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.
Highlights
Soils with highly salinity-alkalinity are widely distributed in the world, and account for 10% of the arable land
A total of six of putative protein encoding the soluble inorganic pyrophosphatase (sPPase) in rice were retrieved by the Rice Data database3 and are represented by Os01g0974800, Os01g0866500, Os02g0704900, Os04g0687100, Os05g0114000, and Os05g0438500, respectively
These six proteins share more than 75% of homogeneity, the OsPPa6 (Os02g0768600) only shares less than 30% of homology with these six putative proteins (Supplementary Figure S1), indicating that the OsPPa6 gene exhibits structural specificity in the sPPase family
Summary
Soils with highly salinity-alkalinity are widely distributed in the world, and account for 10% of the arable land. Statistics shows that saline-alkaline soils of nearly 8.3 million ha are universally used for rice planting (Haefele et al, 2014). Study shows that most of the translocation and distribution of ions are closely associated with the H+-translocating inorganic pyrophosphatase (H+-PPase) widely presenting in plants (Davies et al, 1992; Tsai et al, 2014). To better understand whether the soluble inorganic pyrophosphatase (sPPase) has a similar regulatory role in the transport and translocation of ions to the H+-PPase, evaluating the genes encoding sPPase is imperative to better exploit these genes in cultivating alkaline tolerant crop
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