Aquaporins play important regulatory roles in improving plant abiotic stress tolerance. To better understand whether the OsPIP1 genes collectively dominate the osmotic regulation in rice under salt stress, a cluster editing of the OsPIP1;1, OsPIP1;2 and OsPIP1;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 OsPIP1;1 and OsPIP1;3 genes, and inserted a single T base in the OsPIP1;2 gene, respectively. While line 18 demonstrated an insertion of a single A base in the OsPIP1;1 gene and a single T base in both the OsPIP1;2 and OsPIP1;3 genes, respectively. Multiplex editing of the OsPIP1 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 OsPIP1 genes obviously suppressed the expressions of the OsPIP1, OsSOS1, OsCIPK24 and OsCBL4 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 OsPIP1 genes are essential modulators collectively contributing to the enhancement of rice salt stress tolerance, and multiplex editing of the OsPIP1 genes provides insight into the osmotic regulation of the PIP genes.

Plant mitochondrial phosphate transporters regulate phosphate transport and ATP synthesis. Determining whether they function in abiotic stress response process would shed light on their response to salt stress. We used the CRISPR/Cas9 gene-editing system to mutagenize two mitochondrial phosphate transporters, OsMPT3;1 and OsMPT3;2, to investigate their regulatory roles under salt stress. Two cas9 (CRISPR-associated protein 9)-free homozygous mutants, mpt33 and mpt30, were confirmed to be stable. Both OsMPT3;1 and OsMPT3;2 were markedly induced by salt stress, and their mutagenesis strongly inhibited growth and development, especially under salt stress. Mutagenesis sharply reduced the accumulation of ATP, phosphate, calcium, soluble sugar, and proline and increased osmotic potential, malondialdehyde, and Na⁺/K⁺ ratio under salt stress. Both mutants demonstrate normal growth and development in the presence of ATP, revealing high sensitivity to exogenous ATP under salt stress. The mutants showed lowered rates of Na⁺ efflux but also of K⁺ and Ca²⁺ influx under salt stress. Mutagenesis of OsMPT3;2 altered the enrichment profiles of differentially expressed genes involved mainly in synthesis of secondary metabolites, metabolism of glycolysis, pyruvate, tricarboxylic acid cycle, in response to salt stress. The mutant displayed significant accumulation differences in 14 metabolites involved in 17 metabolic pathways, and strongly up-regulated the accumulation of glutamine, a precursor in proline synthesis, under salt stress. These findings suggest that the OsMPT3 gene modulates phosphate transport and energy supply for ATP synthesis and triggers changes in accumulation of ions and metabolites participating in osmotic regulation in rice under salt stress, thus increasing rice salt tolerance. This study demonstrates the effective application of CRISPR/Cas9 gene-editing to the investigation of plant functional genes.