NF-YC12 is a key multi-functional regulator of accumulation of seed storage substances in rice

Abstract

Starch and storage proteins, the primary storage substances of cereal endosperm, are a major source of food for humans. However, the transcriptional regulatory networks of the synthesis and accumulation of storage substances remain largely unknown. Here, we identified a rice endosperm-specific gene, NF-YC12, that encodes a putative nuclear factor-Y transcription factor subunit C. NF-YC12 is expressed in the aleurone layer and starchy endosperm during grain development. Knockout of NF-YC12 significantly decreased grain weight as well as altering starch and protein accumulation and starch granule formation. RNA-sequencing analysis revealed that in the nf-yc12 mutant genes related to starch biosynthesis and the metabolism of energy reserves were enriched in the down-regulated category. In addition, starch and protein contents in seeds differed between NF-YC12-overexpression lines and the wild-type. NF-YC12 was found to interact with NF-YB1. ChIP-qPCR and yeast one-hybrid assays showed that NF-YC12 regulated the rice sucrose transporter OsSUT1 in coordination with NF-YB1 in the aleurone layer. In addition, NF-YC12 was directly bound to the promoters of FLO6 (FLOURY ENDOSPERM6) and OsGS1;3 (glutamine synthetase1) in developing endosperm. This study demonstrates a transcriptional regulatory network involving NF-YC12, which coordinates multiple pathways to regulate endosperm development and the accumulation of storage substances in rice seeds.

Keywords: Grain filling; NF-Y factor; rice (Oryza sativa); seed specific expression; storage substance accumulation; transcriptional regulation.

Fig. 1.

Interaction between rice NF-YB1 and NF-YC12. (A) Yeast two-hybrid assay. The full-length and truncated NF-YC12 cDNAs were cloned into a vector bearing the DNA binding domain (BD), and the full length cDNAs of NF-YB1 were cloned into a vector bearing an activation domain (AD). The transformants were grown on DDO (SD/–Leu/–Trp), QDO (SD/–Leu/–Trp/–His/–Ade), and QDO with X-α-Gal plates. (B) BiFC assays of NF-YC12 and NF-YB1. NF-YB1-cCFP and NF-YC12-nCerulean interacted to form a functional CFP in rice protoplast cells. Scale bars are 5 μm. (C). Pull-down assays Showing that there was a direct interaction between GST-NF-YB1 and His-NF-YC12 in vitro. IB, immunoblotting.

Fig. 2.

Rice nf-yc12 knockout mutants generated by CRISPR/Cas9 and their phenotypes. (A) Schematic diagram of the genomic region of NF-YC12 and the sgRNA target site. (B) Mutation sites in nf-yc12-1 and nf-yc12-2, as compared with wild-type (WT) sequences. The target sites are underlined, protospacer-adjacent motif sequences are shown in bold, and inserted or deleted nucleotides are indicated in red. (C) Thousand-grain weights and (D) percentage of grains with chalkiness (PGWC) of WT and nf-yc12 seeds. Data are means (±SD) from three replicates, each of which included at least 200 seeds. Significant differences between the WT and the mutants were determined using Student’s t-test (**P<0.01). (E) Phenotypes of seeds of the WT and nf-yc12 mutants. (a, f, k) Images of 200 grains of mature seeds; scale bars are 10 mm. (b, g, l) Appearance of mature seeds; scale bars are 1 mm. (c, h, m) Cross-sections of mature seeds; scale bars are 1 mm. (d, e, i, j, n, o) SEM images of the central area of mature endosperm at different magnifications: the areas are indicated by the squares in (c, h, m). Scale bars are 20 μm (d, i, n), 5 μm (e, j, o).

Fig. 3.

Seed properties and amyloplast development in wild-type rice and nf-yc12 mutants. (A–D) Quality trait parameters of mature seeds from wild-type (WT) and nf-yc12. Data are means (±SD) of n=3 replicates. Significant differences between the WT and the mutant were determined using Student’s t-test (*P<0.05; **P<0.01). (E) TEM images of the compound starch granules of the WT and nf-yc12 mutant at (a, d) 7 d after pollination (DAP), (b, e) 10 DAP, and (c, f) 14 DAP. Scale bars are 2 μm.

Fig. 4.

Transgenic lines of rice overexpressing NF-YC12. (A) Expression levels of NF-YC12 in the developing seeds at 7 d after pollination (DAP) of the wild-type (WT) and two overexpression lines (OE1 and OE2), as determined by real-time quantitative RT-PCR. Data are means (±SD) for n=3 replicates. Significant differences between the WT and OE lines were determined using two-tailed Student’s t-tests (*P<0.05; **P<0.01). (B) Expression levels of representative protein in the WT and OE lines. Total protein extracted from developing seeds at 10 DAP was used for western blot analysis with an anti-FLAG antibody. (C) Representative images of the shapes of the grains of the WT and OE lines; the scale bar is 2 mm. (D–F) Thousand-grain weight (D), grain width (E), grain length (F), and contents of (G) starch, (H) prolamin, and (I) glutelin of the WT and OE lines. Data are means (±SD) of n=20 replicates (D–F) and n=3 replicates (G–I). Significant differences between the WT and OE lines were determined using Student’s t-test (*P<0.05; **P<0.01).

Fig. 5.

Transcriptional characterization of rice NF-YC12. (A) qRT-PCR analysis of NF-YC12 In different organs: R, roots; S, stems; L, leaves; P, panicles; Em, embryo; S1, caryopses collected at 1 d after pollination (DAP); S3, caryopses collected at 3 DAP; En5–En21, endosperm collected at 5–21 DAP. (B) In situ hybridization of sectioned caryopses collected at 5, 7, and 10 DAP using antisense and sense probes. NF-YC12 was highly expressed in the aleurone layer (AL) and the starchy endosperm (SE). DV, dorsal vascular bundle; NP, nucellar projection; PE, pericarp. Scale bars are 100 μm.

Fig. 6.

Transcriptomic analyses of the rice nf-yc12 mutant. (A) A selection of enriched gene ontology (GO) terms of the differentially expressed genes (DEGs) as determined by RNA-seq using endosperm at 7 d after pollination (DAP). Wallenius’ non-central hyper-geometric distribution was implemented using the R package GOseq (Young et al., 2010). Only GO terms with a corrected P-value <0.05 and including at least five annotated genes were kept. The length of the bars represents the negative logarithm (base 10) of the corrected P-value. (B) qRT-PCR analysis confirming the down-regulated genes in the endosperm of the nf-yc12 mutant. The relative expressions of genes involved in starch biosynthesis and metabolic process were calculated. The expression of each gene in the wild-type (WT) endosperm at 7 DAP was set as a reference value of 1. Data are means (±SD) from n=3 replicates. Significant differences between the WT and the mutant were determined using Student’s t-test (*P<0.05; **P<0.01).

Fig. 7.

Overview of ChIP-seq data and identification of NF-YC12 direct target genes in rice. (A) Enriched gene ontology (GO) terms of the genes bound by NF-YC12 as determined by ChIP-seq analysis. Only GO terms with a corrected P-value <0.05 and including at least five annotated genes were kept. The length of the bars represents the negative logarithm (base 10) of the corrected P-value. (B) Motif analysis of NF-YC12 binding peaks by DREME. The ‘CCAATA’ (CCAAT-box) motif was identified as one of the top five enriched motifs. The E-value is the enrichment P-value multiplied by the number of candidate motifs tested. The enrichment P-value was calculated using Fisher’s exact test for enrichment of the motif in the positive sequences. (C) Venn diagram showing the number of overlapping genes between the NF-YC12-bound gene set (ChIP-seq data) and the NF-YC12-regulated gene set (RNA-seq). (D) ChIP-PCR verification of NF-YC12-bound regions. The data are the mean values (±SD) of fold-enrichment from n=3 technical replicates. (E) The interaction between NF-YC12 and the promoters of target genes as determined by yeast one-hybrid analysis. EV, empty vector; SC2,: SD/–Leu/–Trp; SC3, SD/–Leu/–Trp/–His. (F) qRT-PCR analysis of expression levels of the target genes in nf-yc12 Compared with the wild-type (WT). Ubiquitin was used as the reference gene.

Fig. 8.

Schematic diagram of the regulatory network of NF-YC12 in rice endosperm. NF-YC12 plays upstream regulatory roles in sucrose loading, endosperm development, and the accumulation of storage substances. It modulates starch synthesis through direct regulation of FLO6, which is a key regulator involved in starch synthesis, and through indirectly regulating other starch synthesis genes, including AGPase and SS. At the same time, NF-YC12 also influences accumulation of storage proteins through directly regulating the amino acid metabolic enzyme OsGS1;3 and other as yet undetermined seed storage-protein synthases. In addition, NF-YC12 interacts with NF-YB1, and they co-regulate sucrose loading through directly regulating SUTs in the aleurone layer.