Mutation of iPGAM using the CRISPR/Cas9 system affects pollen vitality and chlorophyll synthesis in Nicotiana tabacum

Abstract

2, 3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGAM), is a key enzyme that catalyzes the reversible interconversion of 3-phosphoglycerate and 2-phosphoglycerate in glycolysis. iPGAM has been found to be involved in several physiological processes, including stomatal movement, chlorophyll synthesis, pollen production, chloroplast development and photosynthesis in different plants. However, the roles iPGAM plays in model plant tobacco are still elusive. In the current study, two copies of the iPGAM gene in Nicotiana tabacum were cloned, NtiPGAMa and NtiPGAMb, from the variety “Honghuadajinyuan”. Expression profiles and subcellular localization analyses showed that NtiPGAMa and NtiPGAMb were highly expressed in the anthers and were localized to the nucleus, cytosol and cell membrane. Using the CRISPR/Cas9 system, three mutant lines with mosaic leaves were obtained by targeting the second exon of the iPGAM gene. Based on Sanger sequencing, the mutant lines were chimeric plants and displayed mutation rates ranging from 82.3% to 92.5%. The iPGAM mutations showed significantly decreased photosynthetic pigments, i.e., chlorophyll a, chlorophyll b and carotenoids, thinning or loss of chloroplast thylakoid lamellar structures. RT-PCR analysis showed that the expressions of genes associated with chlorophyll synthesis and photosynthesis were significantly down-regulated in the iPGAM mutant plants compared to wild type. The pollen development was greatly impaired in mutant plants, resulting in reduced and deformed pollens with grains inviable. Our findings indicate that iPGAM is critical for pollen vitality, chlorophyll synthesis and chloroplast morphogenesis in N. tabacum.