A Novel Mutation of OsPPDKB, Encoding Pyruvate Orthophosphate Dikinase, Affects Metabolism and Structure of Starch in the Rice Endosperm

Abstract

Starch, as a main energy storage substance, plays an important role in plant growth and human life. Despite the fact that several enzymes and regulators involved in starch biosynthesis have been identified, the regulating mechanism of starch synthesis is still unclear. In this study, we isolated a rice floury endosperm mutant M14 from a mutant pool induced by ⁶⁰Co. Both total starch content and amylose content in M14 seeds significantly decreased, and starch thermal and pasting properties changed. Compound starch granules were defected in the floury endosperm of M14 seeds. Map-based cloning and a complementation test showed that the floury endosperm phenotype was determined by a gene of OsPPDKB, which encodes pyruvate orthophosphate dikinase (PPDK, EC 2.7.9.1). Subcellular localization analysis demonstrated that PPDK was localized in chloroplast and cytoplasm, the chOsPPDKB highly expressed in leaf and leaf sheath, and the cyOsPPDKB constitutively expressed with a high expression in developing endosperm. Moreover, the expression of starch synthesis-related genes was also obviously altered in M14 developing endosperm. The above results indicated that PPDK played an important role in starch metabolism and structure in rice endosperm.

Keywords: amyloplast development; floury endosperm; pyruvate orthophosphate dikinase; rice; starch.

Figure 1

Phenotype analyses of M14 seeds. (A,B) The appearance of brown rice of WT and M14. (C,D) Transverse sections of WT and M14 seed. Bars = 1 mm (A–D). (E–H) Scanning electron microscope observation of WT and M14 seeds. (E,F) represent the region of “1” and “2” in WT endosperm, respectively, and (G,H) represent the region of “1” and “2” in M14 endosperm, respectively. Bars = 5 μm (E–H). (I) Seed length, width, and thickness of WT and M14 (n = 20). (J) 1000-seed weight of WT and M14. Data are given as means ± SD (n = 3). ** and * indicate significant differences between WT and M14 at p < 0.01 and p < 0.05 by Student’s t test.

Figure 2

The formation of abnormal compound starch granules in M14 endosperm. (A–F) Semi-thin sections of WT (A–C) and M14 (D–F) kernels at 9 DAF are stained with I₂-KI. (A,D) and (B,E) represent the outer and inner of endosperm, respectively. (C,F) represent the magnified region indicated by red outline in WT and M14 endosperm, respectively. Red arrowheads in (F) indicate abnormal weakly stained starch granules. AL, aleurone layer. Bars = 20 μm.

Figure 3

Components of seed and starch and pasting properties of starch. (A–C) Starch, amylose and protein contents in WT and M14. All data are given as means ± SD (n = 3). ** indicate significant differences between WT and M14 at p < 0.01 by Student’s t test. (D) Differences in chain length distributions of amylopectin between WT and M14. (E) Pasting properties of starch from WT (black line) and M14 (red line) endosperm. The gray line indicates the temperature changes during the measurements.

Figure 4

Map-based cloning and complementation of M14. (A) Fine mapping of the OsPPDKB locus (red arrow). The OsPPDKB locus is mapped to a 71.4 kb region on chromosome 5, which contains eight predicted ORFs. (B) Structure of the OsPPDKB gene and positions of mutant site and qRT-PCR primers. The sequence of P1 (124 bp) was amplificated by specific primers P1-F and P1-R and the sequence of P2 (118 bp) was amplificated by specific primers P2-F and P2-R. (C,D) Complementation test of the OsPPDKB gene completely rescues the seed appearance (C) and compound starch granule arrangement (D) of M14. Bars = 2 mm (C) and 10 μm (D).

Figure 5

Expression pattern of OsPPDKB. (A) Expression analysis of the chOsPPDKB in various tissues and developing kernels of WT. (B) Expression analysis of the cyOsPPDKB in various tissues and developing kernels of WT. R, root; S, stem; L, leaf; LS, leaf sheath; P, panicle. Actin1 is used as an internal control. All data are given as means ± SD (n = 3).

Figure 6

Subcellular localization of OsPPDKB-GFP fusion proteins in Nicotiana benthamiana leaf. (A) The free GFP serves as a control. (B) The chOsPPDKB-GFP fusion protein is localized in the chloroplasts. (C) The cyOsPPDKB-GFP fusion protein is localized in the cytoplasm. Bars = 20 μm.

Figure 7

Expression of starch biosynthesis-related genes in WT and M14. Total RNA extracted from 9 DAF endosperm is used for qRT-PCR analysis. ADP glucose pyrophosphorylase small subunit (AGPS1 and AGPS2) and large subunit (AGPL1, AGPL2, AGPL3, and AGPL4); granule-bound starch synthase I (GBSSI); soluble starch synthase I, IIa, IIIa, IVa, and IVb (SSI, SSIIa, SSIIIa, SSIVa, and SSIVb); branching enzyme I, IIa, and IIb (BEI, BEIIa, and BEIIb); isoamylase isozymes (ISA1 and ISA2); pullulanase (PUL); plastidial phosphorylase (Pho1). The expression level of every target gene in WT is set at 1.0. All data are given as means ± SD (n = 3). ** and * indicate significant differences between WT and M14 at p < 0.01 and p < 0.05 by Student’s t test.

Figure 8

Accumulation of proteins involved in starch synthesis in WT and M14 endosperm at 9 DAF. Anti-GBSSI, anti-SSI, anti-BEI, anti-BEIIa and anti-BEIIb are used for the immunoblot analysis. Anti-HSP82 is used for the loading control.