Abstract
OsIPK1 encodes inositol 1,3,4,5,6-pentakisphosphate 2-kinase, which catalyzes the conversion of myo-inositol-1,3,4,5,6-pentakisphosphate to myo-inositol-1,2,3,4,5,6-hexakisphosphate (IP6) in rice. By clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein (Cas9)-mediated mutagenesis in the 3rd exon of the gene, three OsIPK1 mutations, i.e., osipk1_1 (a 33-nt deletion), osipk1_2 (a 1-nt deletion), and osipk1_3 (a 2-nt deletion) were identified in T0 plants of the rice line Xidao #1 (wild type, WT). A transfer DNA free line with the homozygous osipk1_1 mutation was developed; however, no homozygous mutant lines could be developed for the other two mutations. The comparative assay showed that the osipk1_1 mutant line had a significantly lower level of phytic acid (PA, IP6; −19.5%) in rice grain and agronomic traits comparable to the WT. However, the osipk1_1 mutant was more tolerant to salt and drought stresses than the WT, with significantly lower levels of inositol triphosphate (IP3), reactive oxygen species (ROS) and induced IP6, and higher activities of antioxidant enzymes in seedlings subjected to these stresses. Further analyses showed that the transcription of stress response genes was significantly upregulated in the osipk1_1 mutant under stress. Thus, the low phytic acid mutant osipk1_1 should have potential applications in rice breeding and production.
Highlights
Phytic acid (PA), identified as Myo-inositol-1,2,3,4,5,6-hexakisphosphate (IP6 ), is thought to be the main storage form of nutrient phosphorous (P) (~80%) constituting~1.6% of the dry biomass in crop grains [1]
Twenty-one hygromycin phosphotransferase (HPT)-positive T0 rice seedlings were selected from transformation with the vector of clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), pH_ipk1, and eight rice seedlings were verified mutated at the position of target
To explore how mutation of osipk1_1 alleviated salt and drought stress, we measured the contents of IP3, IP6, stress-related free amino acid proline (Pro), Reactive Oxygen Species (ROS) (H2O2), malondialdehyde (MDA), and anti-oxidant enzyme activities (peroxidase (POD), catalase (CAT), and superoxide dismutase (SOD)) in seedlings subjected to stresses (Figures 5–7)
Summary
Phytic acid (PA), identified as Myo-inositol-1,2,3,4,5,6-hexakisphosphate (IP6 ), is thought to be the main storage form of nutrient phosphorous (P) (~80%) constituting. The undigested phytic acid phosphorus (PA-P) in animal wastes has gradually become one of the main causes of P pollution [5] Because of these PArelated environmental and nutritional adverse effects, mutagenesis and biotechnological approaches have been used to produce low phytic acid (lpa) mutant lines in cereals [6] such as maize [3,7], wheat [8], and rice [9,10,11,12,13]. We generated osipk mutants by CRISPR/Cas9-mediated mutagenesis and analyzed seed accumulation of phosphorus (total phosphorus, TP; phytic acid phosphorus, PA-P; inorganic phosphorus, Pi), agronomic traits, seed germination, and stress tolerance with the aim to evaluate and explore the probability of producing the LPA rice with no adverse effect on agronomic traits and seed viability, and with better tolerance to salt and drought stresses
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