TALEN-Based mutagenesis of lipoxygenase LOX3 enhances the storage tolerance of rice (Oryza sativa) Seeds

Abstract

The deterioration of rice grain reduces the quality of rice, resulting in serious economic losses for farmers. Lipoxygenases (LOXs) catalyze the dioxygenation of polyunsaturated fatty acids with at least one cis,cis-1,4-pentadiene to form hydroperoxide, which is a major factor influencing seed longevity and viability. Recently, genome editing, an essential tool employed in reverse genetics, has been used experimentally to investigate basic plant biology or to modify crop plants for the improvement of important agricultural traits. In this study, we performed targeted mutagenesis in rice using transcription activator-like effector nucleases (TALENs) to improve seed storability. A modified ligation-independent cloning method (LIC) was employed to allow for the quick and efficient directional insertion of TALEN monomer modules into destination vectors used in plants. We demonstrated the feasibility and flexibility of the technology by developing a set of modular vectors for genome editing. After construction and validation, the TALEN pairs were used to create stable transgenic rice lines via Agrobacterium-mediated transformation. One heterozygous mutant (4%) was recovered from 25 transgenic NPTII-resistant lines, and the mutation was transmitted to the next generation. Further molecular and protein level experiments verified LOX3 deficiency and demonstrated the improvement of seed storability. Our work provides a flexible genome editing tool for improving important agronomic traits, as well as direct evidence that Lox3 has only a limited impact on seed longevity.