of two chickpea genotypes differing in salt-stress response, Aydın-92 and ILC 482, under NaCl stress and ZnO NP treatments. Salinity stress markedly inhibited root and shoot development, reduced photosynthetic performance, impaired membrane stability, and increased oxidative stress markers. ZnO NP application partially mitigated these adverse effects in a dose- and genotype-dependent manner, particularly by improving growth-related traits, maintaining chlorophyll status, reducing membrane injury, and modulating proline accumulation and antioxidant-related responses. The RT-qPCR results showed that several CabHLH genes, including CabHLH-1, CabHLH-5, CabHLH-6, and CabHLH-12, exhibited genotype, tissue, and treatment-dependent expression patterns under salinity and ZnO NP treatments. CabHLH-1 showed contrasting expression responses between the tolerant and sensitive genotypes, whereas CabHLH-5 and CabHLH-6 displayed relatively broad transcriptional responsiveness across tissues and treatments. The combined ZnO NP + NaCl treatments produced dose-, genotype-, and gene-dependent responses. Although 50 mg L^-1 ZnO NP improved several physiological and transcriptional responses under salt stress, this effect was not uniform across all CabHLH genes, and some transcripts showed stronger responses at 25 mg L^-1. Overall, these findings suggest that ZnO NPs may contribute to salinity-stress mitigation in chickpea through coordinated physiological, biochemical, and transcriptional responses, while the identified CabHLH genes should be considered candidate components for future functional validation.