Lose-of-Function of a Rice Nucleolus-Localized Pentatricopeptide Repeat Protein Is Responsible for the floury endosperm14 Mutant Phenotypes

Abstract

Background: The endosperm of rice (Oryza sativa) has been usually used for the study of starch synthesis. Although several related factors have been revealed, other unknown members remain to be identified, given that starch synthesis is a complicated and sophisticated process.

Results: Here, we identified and characterized a new rice seed mutant, floury endosperm14 (flo14), which showed chalked endosperm and seed-lethal phenotypes. Map-based cloning indicated FLO14 encodes a novel P-family PPR protein which contains ten PPR motifs. Afterwards the gene was named OsNPPR3. Subcellular localization showed OsNPPR3 was targeted to nucleolus. Quantitative RT-PCR analysis demonstrated that OsNPPR3 was universally expressed in various tissues, with pronounced levels during rice endosperm development. Molecular analysis further suggested that OsNPPR3 was involved in the regulation of expression levels and splicing of a few genes in mitochondria.

Conclusion: The study demonstrates that the nucleolus-localized PPR protein is responsible for the flo14 mutant phenotypes through affecting nuclear and mitochondrial gene expression and splicing.

Keywords: Nucleolus-targeted; OsNPPR3; PPR protein; Rice (Oryza sativa); flo14 mutant.

Fig. 1

Phenotypic characterization of the flo14 mutant. a Comparison of wild-type (WT) and flo14 mutant (flo14) seeds. b Cross sections of wild-type and flo14 mutant seeds. c Vertical-sections of imbibed embryos of wild type and flo14 mutant. d Tetrazolium assay of wild-type and flo14 mutant seeds. e Young seedlings of wild type and flo14 mutant at 5 days after germination. f Thousand kernel weight of wild-type and flo14 mutant seeds. Data indicate means ± SD (from at least three independent samples) and was compared with wild type by Student’s t-test (* P < 0.05, ** P < 0.01). Scale bars: 1 mm in (a and b), 1 cm in (c and d), 500 μm in (e)

Fig. 2

Starch granules development in the flo14 mutant. a-d Scanning electron microscope (SEM) analysis of the endosperm in wild-type (WT) (a, c) and flo14 mutant (flo14) (b, d) seeds. e-h Semi-thin sections of wild-type and flo14 mutant seeds. The central part of 12 days after flowering (DAF) in the wild-type endosperm cells (e-f) and the central part of mutant flo14 endosperm cells (g-h). i Starch content of wild type and flo14 mutant (n = 3 each). j Amylose content of wild type and flo14 mutant (n = 3 each). Data are shown as means ± SD (from at least three independent samples) and was compared with wild type by Student’s t-test (* P <0.05). NS: no obviously changed. Scale bars: 0.5 mm in (a, b), 15 μm in (c, d), 100 μm in (e, g), 200 μm in (f, h)

Fig. 3

Map-based cloning of the gene responsible for the OsNPPR3 phenotypes. a Fine mapping of the OsNPPR3 locus. The OsNPPR3 locus was mapped to a 165-Kb region by markers FY3–3 and FY3–6 on Chromosome 3 (Chr.3), which contains 15 predicted genes. The number of recombinants is indicated below the map. The candidate gene is indicated by red arrow. b The PPR gene structure and its protein. The lines indicate 5′-UTR and 3′-UTR, respectively. The blue box means exon. ATG and TAA represent start codon and stop codon. The PPR protein contains 10 PPR motifs. A single nucleotide substituted in the coding region of mutant gene leads to a premature stop codon. c Real-time RT-PCR analysis of OsNPPR3 in developing endosperms at 12 days after flowering (DAF) in the wild type (WT), flo14 mutant (flo14) and three flo14 mutant lines expressing the wild-type OsNPPR3 gene (Com1, Com2 and Com3). The value of Actin I mRNA was used as an internal control for data normalization. d Complementation of the flo14 mutant restored normal seed appearance. e Semi-thin sections of wild type and the complementation of the flo14 mutant (Com) endosperm at 15 days after flowering. Values are means ± SD (n = 3). The asterisks indicate statistical significance compared with the flo14 mutant, as determined by a Student’s t–test (** P < 0.01). Scale bars: 1 mm in (d), 100 μm in (e)

Fig. 4

Knockout of the OsNPPR3 gene by CRISPR system. a Seeds of the wild type (WT), flo14 mutant (flo14), and two independent CRISPR/Cas9 T₁ transgenic lines, which were named CR9–1 and CR9–2. b Verification of the knockout lines by PCR-based sequencing. The representative transgenic lines were generated from Oryza sativa L. japonica variety Dianjingyou1 genetic background. ATG and TAG indicate the start and stop codons, respectively. UTR means untranslated region. Arabic number indicates the base position from the start codon. The target sequences designed for knocking out the Os03g0728200 by CRISPR/Cas9 system and the protospacer-adjacent motif (PAM) are underlined in blue and red, respectively. The missing bases are marked with red dotted lines. The chromatograms of the wild type and two knockout lines (CR9–1 and CR9–2) are shown. Triangle means the site of base deletion in the knockout line. Scale bars:1 mm in (a)

Fig. 5

Homologous comparison and expression analysis. a A neighbor-joining tree of PPR gene and its homologs. The tree was constructed using MEGA and bootstrapped with 1000 replicates. The proteins are named according to their gene/EST names or NCBI accession numbers. OsNPPR3 was indicated by the red frame. b Expression levels of PPR gene in various tissues and different developmental stages endosperm of the wild type (n = 3 each). c Histochemical staining showed that PPR: GUS reporter gene is ubiquitously expressed in the root, stem, leaves, panicles and leaf shoots from the left photo to the right, respectively. d Real-time PCR analysis of starch synthesis genes in 12 days after flowering (DAF) wild-type (WT) and flo14 mutant (flo14) seeds. Actin1 was used as an internal control. Data gives as means ± SD from three independent biological replicates and was compared by Student’s t-test (* P < 0.05, ** P < 0.01). Scale bars: 1 cm for all panels in (c). BE I: branching enzyme I, UGPase I: UTP-glucose-1-phosphate-uridyly-1 transferase, SS I:soluble starch synthase I, SS IIa: soluble starch synthase IIa, SS IIIa: soluble starch synthase IIIa, SS IIIb: soluble starch synthase IIIb, SS IVb: soluble starch synthase IVb, GBSS I: granule-bound starch synthase I, GBSS II: granule-bound starch synthase II, PUL: pullulanase, PPDKB: pyruvate phosphate dikinase B, BE IIa: branching enzyme IIa, BE IIb: branching enzyme IIb, PHOL: starch phosphorylase L, ISA I: Isoamylase I, ISA II: Isoamylase II, AGPS 2b: ADP-glucose pyrophosphorylase 2b, AGPL 1: ADP-glucose pyrophosphorylase large subunit 1, AGPL 2: ADP-glucose pyrophosphorylase large subunit 2, SUS 4: sucrose synthase 4

Fig. 6

Subcellular localization of OsNPPR3. a-e Transient expression of 35S: OsNPPR3-GFP fusion protein located in the nucleolus of rice protoplasts (a, b) and Nicotiana tabacum protoplasts (c-e). Nucleus marker was used as a nucleus indicator (a-d). Nucleolus marker was used as a nucleolus indicator (e). RPBF (Rice Prolamin Box Binding Factor) and RPL23aB (r-Protein family member) were employed as nucleus-targeted and nucleolus-targeted marker, respectively. Scale bars: 1.6 μm in (a), 5 μm in (b, c), 10 μm in (d, e)

Fig. 7

Expression analysis of OsNPPR3 and genes associated with mitochondria. a Real-time RT-PCR analysis of the mitochondrial gene expression in endosperms of wild type (WT) and flo14 mutant (flo14) at 12 days after flowering (DAF). b Immunoblotting analysis of mitochondria related proteins in developing endosperm of wild type and flo14 mutant. c Real-time RT-PCR analysis of the gene expression in the wild type and flo14 mutant. Data are shown as means ± SD from three independent biological replicates and compared by Student’s t-test (* P < 0.05, ** P < 0.01). Actin1 was used as an internal control. rpl2: ribosomal protein L2, rpl16: ribosomal protein L16, rpl7: ribosomal protein L7, rps2: ribosomal protein S2, rps13: ribosomal protein S13, cox1: cytochrome c oxidase subunit 1, cyt c: cytochrome C, ccmb: cytochrome c biogenesis B, ccmFn: cytochrome c biogenesis Fn, ccmFc: cytochrome c biogenesis Fc, nad4: NADH dehydrogenase subunit IV, nad9: NADH dehydrogenase subunit XI, AOX 1a: alternative oxidase 1a, AOX 1b: alternative oxidase 1b, AOX 1c: alternative oxidase 1c

Fig. 8

Transmission electron micrographs of mitochondria. Transmission electron micrographs of mitochondria from 12 days after flowering (DAF) wild-type (WT) (a) and flo14 mutant (flo14) (b) endosperms. Scale bars: 1 μm in (a) and (b)

Fig. 9

The flo14 mutant is defective in the splicing of mitochondria genes. Gene transcripts are labeled at the left. Spliced (S) and unspliced (U) transcripts are shown at the right. RNA were extracted from 12 days after flowering (DAF) endosperms of wild type (WT) and flo14 mutant (flo14). The splicing genes are tagged with red boxes. UBQ was used as a quantitative control. Nad 1–1: NADH dehydrogenase subunit 1–1, nad 1–2: NADH dehydrogenase subunit 1–2, nad 2: NADH dehydrogenase subunit 2, nad 2–1: NADH dehydrogenase subunit 2–1, nad 2–2: NADH dehydrogenase subunit 2–2, nad 3: NADH dehydrogenase subunit 3, nad 4–1: NADH dehydrogenase subunit 4–1, nad 4–2: NADH dehydrogenase subunit 4–2, nad 5: NADH dehydrogenase subunit 5, nad 6: NADH dehydrogenase subunit 6, nad 7: NADH dehydrogenase subunit 7, nad 9: NADH dehydrogenase subunit 9, atp 1: ATP synthase F0 subunit 1, atp 4: ATP synthase F0 subunit 4, cox1: cytochrome c oxidase subunit 1, cox 2: cytochrome c oxidase subunit II, cox 3–1: cytochrome c oxidase subunit 3–1, cox 3–2: cytochrome c oxidase subunit 3–2, ccmFc: cytochrome c biogenesis Fc (two pairs of primers were used), rpl 2: 50S ribosomal protein L2, rpl 5: ribosomal protein L5, rps 2: 30S ribosomal protein S2, rps 3: 30S ribosomal protein S3, rps 13: ribosomal protein S13, orf X: hypothetical protein, cyt b: cytochrome b