Mutations in OsDET1, OsCOP10, and OsDDB1 confer embryonic lethality and alter flavonoid accumulation in Rice (Oryza sativa L.) seed

Abstract

Morphological and biochemical changes accompanying embryogenesis and seed development are crucial for plant survival and crop productivity. Here, we identified a novel yellowish-pericarp embryo lethal (yel) mutant of the japonica rice cultivar Sindongjin (Oryza sativa L.), namely, yel-sdj. Seeds of the yel-sdj mutant showed a yellowish pericarp and black embryo, and were embryonic lethal. Compared with wild-type seeds, the yel-sdj mutant seeds exhibited significantly reduced grain size, grain weight, and embryo weight, and a remarkably lower rate of embryo retention in kernels subjected to milling. However, the volume of air space between embryo and endosperm, density of embryo, and total phenolic content (TPC) and antioxidant activity of mature grains were significantly higher in the yel-sdj mutant than in the wild type. Genetic analysis and mapping revealed that the yel-sdj mutant was non-allelic to the oscop1 null mutants yel-hc, yel-cc, and yel-sk, and its phenotype was controlled by a single recessive gene, LOC_Os01g01484, an ortholog of Arabidopsis thaliana DE-ETIOLATED 1 (DET1). The yel-sdj mutant carried a 7 bp deletion in the second exon of OsDET1. Seeds of the osdet1 knockout mutant, generated via CRISPR/Cas9-based gene editing, displayed the yel mutant phenotype. Consistent with the fact that OsDET1 interacts with CONSTITUTIVE PHOTOMORPHOGENIC 10 (OsCOP10) and UV-DAMAGED DNA BINDING PROTEIN 1 (OsDDB1) to form the COP10-DET1-DDB1 (CDD), seeds of oscop10 and osddb1 knockout mutants also showed the yel phenotype. These findings will enhance our understanding of the functional roles of OsDET1 and the CDD complex in embryogenesis and flavonoid biosynthesis in rice seeds.

Keywords: CDD complex; CRISPR/Cas9; OsDET1; embryo development; rice (Oryza sativa); yellowish-pericarp embryo lethal (yel) mutant.

FIGURE 1

Comparison of the grain characteristics of wild-type (WT; Sindongjin [SDJ]) and yel-sdj mutant rice. (A) Evaluation of the morphology of WT (upper panel) and yel-sdj mutant (lower panel) grains. (B) Rate of embryo retention in kernels after milling for 5 s. Data represent the mean ± standard deviation (SD) of five biological replicates. (C,D) Longitudinal cross section of WT (C) and yel-sdj mutant (D) grains using computed tomography (CT). White rectangles indicate the area coinciding with the air space found between the embryo and endosperm (scale bar = 1 mm). The color scale represents embryo density. EN, endosperm; EM, embryo; EAS, endosperm adjacent to the scutellum. (E–G) Volume of air space between the embryo and endosperm (E), content of total phenolics (F), and antioxidant activity (G) in WT and yel-sdj mutant grains. Data represent the mean ± SD of three biological replicates. Asterisks indicate statistical significance, as determined by Student’s t-test (*p < 0.05, ***p < 0.001).

FIGURE 2

Map-based cloning of the gene responsible for the yel-sdj mutant phenotype. (A) Schematic showing the physical position of the causal locus on rice chromosome 1, as identified by map-based cloning. (B) Gene structure of OsDET1. Black lines, white solid boxes, and black solid boxes indicate introns, untranslated regions, and exons, respectively. The 7 bp deletion is indicated with a black arrow. ATG and TGA indicate the initiation and termination codons, respectively. (C) Comparison of the predicted amino acid sequence of the mutated region of gene between the WT (SDJ) and yel-sdj mutant. The 7 bp deletion resulted in a frameshift (red arrow) and premature stop (red asterisk) in yel-sdj. Amino acids in the frameshift region are indicated in red.

FIGURE 3

Grain phenotypic analysis and sequence analysis of CRISPR/Cas9-induced knockout yel mutants generated by targeting the OsDET1 gene. (A) Grain phenotype of Dongjin (WT) and transgenic seeds. Grains showing the yel phenotype were randomly selected from each positive T₀ transgenic plant. (B) Comparison of the OsDET1 nucleotide sequence targeted by CRISPR/Cas9 in WT and mutant plants. Targets 1 and 2 represent the first and second exons, respectively, of OsDET1 (LOC_Os01g01484/Os01g0104600). Red dashes and letters indicate deletions and insertions, respectively, in transgenic lines. The protospacer adjacent motif (PAM) is highlighted in green in the WT and is underlined in mutant lines. Sequences of targets 1 and 2 are highlighted in gray in the WT. Mutation types are shown to the right of each mutated sequence (-, deletion; +, insertion; RC, reverse complementary).

FIGURE 4

Grain phenotypic analysis and sequence analysis of CRISPR/Cas9-induced knockout yel mutants generated by targeting OsCOP10 and OsDDB1 genes. (A,B) Grain phenotype of Dongjin (WT) seeds and CRISPR mutant seeds generated by targeting OsCOP10 (A) and OsDDB1 (B). Grains showing the yel phenotype were randomly selected from each positive T₀ transgenic plant. (C) Sequence comparison of OsCOP10 (LOC_Os07g38940/Os07g0577400) and OsDDB1 (LOC_Os05g51480/Os05g0592400) target regions in the WT and mutants. The first exon of OsCOP10 and second exon of OsDDB1 were targeted to induce mutations. Red dashes and letters indicate deletions and insertions, respectively, in transgenic lines. Black and green letters indicate the target sequence and PAM, respectively. Mutation types are shown to the right of each mutated sequence (-, deletion; +, insertion).

FIGURE 5

Expression analysis of OsDET1 and yel phenotype-associated genes. (A) Quantitative real-time PCR (qRT-PCR) analysis of OsDET1 in various organs of SDJ (WT). (B) Relative expression levels of yel phenotype-associated genes in the developing seeds of SDJ (WT) and yel-sdj mutant at 7 days after pollination (DAP). Expression level of genes was normalized relative to that of ACTIN. Data represent mean ± SD of three biological replicates. Asterisks indicate statistical significance, as determined by Student’s t-test (*p < 0.05, **p < 0.01).