Mutagenesis Reveals That the OsPPa6 Gene Is Required for Enhancing the Alkaline Tolerance in Rice

Abstract

Alkaline stress (AS) is one of the abiotic stressful factors limiting plant’s growth and development. Inorganic pyrophosphatase is usually involved in a variety of biological processes in plant in response to the abiotic stresses. Here, to clarify the responsive regulation of inorganic pyrophosphatase in rice under AS, the mutagenesis of the OsPPa6 gene encoding an inorganic pyrophosphatase in rice cv. Kitaake ( Oryza sativa L. ssp. japonica) was performed by the CRISPR/Cas9 system. Two homozygous independent mutants with cas9-free were obtained by continuously screening. qPCR reveals that the OsPPa6 gene was significantly induced by AS, and the mutagenesis of the OsPPa6 gene apparently delayed rice’s growth and development, especially under AS. Measurements demonstrate that the contents of pyrophosphate in the mutants were higher than those in the wild type under AS, however, the accumulation of inorganic phosphate, ATP, chlorophyll, sucrose, and starch in the mutants were decreased significantly, and the mutagenesis of the OsPPa6 gene remarkably lowered the net photosynthetic rate of rice mutants, thus reducing the contents of soluble sugar and proline, but remarkably increasing MDA, osmotic potentials and Na + /K + ratio in the mutants under AS. Metabonomics measurement shows that the mutants obviously down-regulated the accumulation of phosphorylcholine, choline, anthranilic acid, apigenin, coniferol and dodecanoic acid, but up-regulated the accumulation of L-valine, alpha-ketoglutarate, phenylpyruvate and L-phenylalanine under AS. This study suggests that the OsPPa6 gene is an important osmotic regulatory factor in rice, and the gene-editing of CRISPR/Cas9-guided is an effective method evaluating the responsive regulation of the stress-induced gene, and simultaneously provides a scientific support for the application of the gene encoding a soluble inorganic pyrophosphatase in molecular breeding.

Creation of novel alleles of fragrance gene OsBADH2 in rice through CRISPR/Cas9 mediated gene editing

“…Genome editing for multiple susceptible genes have been targeted to confer broad spectrum resistance against blight disease in rice [ 55 – 57 ]. Subsequently, CRISPR/Cas9 was applied for improving important quantitative and qualitative traits in rice including editing of OsSAPK2 regulating drought responses [ 58 ], OsGIF1 regulating size of stem, leaves and grain [ 59 ], OsMGD2 regulating grain quality [ 60 ], OsARM1 controlling tolerance against for arsenic toxicity [ 61 ], OsPPa6 contributing for tolerance against alkalinity [ 62 ] and OsBADH2 leading to aroma in rice [ 35 ]. Based on the above evidences, this study was undertaken with an aim of demonstrating the efficacy of CRISPR/Cas9 in accelerated development of aromatic rice genotypes and secondly to create different mutations in OsBADH2 leading to accumulation of aroma in rice.…”

CRISPR/Cas: A powerful tool for gene function study and crop improvement

“…CRISPR/Cas-generated mutants of G protein genes gs3 and dep1 enhanced rice tolerance to various abiotic stresses, especially for salinity stress [101] . Knockout of ppa6 gene enhanced rice tolerance to alkaline stress [102] .…”

Recent Molecular Aspects and Integrated Omics Strategies for Understanding the Abiotic Stress Tolerance of Rice

“…In contrast, the overexpression of OsAL7.1 and OsAL11 reduced the stress tolerance at the adult stage [ 222 ]. Wang et al [ 104 ] used the CRISPR/Cas9 system to obtain a mutant of the inorganic pyrophosphatase OsPPa6 gene. In the mutant, inorganic phosphorus, ATP, chlorophyll, sucrose, starch, net photosynthetic rate, soluble sugar, and proline were significantly reduced, while the MDA, osmotic potential, and Na + /K + ratio significantly increased, indicating that the OsPPa6 gene is an important osmoregulatory factor in rice.…”