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, 16 (11), 1878-1891

OsPK2 Encodes a Plastidic Pyruvate Kinase Involved in Rice Endosperm Starch Synthesis, Compound Granule Formation and Grain Filling

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OsPK2 Encodes a Plastidic Pyruvate Kinase Involved in Rice Endosperm Starch Synthesis, Compound Granule Formation and Grain Filling

Yicong Cai et al. Plant Biotechnol J.

Abstract

Starch is the main form of energy storage in higher plants. Although several enzymes and regulators of starch biosynthesis have been defined, the complete molecular machinery remains largely unknown. Screening for irregularities in endosperm formation in rice represents valuable prospect for studying starch synthesis pathway. Here, we identified a novel rice white-core endosperm and defective grain filling mutant, ospk2, which displays significantly lower grain weight, decreased starch content and alteration of starch physicochemical properties when compared to wild-type grains. The normal starch compound granules were drastically reduced and more single granules filled the endosperm cells of ospk2. Meanwhile, the germination rate of ospk2 seeds after 1-year storage was observably reduced compared with wild-type. Map-based cloning of OsPK2 indicated that it encodes a pyruvate kinase (PK, ATP: pyruvate 2-O-phosphotransferase, EC 2.7.1.40), which catalyses an irreversible step of glycolysis. OsPK2 has a constitutive expression in rice and its protein localizes in chloroplasts. Enzyme assay showed that the protein product from expressed OsPK2 and the crude protein extracted from tissues of wild-type exhibits strong PK activity; however, the mutant presented reduced protein activity. OsPK2 (PKpα1) and three other putative rice plastidic isozymes, PKpα2, PKpβ1 and PKpβ2, can interact to form heteromer. Moreover, the mutation leads to multiple metabolic disorders. Altogether, these results denote new insights into the role of OsPK2 in plant seed development, especially in starch synthesis, compound granules formation and grain filling, which would be useful for genetic improvement of high yield and rice grain quality.

Keywords: amyloplast development; grain filling; pyruvate kinase; rice; starch biosynthesis.

Figures

Figure 1
Figure 1
Phenotype of the ospk2 mutant. (a–b) Appearance comparison of seeds (a) and brown rice (b) of wild‐type (WT) (above) and ospk2 (below). (c) Transverse sections of WT (above) and ospk2 (below) brown rice. (d–g) Scanning electron microscopy images of transverse sections of the WT (d, e) and ospk2 mutant (f, g) grains. Scale bars, 1 mm in (a–c); 20 μm in (d, f) and 50 μm in (e, g). (h) Weight of dry grains of WT and ospk2 at various stages of grain filling. DAF, days after fertilization. (i–l) Weight of 1000‐grains (i), grain length (j), grain width (k) and grain thickness (l) of WT and ospk2. Data in (h–l) are means ± SD from three biological replicates, and each replication in (i) and (h, j–l) not less than 200 and 50 seeds, respectively. Asterisks in (i–l) indicate statistical significance between the WT and the mutant, as determined by a Student's t‐test (**< 0.01).
Figure 2
Figure 2
Starch granules formation in endosperm cells. (a–c) Semithin sections of wild‐type endosperm at 10 days after fertilization (DAF). (d) SEM analysis of starch or insoluble glucan granules purified from mature kernels of the wild‐type. (e–g) Semithin sections of ospk2 mutant endosperm at 10 DAF. (h) SEM analysis of starch or insoluble glucan granules purified from mature kernels of ospk2. (a, e) represent the peripheral region of endosperm of wild‐type and ospk2 mutant, respectively; (b, c) represent the central region of endosperm of wild‐type; (f, g) represent the central region of endosperm ospk2 mutant; (c, g) are enlarged sections of the white circle region from (b) and (f), respectively. Stars in (a, e) indicate the aleurone layer cells. Arrowheads in (g) indicate smaller, weak staining or abnormal starch granules in cytosol of ospk2. Arrowheads in (h) represent smaller or irregular shapes starch granules in ospk2. Scale bars: 10 μm in (a–h).
Figure 3
Figure 3
Electron micrographs depicting amyloplast development in endosperm cells of wild‐type (a–c) and ospk2 (d–i). (a, d, g) display an amyloplast in endosperm cells of wild‐type and ospk2 at 3 days after fertilization (DAF). (b, e, h) Amyloplast in endosperm cells of wild‐type and ospk2 at 6 DAF. (c, f, i) Amyloplast in endosperm cells of wild‐type and ospk2 at 9 DAF. (d–f) Represent few well‐developed amyloplasts in the endosperm cells of ospk2 mutant. (g–i) Represent the majority phenomenon of starch granules in ospk2 endosperm cells. Arrowheads in (a–d) show the stroma inside the amyloplast. Arrowheads in (h, i) indicate smaller, irregular shape, abnormal granules in ospk2. Bars: 2 μm (a–f), 5 μm (g–i).
Figure 4
Figure 4
Grain characteristics and starch physicochemical characteristics in the ospk2 mutant. (a–d) The per cent contents of total starch, amylose, protein and lipid in endosperm of wild‐type (WT) and ospk2. (e) Differences in the amylopectin chain length distributions between the WT and ospk2. (f) Pasting properties of endosperm starch of WT (red line) and ospk2 mutant (blue line). The viscosity value at each temperature is the average of three replicates. The grey line indicates the temperature changes during the measurements. (g) The gel consistency of endosperm starch of WT and ospk2 mutant. (h) Gelatinization characteristics of starch in urea solutions. Starch powder of WT and ospk2 was mixed with varying concentrations (1–9 m) of urea solution. Asterisks indicate the starch of ospk2 endosperm is more difficult to gelatinize in 4–9 m urea solution than that of WT. The most significant difference was observed for 4 m urea (right panel). Values in (a–d, g) are means ± SD from three biological replicates. Asterisks indicate statistical significance between WT and mutant, as determined by a Student's t‐test (*< 0.05; **< 0.01).
Figure 5
Figure 5
Map‐based cloning and complementation of the ospk2 mutant. (a) Fine mapping of the OsPK2 locus. The OsPK2 locus was mapped to a 90.8 kb region by markers In18 and In20 on chromosome 7 (Chr.7) which contained nine predicted genes. The molecular markers and the number of recombinants are shown. cM, centimorgan; ORF, open reading frame. (b) Gene structure and mutation site in OsPK2. Base pair change (C to T) detected in ospk2 in the 4th exon of Os07g0181000 causing a Ser‐296 to Leu‐296 change. ATG and TGA represent the start and stop codons, respectively. (c) Complementation of the ospk2 mutation in transgenic lines (S1) and overexpression of OsPK2 in ospk2 (OX‐1) showing the restored wild‐type seed appearance and normal starch granules in endosperm, whereas RNAi (Ri‐1) and CRISPR/Cas9 mediated editing (CP‐19) of OsPK2 in ZH11 background produce abnormal seed appearance and seeds became chalky. Bars: 20 μm. (d, e) Total starch content and 1000‐brown grains weight of grains in complementation (S1), overexpression (OX‐1), RNAi (Ri‐1) and CRISPR/Cas9 mediated editing lines (CP‐19). Data are shown as means ± SD from three biological replicates, each replication in (e) is not less than 200 seeds. Significant difference analysed by multiple‐comparison. Different letters indicate statistically significant difference (small letter, < 0.05 and capital letter, < 0.01).
Figure 6
Figure 6
Expression pattern, subcellular localization and enzyme assay of OsPK2. (a) OsPK2 expression level in various tissues and in developing endosperms of the WT and ospk2. RNA was isolated from root, stem, leaf and panicle at the heading stage and developing endosperms at 5, 7, 10, 15 and 20 days after fertilization (DAF). Values are means ± SD from three biological replicates. Asterisks indicate statistical significance between wild‐type and mutant, as determined by a Student's t‐test (*P < 0.05, **< 0.01). (b) GUS staining in root (i, ii), stem (iii), leaf (iv), leaf sheath (v), panicle (vi), glume (vii), spikelet (viii) and brown rice (ix) driven by the OsPK2 promoter. Bar: 2 mm (i, vii–ix); 1 cm (ii–v,); 5 mm (vi). (c) Subcellular localization of OsPK2 in rice protoplast cells. From the top panel to the bottom panel: free GFP used as control; OsPK2 full‐length coding region and GFP fusion protein (OsPK2‐GFP) and ospk2 full‐length coding region and GFP fusion protein (ospk2‐GFP). Forty‐eight hours after transformation, protoplast cells were observed using a confocal laser scanning microscope. GFP signals, chlorophyll autofluorescence, bright‐field images and the merged images of GFP and chlorophyll signals are shown in each panel. Bars: 5 μm. (d) Enzyme activity assay of OsPK2 and ospk2 protein expressed in baculovirus system and purified by His‐tag. PK activity was determined by the decreasing of NADH (measured the absorbance value at 340 nm for 2 min). (e) PK activity of fresh seeds (10 DAF) from wild‐type and ospk2. (f) Pyruvate content of seeds (10 DAF) from wild‐type and ospk2. Data in (d–f) are shown as mean ± SD from three biological replicates. Asterisks indicate statistical significance as determined by a Student's t‐test (*< 0.05, **< 0.01).
Figure 7
Figure 7
The interactions between OsPK2 and other PKps (a) yeast two‐hybrid assays showed interactions between OsPK2 (PKpα1, α1) and three putative pyruvate kinase plastidic isozymes, PKpα2 (α2), PKpβ1 (β1) and PKpβ2 (β2). Serial dilutions (10‐fold) of yeast cells expressing the indicated proteins were plated onto nonselective medium (SD/‐Leu/‐Trp) (left) or selective medium (SD/‐Leu/‐Trp/‐Ade/‐His) (right). The interactions between pGADT7‐T (T) and pGBKT7‐53 (53), pGADT7 (AD) and pGBKT7 (BD) were used as the positive and negative controls, respectively. (b) BiFC assay showed that OsPK2 (PKpα1, α1) can interact with PKpα2 (α2), PKpβ1 (β1) and PKpβ2 (β2), and PKpβ1 (β1) also can interact with PKpβ2 (β2) in the chloroplast of tobacco cells. Bars = 30 μm.
Figure 8
Figure 8
Expression analyses of genes related to glycolysis/gluconeogenesis, pyruvate/phosphoenolpyruvate metabolism, fatty acid metabolism and starch synthesis. (a) Enzyme genes of glycolysis/gluconeogenesis metabolism. PDH (Os04g0119400), a putative pyruvate dehydrogenase; GPI (Os06g0256500), glucose‐6‐phosphate isomerase; TIM (Os09g0535000), similar to the chloroplast precursor of triosephosphate isomerase and OsFBP1 (Os01g0866400), a putative fructose‐1, 6‐bisphosphatase. (b) Genes related to pyruvate/PEP metabolism. ME6 (Os01g0188400) encodes a putative chloroplastic NADP‐dependent malic enzyme; PEPC‐1 (Os02g0244700) and PEPC‐2 (Os01g0208700), putative phosphoenolpyruvate (PEP) carboxylases; PEPCK (Os10g0204400), a putative PEP carboxykinase; OsAlaAT1 (Os10g0390500) and ALT‐2 (Os07g0108300) encode alanine transaminases. (c) Genes involved in fatty acid synthesis and degradation. OsKASI (Os06g0196600) encodes a β‐ketoacyl‐[acyl carrier protein] synthase;. THIS1 (Os01g0751600) and Lipase‐II (Os07g0668700) as lipolytic enzymes. (d) Genes associated with starch metabolism, including AGP genes (OsAGPL1, OsAGPL2, OsAGPS1 and OsAGPS2b), starch branching enzyme genes (OsBEI, OsBEIIb), amylopectin synthesis genes (OsSSI, OsSSIIa, OsSSIIIa and OsSSIVb), amylose starch synthase gene (OsGBSSI), starch debranching enzyme genes (ISA1,ISA2,ISA3,PUL), sucrose synthase genes (Susy1, Susy2 and Susy3), OsPHOL and OsPPDKB. In (a–d), RNA was isolated from wild‐type (WT) and ospk2 grains of 12 days after fertilization. Expression levels are represented as relative to the corresponding genes in WT (set as reference value of 1) and data are shown as means ± SD from three biological replicates. Asterisks indicate statistical significance between the WT and the mutant, as determined by a Student's t‐test (*< 0.05; **< 0.01).

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