A cluster of mutagenesis revealed an osmotic regulatory role of the OsPIP1 genes in enhancing rice salt tolerance

Aquaporins play important regulatory roles in improving plant abiotic stress tolerance.To better understand whether the Os PIP1 genes collectively dominate the osmotic regulation in rice under salt stress,a cluster editing of the Os PIP1;1,Os PIP1;2 and Os PIP1;3 genes in rice was performed by CRISPR/Cas9 system.Sequencing showed that two mutants with Cas9-free,line 14 and line 18 were successfully edited.Briefly,line 14 deleted a single C base in both the Os PIP1;1 and Os PIP1;3 genes,and inserted a single T base in the Os PIP1;2 gene,respectively.While line 18 demonstrated an insertion of a single A base in the Os PIP1;1gene and a single T base in both the Os PIP1;2 and Os PIP1;3 genes,respectively.Multiplex editing of the Os PIP1 genes significantly inhibited photosynthetic rate and accumulation of compatible metabolites,but increased MDA contents and osmotic potentials in the mutants,thus delaying rice growth under salt stress.Functional loss of the Os PIP1 genes obviously suppressed the expressions of the Os PIP1,Os SOS1,Os CIPK24 and Os CBL4 genes,and increased the influxes of Na+and effluxes of K^(+)/H^(+)in the roots,thus accumulating more Na+in rice mutants under salt stress.This study suggests that the Os PIP1 genes are essential modulators collectively contributing to the enhancement of rice salt stress tolerance,and multiplex editing of the Os PIP1 genes provides insight into the osmotic regulation of the PIP genes.