Low amylose content in rice lowac2 mutant is caused by a new allele of C₂H₂ zinc-finger protein that regulates pre-mRNA splicing of Wx^b

Abstract

The isolated rice mutant, designated lowac2, exhibited low amylose content. Whole-genome resequencing and single-nucleotide polymorphism (SNP) fragment analysis revealed that a mutation in the gene encoding the C₂H₂ zinc-finger protein in the long-arm terminal of Chromosome 6 was responsible for this low amylose phenotype. An SNP from G to A was observed in the 5ʹ splice junction of intron11, resulting in the production of a protein lacking the C-terminal. Mutation of the C₂H₂ zinc-finger protein specifically affected the splicing efficiency of intron1 in Waxy^b (Wx^b). This reduced the levels of granule-bound starch synthase I, which is encoded by Wx^b. Mutations in lowac2 increased the mRNA expression levels of several starch biosynthetic enzymes, especially starch synthase IIa and starch branching enzyme I. This was consistent with the presence of amylopectin with reduced short glucan chains in lowac2 seeds compared to wild-type seeds. Furthermore, the crossed lines possessing a gene combination between Wx^a from indica variety and lowac2 also showed a decrease in amylose content and Wx^a expression; however, this did not affect splicing in Wx^a. The new allele of the C₂H₂ zinc-finger protein found in this study affects both Wx genes differently.