Quantitative Proteomic Analysis Deciphers the Molecular Mechanism for Endosperm Nuclear Division in Early Rice Seed Development

Abstract

Understanding the molecular mechanisms underlying early seed development is important in improving the grain yield and quality of crop plants. We performed a comparative label-free quantitative proteomic analysis of developing rice seeds for the WT and osctps1-2 mutant, encoding a cytidine triphosphate synthase previously reported as the endospermless 2 (enl2) mutant in rice, harvested at 0 and 1 d after pollination (DAP) to understand the molecular mechanism of early seed development. In total, 5231 proteins were identified, of which 902 changed in abundance between 0 and 1 DAP seeds. Proteins that preferentially accumulated at 1 DAP were involved in DNA replication and pyrimidine biosynthetic pathways. Notably, an increased abundance of OsCTPS1 was observed at 1 DAP; however, no such changes were observed at the transcriptional level. We further observed that the inhibition of phosphorylation increased the stability of this protein. Furthermore, in osctps1-2, minichromosome maintenance (MCM) proteins were significantly reduced compared with those in the WT at 1 DAP, and mutations in OsMCM5 caused defects in seed development. These results highlight the molecular mechanisms underlying early seed development in rice at the post-transcriptional level.

Keywords: CTP synthase; MCM proteins; early seed development; protein stability; proteomic profiling; rice.

Figure 1

Protein accumulation of OsCTPS1 in the developing embryo sac at 1 day after pollination (DAP). (A) The OsCTPS1-sGFP fusion construct driven by the maize ubiquitin promoter was stably transformed. Embryo sacs from the transgenic plants were harvested at 0 DAP (a–d) and 1 DAP (e–h). c and d, enlargement images of yellow and orange boxed areas in panel b, respectively. g and h, enlargement images of yellow and orange boxed areas in panel f, respectively. Scale bars, 100 µm (a,e) and 50 µm (b–d,f–h). (B) Genomic structure of OsCTPS1 and protein structure of OsCTPS1. The target site of the CRISPR/Cas9 system is also presented in the second exon region. The osctps1-2 mutant showed premature termination, green boxs, UTR regions; yellow boxs, exon regions; *, premature termination codon. (C) Transcript levels of OsCTPS1 in WT and osctps1-2 mutants at 0 and 1 DAP. Each sample contains more than 10 ovaries or seeds; n = 4. NSD, no significant difference (p > 0.05). (D) Phenotype of WT and osctps1-2 developing seeds at 10 DAP. Bar = 2 mm. (E) Phenotype of mature seeds of WT and osctps1-2. Bar = 2 mm.

Figure 2

Comparison of differentially accumulated proteins between WT and osctps1-2 in 0 and 1 DAP seeds. (A) Nuclear division phenotype in WT and osctps1-2 at 1 DAP. Bars = 30 µm. (B) SDS-PAGE of total proteins isolated from 0 and 1 DAP seeds of WT and osctps1-2 mutants. Loaded samples contained 30 µg of total proteins. (C) Western blot analysis using β-tubulin and Rubisco antibodies. Label-free quantitative proteomic analysis of 0 and 1 DAP seeds. (D) Schematic representation of the total number of proteins identified and the proteins validated by the narrow-down approaches. (E,F) A volcano plot of differently modulated proteins in WT and osctps1-2 mutant seeds at 0 DAP (E) and 1 DAP (F).

Figure 3

Comparison of differentially accumulated proteins between WT and osctps1-2 in 0 and 1 DAP seeds. (A) Hierarchical clustering analysis of differentially modulated proteins identified using a label-free quantitative proteomic approach in 0 and 1 DAP samples. (B,C) Bar charts showing the representative gene ontology biological process (GOBP) term of significantly modulated proteins in WT and osctps1-2 at 0 (B) and 1 DAP (C) using DAVID bioinformatics resources (GO enrichment).

Figure 4

Quantitative proteomic analysis of 0 and 1 DAP of WT seeds. (A) Heat map images of significantly modulated proteins at 0 and 1 DAP in WT. (B) KEGG analysis of differentially accumulated proteins at 0 and 1 DAP seeds. Cluster1 included more accumulated proteins at 0 DAP seeds, and cluster2 included highly accumulated proteins in 1 DAP seeds. (C) The chromatographic peak of OsCTPS1 peptide 1 (ATLFDALQDTVR). (D) LFQ intensity of OsCTPS1 peptide 1 (ATLFDALQDTVR) at 0 and 1 DAP seeds in WT and osctps1-2; n = 3. (E) The chromatographic peak of OsCTPS1 peptide 2 (YTGLSDSYLSVLK). (F) LFQ intensity of OsCTPS1 peptide 2 (YTGLSDSYLSVLK) at 0 and 1 DAP seeds in WT and osctps1-2; n = 3.

Figure 5

Regulation of OsCTPS1 protein stability. (A) Effect of treatment with mock or different signalling inhibitors on the stability of OsCTPS1-sGFP protein. Protoplasts were isolated from the OsCTPS1-sGFP OX transgenic plants and then incubated with 30 μM MG132, 2 μM staurosporine, 2 μM okadaic acid, 100 μM E64d, and 116.86 μM 6-diazo-5-oxo-L-norleucine (DON). After treatments, GFP fluorescence intensity was measured every 30 min for 15 h; n = 3. (B) MapMan overviews of regulation about differentially accumulated proteins in WT and osctps1-2 in 1 DAP seeds.

Figure 6

Comparison of differentially accumulated proteins between WT and osctps1-2 in 1 DAP seeds and characterization of OsMCM5 functions in seed development. (A) Heatmap images of protein expression involved in cell organization and DNA synthesis at 1 DAP in WT and osctps1-2 mutants. Cell organization and DNA synthesis groups were selected for functions in endosperm nuclear division. (B) Expression patterns of rice MCM family genes in rice. (C) Schematic representation of the gene structure of OsMCM5. The red arrow indicates the target site of CRISPR/Cas9, green boxs, UTR regions; yellow boxs, exon regions. (D) Sequence alignment of the sgRNA target sequence with altered bases in three independent mutant lines. The target sequence is underlined, and the NGG PAM site is highlighted. Altered DNA sequences are indicated in red. (E) Different seed types caused by the mutation. More than 30 seeds were observed to check the seed phenotypes. The osmcm5 mutant seeds were divided into four main types. (F–I) Phenotypes of WT (F) and osmcm5 mutant seeds (G–I). Scale bars, 2 mm.