*3.4. Identification of CBL10 Interacting Proteins from the Cotton AD Library under Drought and Salt Stress Using the Y2H System*

To confirm the potential role of *GhSAMS2* in abiotic stress tolerance in upland cotton, we searched for prey proteins interacting with *GhCBL10*, the salt responsive gene, using the Yeast two-hybrid (Y2H) system. The summary of the experiments, including the self-auto-activation state, the toxicity test, the verification of the interactions, and the mating efficiency determination of the *GhCBL10* bait gene in the Y2H system, is shown in Figure S2. The zygotes that resembled a cloverleaf with a three-lobed structure (Figure S2e) confirmed the successful mating between *GhCL10* and the prey proteins contained in the cotton AD library. The *GhCBL10* bait protein did not auto-activate the reporter genes in the Y2HGold cells in the absence of a prey protein, confirming the suitability of the results. We identified 23 prey proteins that showed interaction with *GhCBL10* (Table 3). Of them, only four proteins, *PRA1 B1*, *DSP8*, *CAB-151*, and *SAMS2*, could activate the expression of the reporter genes in diploid yeast cells. The SAMS2 gene showed the highest interaction frequency with the CBL10 bait protein, supporting its importance for upland cotton's tolerance to abiotic stress.

**Figure 3.** Heat maps showing *GhSAMS* genes' differential expression in *G. hirsutum* under drought and salt stress conditions. RT-qPCR analysis of the expression of *GhSAMS* genes under drought stress in the leaf (**a**) and roots (**b**). RT-qPCR analysis of the expression of *GhSAMS* genes under salt stress in the leaf (**c**) and roots (**d**). The higher expression level, the lower expression level, and no expression of the *GhSAMS* genes at a particular time are depicted by red, green, and black colors, respectively.
