Functional characterization of CiCIPK8 and CiCIPK11 reveals their roles in osmotic stress via CBL-CIPK signaling in pecan (Carya illinoinensis).

Osmotic stress severely limits plant growth and agricultural productivity, primarily by disrupting cellular water balance. Although the CBL-CIPK signaling network is recognized as a central mediator of abiotic stress responses, the functions of CiCIPKs from pecan (Carya illinoinensis) in osmotic stress tolerance remain largely unclear. In this study, two osmotic stress-responsive genes, CiCIPK8 and CiCIPK11, were cloned and functionally characterized via heterologous expression in Arabidopsis. Under mannitol-induced osmotic stress, transgenic lines exhibited significantly enhanced seed germination and root elongation compared with the wild-type (WT) plants. Physiological analyses revealed that overexpression of CiCIPK8 and CiCIPK11 improved osmotic stress tolerance by increasing antioxidant enzyme activities, reducing oxidative damage, and promoting the accumulation of compatible osmolytes, including proline, soluble proteins, and soluble sugars. Transcriptomic profiling, combined with quantitative reverse transcription-polymerase chain reaction (qRT-PCR) validation, demonstrated that CiCIPK8 and CiCIPK11 coordinately regulate multiple stress-responsive pathways, including plant hormone signal transduction, mitogen-activated protein kinase (MAPK) cascades, cell wall dynamics, and metabolic regulation. In addition, yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays demonstrated that CiCIPK8 interacted with CiCBL1 and CiCBL8, whereas CiCIPK11 specifically interacted with CiCBL1, suggesting the involvement of a Ca2+-dependent CBL-CIPK signaling module. In conclusion, these findings reveal that CiCIPK8 and CiCIPK11 function as positive regulators of osmotic stress tolerance by integrating ROS scavenging, osmotic adjustment, and transcriptional reprogramming. This study provides valuable insights into stress signaling in pecan and offers candidate targets for the molecular breeding of osmotic stress-tolerant woody crops.