Genome-wide identification of plasma-membrane intrinsic proteins in pumpkin and functional characterization of CmoPIP1-4 under salinity stress

Plasma membrane intrinsic proteins ( PIPs ) play a crucial role in the salinity and drought tolerance of plants. PIPs operate as vital water conduits for regulating water homeostasis during salt stress. Pumpkin is an important Cucurbitaceae crop, and no research on PIPs in pumpkin has been carried out. The current study, presents the first genome-wide characterization of the PIP gene family and functional characterization of CmoPIP1–4 with knockout mutants in pumpkin. According to their phylogeny, 21 CmoPIP genes have been identified and divided into 2 subgroups: CmoPIP1s and CmoPIP2s . Functional studies on their NPA motifs, ar/R filter, and Froger's positions predicted that these CmoPIP proteins would be substrate specific. The CmoPIP1s expression was found to be responsive to salt stress in leaves and roots. CmoPIP1–4 plays an important role in the salt stress tolerance of pumpkin, and the overexpression of CmoPIP1–4 confers salinity tolerance to yeast. However, hyper sensitivity to salt was observed in CmoPIP1–4 CRISPR plants phenotypically (weaker health, lower relative water contents and reduced photosynthetic activities), physiologically (higher Na + /K + ratios, malondialdehyde, ROS and membrane stability index, lower the contents of proline, glycine betaine and also decreased antioxidative enzyme activities), and molecularly (lower expression levels of salt-associated genes CmoSOS1 , CmoHKT1;1 , CmoNHX4 , and a lower expression levels antioxidative genes CmoSOD1/2, CmoPOD1/2 and CmoCAT1/2 ). This study highlights that CmoPIP1–4 is a key player in the stress-signaling pathway in pumpkin plants and could be valuable for developing stress-resistant cucurbit crops in the future. • Twenty-one plasma membrane intrinsic proteins ( PIPs ) were genome-wide characterized in Cucurbita moschata . • Salinity stress decreases the expression of most CmoPIPs . • Overexpression of CmoPIP1-4 conferred salt tolerance in yeast. • A salt-sensitive phenotype was observed after the CmoPIP1-4 knockout in pumpkin roots.