Allelic Variants of CRISPR/Cas9 Induced Mutation in an Inositol Trisphosphate 5/6 Kinase Gene Manifest Different Phenotypes in Barley

Inositol trisphosphate 5/6 kinases (ITPK) constitute a small group of enzymes participating in the sequential phosphorylation of inositol phosphate to inositol hexakisphosphate (IP6), which is a major storage form of phosphate in cereal grains. The development of lines with reduced IP6 content could enhance phosphate and mineral bioavailability. Moreover, plant <i>ITPK</i>s participate in abiotic stress signaling. To elucidate the role of <i>HvITPK1</i> in IP6 synthesis and stress signaling, a barley <i>itpk1</i> mutant was created using programmable nuclease Cas9. Homozygous single bp insertion and deletion mutant lines were obtained. The mutants contained altered levels of phosphate in the mature grains, ranging from 65% to 174% of the wild type (WT) content. Homozygous mutant lines were tested for their response to salinity during germination. Interestingly, insertion mutant lines revealed a higher tolerance to salinity stress than deletion mutants. Mature embryos of an insertion mutant <i>itpk1-2</i> and deletion mutant <i>itpk1-33</i> were cultivated in vitro on MS medium supplemented with NaCl at 50, 100, and 200 mM. While both mutants grew less well than WT on no or low salt concentrations, the <i>itpk1-2</i> mutant was affected less than the WT and <i>itpk33</i> when grown on the highest NaCl concentration. The expression of all <i>ITPK</i>s was induced in roots in response to salt stress. In shoots, the differential effect of high salt on <i>IPTK </i>expression in the two <i>iptk1</i> mutants was consistent with their different sensitivities to salt stress. The results extend the evidence for the involvement of <i>ITPK</i> genes in phosphate storage and abiotic stress signaling.