Forskolin Improves Salt Tolerance of Gossypium Hirsutum L. by Upregulation of GhLTI65

Cyclic AMP (cAMP) is a pivotal signaling molecule existing in almost all living organisms. It is an intracellular non-protein small molecule that regulates the activity of intracellular enzymes and non-enzyme proteins when its concentration changes in response to the binding of extracellular signals to cell surface receptors. As such, cAMP is referred to as a second messenger with an important role in cell signaling and modulation a variety of cellular responses. cAMP can help plants cope with abiotic stress, but the mechanism by which it improves salt tolerance has not been elucidated. In our study, we treated cotton with Forskolin (a cAMP activator) to increase cAMP levels, and found that it improved cotton seed tolerance to salt stress, thereby improving the germination rate of seeds in saline soil. Through transcriptome analysis, we characterized the responses of ABA, proteins, and pathways to salt stress after cAMP signaling, and four genes related to salt tolerance and signal transduction were identified. Three of them, GhLTI65, GhGOLS2, and GhNAC002 were silenced by virus induced gene silencing (VIGs), and the plant tolerance to salt stress was significantly reduced as compared with the control plants. Among these, we found that GhLTI65 (RD29B) was not only involved in the salt stress response, but played a role in the cAMP signaling cascade. We further performed yeast two-hybrid assays and bimolecular fluorescence complementation (BiFC) to determine that GhLTI65 interacts with the SNF1-related protein kinase GhPV42A in the cytoplasm, and that their co-expression is regulated by ABA-related signaling. We propose that ABA and cAMP signaling increase the expression of GhPV42A by integrating related mitogen-activated protein kinase (MAPK)cascade signaling, and that GhPV42A further interacts with GhLTI65 to increase its expression, thereby helping plants to receive signals and to respond to salt stress.