Genome-wide analysis of miR172-mediated response to heavy metal stress in chickpea (Cicer arietinum L.): physiological, biochemical, and molecular insights.

Background: Chickpea (Cicer arietinum L.), a critical diploid legume in the Fabaceae family, is a rich source of protein, vitamins, and minerals. However, heavy metal toxicity severely affects its growth, yield, and quality. MicroRNAs (miRNAs) play a crucial role in regulating plant responses to both abiotic and biotic stress, including heavy metal exposure, by suppressing the expression of target genes. Plants respond to heavy metal stress through miRNA-mediated regulatory mechanisms at multiple physiological, biochemical, and molecular levels. Although the Fabaceae family is well represented in miRNA studies, chickpeas have been notably underrepresented. This study aimed to investigate the effects of heavy metal-induced stress, particularly from 100 µM concentrations of cadmium (Cd), chromium (Cr), nickel (Ni), lead (Pb), and 30 µM arsenic (As), on two chickpea varieties: ILC 482 (sensitive) and Azkan (tolerant). The assessment focused on physiological, biochemical, and molecular parameters. Furthermore, a systematic characterization of the miR172 gene family in the chickpea genome was conducted to better understand the plant's molecular response to heavy metal stress.

Results: Variance analysis indicated significant effects of genotype (G), treatment (T), and genotype-by-treatment (GxT) interactions on plant growth, physiological, and biochemical parameters. Heavy metal stress negatively impacted plant growth in chickpea genotypes ILC 482 and Azkan. A reduction in chlorophyll content and relative leaf water content was observed, along with increased cell membrane damage. In ILC 482, the highest hydrogen peroxide (H₂O₂) levels in shoot tissue were recorded under As, Cd, and Ni treatments, while in Azkan, peak levels were observed with Pb treatment. Malondialdehyde (MDA) levels in root tissue were highest in ILC 482 under Cd and Ni exposure and in Azkan under As, Cr, and Cd treatments. Antioxidant enzyme activities, including superoxide dismutase (SOD), catalase (CAT), peroxidase (POD), and ascorbate peroxidase (APX), were significantly elevated under heavy metal stress in both genotypes. Gene expression analysis revealed upregulation of essential antioxidant enzyme genes, such as SOD, CAT, and APX, with APX showing notable increases in both shoot and root tissues compared to the control. Additionally, seven miR172 genes (miR172a, miR172b, miR172c, miR172d, miR172e, miR172f, and miR172g) were identified in the chickpea genome, distributed across five chromosomes. All genes exhibited conserved hairpin structures essential for miRNA functionality. Phylogenetic analysis grouped these miR172 genes into three clades, suggesting strong evolutionary conservation with other plant species. The expression analysis of miR172 and its target genes under heavy metal stress showed varied expression patterns, indicating their role in enhancing heavy metal tolerance in chickpea.

Conclusions: Heavy metal stress significantly impaired plant growth and physiological and biochemical parameters in chickpea genotypes, except for cell membrane damage. The findings underscore the critical role of miR172 and its target genes in modulating chickpea's response to heavy metal stress. These insights provide a foundational understanding for developing stress-tolerant chickpea varieties through miRNA-based genetic engineering approaches.