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

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.


Introduction
In cereal endosperm, starch is synthesized in amyloplasts and forms starch granules. Mature seeds of cereals such as rice, maize and wheat contain large amounts of starch. It provides a substrate for seed germination and seedling growth. In addition, endosperm starch makes an important contribution to the human diet throughout the world [1].
Starch consists of two major components, amylose and amylopectin. Amylose is a linear polymer made of α-1,4-linked D-glucopyranosyl units, whereas amylopectin is a highly branched macromolecule [2]. The enzymology of starch synthesis has been well studied. ADP-glucose (ADPG) is produced by ADP-glucose pyrophosphorylase (AGPase) and is then used as a substrate to initiate amylose and amylopectin synthesis and elongation. Granule-bound starch synthase I (GBSSI) is an amylose-specific starch synthase, whereas starch synthases (SSI, SSII, SSIII, and SSIV), starch branching enzymes (BEI and BEII), and starch debranching enzymes (isoamylase (ISA) and pullulanase (Pul)) are responsible for amylopectin synthesis [1]. In rice, the mutations in the above starch synthesis key enzymes cause abnormal characteristics of endosperm starch with opaque-seed phenotype including shrunken, floury and white-core endosperm. For example, mutations in AGPase small subunit OsAGPS2 and large subunit OsAGPL2 decrease the synthesis of ADPG and form small and round starch granules [3]. Mutation in ISA1 produces a sugary endosperm and accumulates abundant phytoglycogen instead of normal starch granules [4]. The SSIIIa-deficient mutant causes a floury endosperm and reduces the content of long branch-chains of amylopectin [5,6]. The amylose-extender (ae) mutant lacking BEIIb produces a white-core endosperm and alters the fine structure of amylopectin [7].
In addition, some mutations in genes that are not directly involved in starch metabolism also affect starch synthesis. FLO2 (FLOURY ENDOSPERM2), encoding a tetratricopeptide repeat motif-containing protein, affects the starch quality by regulating the expression of starch synthesis-related genes [8,9]. FLO6 contains a CBM48 domain in the C-terminal and regulates the starch synthesis and starch granule development by interacting with ISA1 [10]. A domain of unknown function 1338 (DUF1338) superfamily member FLO7 influences the amylose content and amylopectin structure in the outer endosperm [11]. FLO8, encoding the UDP-glucose pyrophosphorylase 1 (Ugp1), modulates the starch synthesis and structure [12]. Besides the FLOURY genes, other genes can also cause floury endosperm phenotype after loss of function, such as Grain Incomplete Filling1 (GIF1) encoding a cell wall invertase, Fructose-6-phosphate 1-phosphotransferase (PFP) encoding a fructose-6-phosphate 1-phosphotransferase β subunit, and Glutelin Precursor Accumulation3 (GPA3) encoding a plant-specific kelch-repeat protein [13][14][15]. Therefore, the genes involved in carbon partitioning, carbon flux and protein body development may also affect the development of rice kernel and starch metabolism.
Pyruvate orthophosphate dikinase (PPDK, EC 2.7.9.1) catalyzes the interconversion of pyruvate, ATP, and Pi with phosphoenolpyruvate, AMP, and PPi [16]. PPDK in C 4 plants is most well known as a photosynthetic enzyme, which catalyzes the formation of CO 2 acceptor phosphoenolpyruvate from pyruvate [17]. The activity of chloroplastic PPDK can be controlled by reversible phosphorylation of a threonine residue [18]. Maize, a typical C 4 species, contains three PPDK genes, C 4 ppdkZm1, cyppdkZm1 and cyppdkZm2 [19]. During the accumulation of zein protein in developing endosperm, cyPPDKZm1 can be regulated by the O 2 (Opaque2) gene [20]. In most C 3 tissues, PPDK is either not present or at very low abundance [21]. In rice, there are only two genes encoding PPDK, one encoding OsPPDKA and another producing two transcripts of C 4 -type chloroplastic PPDK (chOsPPDKB) and cytosolic PPDK (cyOsPPDKB) [22,23]. A corresponding histochemical analysis of β-glucuronidase (GUS) gene driven by cytosolic OsPPDKB promoter is conducted in rice. The GUS activity shows a high level in developing kernels, but a low level in the flowers and roots [24]. The T-DNA insertional mutant of OsPPDKB shows a white-core endosperm, indicating the essential role of OsPPDKB in modulating the carbon flow for starch and lipid synthesis [16]. Recently, a new cyOsPPDKB mutant Suweon 542 with a milky-white floury endosperm has been identified and characterized [25]. However, the effect of OsPPDKB mutation on starch development, starch physicochemical properties and starch metabolism are still unknown.
In the present study, we characterized a floury endosperm mutant, M14. Map-based cloning and complementation test revealed that the mutant phenotype was caused by OsPPDKB, which is a new allele of FLO4 [16] from rice variety Kitaake. Meanwhile, the starch development, starch physicochemical properties and expression of starch synthesis-related genes in M14 and its wild type (WT) were investigated and compared. Our data showed a vital importance of OsPPDKB in starch metabolism and structure in rice endosperm development.

Phenotypic Analysis of M14
The M14 with a floury endosperm phenotype was identified from a 60 Co-irradiated mutant pool of japonica rice variety Kitaake, and displayed no visible differences from WT plants at the vegetative stage. The brown rice of M14 exhibited an opaque endosperm when compared with WT seeds ( Figure 1A,B).
The seed cross section displayed that the inner endosperm of M14 appeared floury, while the peripheral endosperm was as transparent as the WT ( Figure 1C,D). Scanning electron microscope (SEM) analysis showed that the floury endosperm of M14 comprised small, round and loosely packed compound starch granules, which was different from the transparent endosperm of M14 and WT with densely packed, polyhedral compound starch granules ( Figure 1E-H). Compared with WT, the seed length, width and thickness of M14 significantly decreased ( Figure 1I). Therefore, the 1000-seed weight of M14 was markedly reduced by 11.4% ( Figure 1J).
The M14 with a floury endosperm phenotype was identified from a 60 Co-irradiated mutant pool of japonica rice variety Kitaake, and displayed no visible differences from WT plants at the vegetative stage. The brown rice of M14 exhibited an opaque endosperm when compared with WT seeds ( Figure 1A,B). The seed cross section displayed that the inner endosperm of M14 appeared floury, while the peripheral endosperm was as transparent as the WT ( Figure 1C,D). Scanning electron microscope (SEM) analysis showed that the floury endosperm of M14 comprised small, round and loosely packed compound starch granules, which was different from the transparent endosperm of M14 and WT with densely packed, polyhedral compound starch granules ( Figure 1E-H). Compared with WT, the seed length, width and thickness of M14 significantly decreased ( Figure 1I). Therefore, the 1000-seed weight of M14 was markedly reduced by 11.4% ( Figure 1J). (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.

The Abnormal Morphology of Compound Starch Granules in M14 Endosperm
To observe the morphology of compound starch granules, the developing endosperm of WT and M14 at 9 days after fertilization (DAF) was prepared into semi-thin sections. I2-KI staining displayed that amyloplasts in the outer endosperm of M14 and WT developed well and filled with densely packed, polyhedral starch granules (Figure 2A,D). The inner endosperm of WT showed normal compound starch granules ( Figure 2B,C), however, that of M14 contained a large number of single and abnormal weakly stained starch granules ( Figure 2E,F), indicating that M14 mutation changed the formation of normal compound starch granules in rice endosperm. 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.

The Abnormal Morphology of Compound Starch Granules in M14 Endosperm
To observe the morphology of compound starch granules, the developing endosperm of WT and M14 at 9 days after fertilization (DAF) was prepared into semi-thin sections. I 2 -KI staining displayed that amyloplasts in the outer endosperm of M14 and WT developed well and filled with densely packed, polyhedral starch granules (Figure 2A

Physicochemical Properties of Starch from M14 Seeds
The seeds of WT had a starch content of 79.1%, whereas the M14 had a significantly decreased starch content (73.6%) ( Figure 3A). The amylose content decreased in M14 (13.8%) compared with WT (15.8%) ( Figure 3B), but protein content slightly increased (10.5% vs. 10.0%) ( Figure 3C). The amylopectin fine structure analysis showed that the short branch-chains with degree of polymerization (DP) 6-12 increased in M14, whereas the intermediate branch-chains with DP 13-50 decreased compared with WT ( Figure 3D). The thermal properties of WT and M14 starches were measured by differential scanning calorimetry (DSC), and their thermal parameters are shown in Table 1. The gelatinization onset temperature, peak temperature, and conclusion temperature of M14 starch were all about 2 °C lower than those of WT starch. The pasting properties of WT and M14 starches determined by rapid visco analyzer (RVA) are presented in Figure 3E, and their pasting parameters are given in Supplemental Table S1. The M14 starch had significantly lower peak viscosity, hot viscosity, and final viscosity than WT starch. In conclusion, the physicochemical characteristics of M14 starch changed significantly compared with WT starch.

Physicochemical Properties of Starch from M14 Seeds
The seeds of WT had a starch content of 79.1%, whereas the M14 had a significantly decreased starch content (73.6%) ( Figure 3A). The amylose content decreased in M14 (13.8%) compared with WT (15.8%) ( Figure 3B), but protein content slightly increased (10.5% vs. 10.0%) ( Figure 3C). The amylopectin fine structure analysis showed that the short branch-chains with degree of polymerization (DP) 6-12 increased in M14, whereas the intermediate branch-chains with DP 13-50 decreased compared with WT ( Figure 3D). The thermal properties of WT and M14 starches were measured by differential scanning calorimetry (DSC), and their thermal parameters are shown in Table 1. The gelatinization onset temperature, peak temperature, and conclusion temperature of M14 starch were all about 2 • C lower than those of WT starch. The pasting properties of WT and M14 starches determined by rapid visco analyzer (RVA) are presented in Figure 3E, and their pasting parameters are given in Supplemental Table S1. The M14 starch had significantly lower peak viscosity, hot viscosity, and final viscosity than WT starch. In conclusion, the physicochemical characteristics of M14 starch changed significantly compared with WT starch. Table 1. Thermal properties of starches a . To

Map-Based Cloning of the Gene Responsible for the M14 Mutation
For genetic analysis, an F2 population was produced from reciprocal crosses between WT and M14. All F1 seeds displayed WT phenotype, and the F2 population exhibited a 3:1 segregation ratio (497 normal: 156 floury; χ 2 = 0.43 < χ 2 0.05,1 = 3.84). Therefore, the floury phenotype in the M14 was controlled by a recessive nuclear gene. In order to identify the mutation locus controlling the M14 phenotype, the approach of map-based cloning was adopted. An F2 mapping population with approximately 5000 progeny was created by crossing the M14 with the wide-compatibility indica variety Dular. For bulk-segregant analysis (BSA), two DNA pools were generated from 10 F2 floury endosperm and 10 normal endosperm individuals, respectively. These two pools were genotyped with 170 selected insertion-deletion (InDel) markers for rough mapping. The M14 mutation locus was initially mapped between InDel marker IZ5-1 and IZ5-4 on the long arm of chromosome 5. On this basis, 854 recessive individuals were selected and detected by the two pairs of primers to screen recombinants. To further narrow down the locus, six new InDel markers were developed. The number of recombinants gradually decreased as primers were closer to the target gene ( Figure 4A). The M14 mutation locus was finally mapped to a 71.4 kb region between InDel markers IZ5-5 and IZ5-7. The 71.4 kb region on the BAC clone OJ1174H11 contained 8 putative ORFs (http://rapdb.dna.affrc.go.jp) ( Figure 4A). The genome DNA of 8 ORFs was all sequenced and only one nucleotide substitution of thymidine (T) to cytosine (C) was found in the Os05g0405000, which encodes pyruvate orthophosphate dikinase (PPDK) ( Figure 4B). Amino acid sequence analysis indicated that the single nucleotide mutation caused Leu-637 replacement by Phe ( Figure 4B). The

Map-Based Cloning of the Gene Responsible for the M14 Mutation
For genetic analysis, an F 2 population was produced from reciprocal crosses between WT and M14. All F 1 seeds displayed WT phenotype, and the F 2 population exhibited a 3:1 segregation ratio (497 normal: 156 floury; χ 2 = 0.43 < χ 2 0.05,1 = 3.84). Therefore, the floury phenotype in the M14 was controlled by a recessive nuclear gene. In order to identify the mutation locus controlling the M14 phenotype, the approach of map-based cloning was adopted. An F 2 mapping population with approximately 5000 progeny was created by crossing the M14 with the wide-compatibility indica variety Dular. For bulk-segregant analysis (BSA), two DNA pools were generated from 10 F 2 floury endosperm and 10 normal endosperm individuals, respectively. These two pools were genotyped with 170 selected insertion-deletion (InDel) markers for rough mapping. The M14 mutation locus was initially mapped between InDel marker IZ5-1 and IZ5-4 on the long arm of chromosome 5. On this basis, 854 recessive individuals were selected and detected by the two pairs of primers to screen recombinants. To further narrow down the locus, six new InDel markers were developed. The number of recombinants gradually decreased as primers were closer to the target gene ( Figure 4A). The M14 mutation locus was finally mapped to a 71.4 kb region between InDel markers IZ5-5 and IZ5-7. The 71.4 kb region on the BAC clone OJ1174H11 contained 8 putative ORFs (http://rapdb.dna.affrc.go.jp) ( Figure 4A). The genome DNA of 8 ORFs was all sequenced and only one nucleotide substitution of thymidine (T) to cytosine (C) was found in the Os05g0405000, which encodes pyruvate orthophosphate dikinase (PPDK) ( Figure 4B). Amino acid sequence analysis indicated that the single nucleotide mutation caused Leu-637 replacement by Phe ( Figure 4B). The expression level of the cyOsPPDKB in M14 was comparable to the WT at 9 DAF endosperm (Supplemental Figure S1).
In rice, the OsPPDKB gene contains two transcripts, chOsPPDKB and cyOsPPDKB, and the cyOsPPDKB expressed at a high level in developing kernels [24]. To confirm that Os05g0405000 was responsible for the M14 mutation, the pUbi:cyOsPPDKB was introduced into the M14 to complement the floury phenotype. Positive transgenic lines expressing pUbi:cyOsPPDKB were identified by PCR analysis (Supplemental Figure S2). The seed phenotype and compound starch granule morphology in three positive transgenic lines were rescued ( Figure 4C,D). Therefore, Os05g0405000 was the gene responsible for the M14 phenotype. expression level of the cyOsPPDKB in M14 was comparable to the WT at 9 DAF endosperm (Supplemental Figure S1). In rice, the OsPPDKB gene contains two transcripts, chOsPPDKB and cyOsPPDKB, and the cyOsPPDKB expressed at a high level in developing kernels [24]. To confirm that Os05g0405000 was responsible for the M14 mutation, the pUbi:cyOsPPDKB was introduced into the M14 to complement the floury phenotype. Positive transgenic lines expressing pUbi:cyOsPPDKB were identified by PCR analysis (Supplemental Figure S2). The seed phenotype and compound starch granule morphology in three positive transgenic lines were rescued ( Figure 4C,D). Therefore, Os05g0405000 was the gene responsible for the M14 phenotype.

Expression Pattern and Subcellular Localization of OsPPDKB
We examined the chOsPPDKB and cyOsPPDKB expression patterns with two pairs of specific primers (P1-F/P1-R and P2-F/P2-R in Figure 4B) by quantitative real-time PCR (qRT-PCR). In WT plants, the chOsPPDKB expressed in the stem, leaf, leaf sheath and panicle, with the highest expression level in leaf and leaf sheath ( Figure 5A). In contrast, the expression of cyOsPPDKB was detected in the root, stem, leaf, leaf sheath and developing kernels, with the highest expression in developing kernels ( Figure 5B). During kernel development, the expression of cyOsPPDKB was high at the early stage and then gradually declined ( Figure 5B). The expression trend of cyOsPPDKB in endosperm was similar to those of OsAGPL3, OsAGPS2b, OsGBSSI, OsSSIIa, OsSSIIIa, OsBEI, OsBEIIb, and OsISA2, demonstrating the coexpression of cyOsPPDKB and these genes [26].

Expression Pattern and Subcellular Localization of OsPPDKB
We examined the chOsPPDKB and cyOsPPDKB expression patterns with two pairs of specific primers (P1-F/P1-R and P2-F/P2-R in Figure 4B) by quantitative real-time PCR (qRT-PCR). In WT plants, the chOsPPDKB expressed in the stem, leaf, leaf sheath and panicle, with the highest expression level in leaf and leaf sheath ( Figure 5A). In contrast, the expression of cyOsPPDKB was detected in the root, stem, leaf, leaf sheath and developing kernels, with the highest expression in developing kernels ( Figure 5B). During kernel development, the expression of cyOsPPDKB was high at the early stage and then gradually declined ( Figure 5B). The expression trend of cyOsPPDKB in endosperm was similar to those of OsAGPL3, OsAGPS2b, OsGBSSI, OsSSIIa, OsSSIIIa, OsBEI, OsBEIIb, and OsISA2, demonstrating the coexpression of cyOsPPDKB and these genes [26].
To identify the subcellular localization of OsPPDKB, two GFP fusions, chOsPPDKB-GFP and cyOsPPDKB-GFP, were transiently expressed in Nicotiana benthamiana leaves. The free GFP protein was widely distributed in the nuclei and cytoplasm ( Figure 6A), while the chOsPPDKB-GFP proteins co-localized exclusively with chlorophyll autofluorescence signals ( Figure 6B) and the cyOsPPDKB-GFP was localized in cytoplasm, which displayed a dispersed distribution throughout the leaves ( Figure 6C).

Int. J. Mol. Sci. 2018, 19, x 7 of 14
To identify the subcellular localization of OsPPDKB, two GFP fusions, chOsPPDKB-GFP and cyOsPPDKB-GFP, were transiently expressed in Nicotiana benthamiana leaves. The free GFP protein was widely distributed in the nuclei and cytoplasm ( Figure 6A), while the chOsPPDKB-GFP proteins co-localized exclusively with chlorophyll autofluorescence signals ( Figure 6B) and the cyOsPPDKB-GFP was localized in cytoplasm, which displayed a dispersed distribution throughout the leaves ( Figure 6C).   To identify the subcellular localization of OsPPDKB, two GFP fusions, chOsPPDKB-GFP and cyOsPPDKB-GFP, were transiently expressed in Nicotiana benthamiana leaves. The free GFP protein was widely distributed in the nuclei and cytoplasm ( Figure 6A), while the chOsPPDKB-GFP proteins co-localized exclusively with chlorophyll autofluorescence signals ( Figure 6B) and the cyOsPPDKB-GFP was localized in cytoplasm, which displayed a dispersed distribution throughout the leaves ( Figure 6C).

Discussion
Starch, as the main storage material, occupies most of the volume of rice endosperm [27]. Defects in rice starch synthesis affect kernel development and weight, and further change the physicochemical properties of starch. In this study, we isolated and identified a rice floury endosperm mutant, M14. Map-based cloning and complementation test revealed that the mutant phenotype was controlled by the gene of OsPPDKB. The function knockout mutations of FLO4/OsPPDKB display a white-core endosperm phenotype [16]. Despite both flo4 and M14 displaying a similar opaque appearance in seeds, the M14 showed a more serious defective phenotype with obviously decreased seed weight, starch content and amylose content ( Figure 1D,J and Figure 3A,B) than flo4 [16]. Similar results are reported in the flo2 mutant. The seeds in flo2 mutant CNY8-1 exhibit an opaque appearance with whole floury endosperm, while the flo2 mutant flo(a) contains only a white-core endosperm in the center of seeds [9,28]. This phenotype difference may be due to genetic background and environmental effects [9].
Amylose is an important factor affecting the physicochemical properties of starch. The mutation of OsPPDKB led to a significant down-regulation of GBSSI expression (Figures 7 and 8), and resulted in the reduction of amylose synthesis, with amylose content of M14 estimated at 13.8%, approximately a 12.6% reduction compared to WT ( Figure 3B). Compared with WT starch, M14 starch exhibited a lower gelatinization peak temperature ( Table 1). The gelatinization peak temperature correlates positively with amylose content [29], which is consistent with the present study. The low amylose content of M14 led to different RVA profiles, including the PV, HV, and FV ( Figure 3E; Supplemental  Table S1). GBSSI, SSIIa and PUL together with BEI and BEIIb affect the starch pasting properties in rice [30][31][32]. Indeed, the expression of these starch synthesis genes significantly reduced in M14 (Figure 7) and, as a result, the pasting properties of M14 starch also changed. Floury endosperm with low seed hardness, low starch damage, and fine particle size is suitable for dry milling, such as the cyOsPPDKB mutant Suweon 542 [25]. In this study, another novel OsPPDKB mutant M14 with floury endosperm was identified, and the physicochemical properties of starch were further analyzed, which would provide a fine flour with a special property for quality breeding.
This seed phenotype of M14 was similar to that of rice ssg1/ssg2/ssg3/beiib, flo6, ssg4 and ss3ass4b mutants having a floury endosperm. The endosperm of the ssg4 mutant has abundant enlarged amyloplasts containing many starch subgranules, and the mutations in SSG1/BEIIb and FLO6 produce small and abnormal starch granules [10,33]. Interestingly, a large number of smaller, single starch granules were observed in the inner of M14 endosperm ( Figure 2E,F), and were similar to those of the ss3ass4b mutant [34]. In addition, the expression of SSIIIa and SSIVb were significantly reduced in M14 endosperm (Figure 7). The difference in starch morphology among M14, ssg1/beiib, flo6 and ssg4 might be due to the different effects of mutation on amylose content and amylopectin structure. In maize, the granule diameter distribution in the PPDK-deficient lines is shifted toward smaller particles [35], which is similar to the results observed in the M14 endosperm. Taken together, OsPPDKB played an important role in the formation of compound starch granules.
A floury seed phenotype is also conditioned by PPDK deficiency in maize [35], although it has clear distinctions from rice. Specifically, a single mutant lacking function of rice OsPPDKB caused the phenotype, whereas maize pdk1/C 4 ppdkZm1 is a minor contributor to total endosperm PPDK, and the floury phenotype is observed only when both the pdk1/C 4 ppdkZm1 and pdk2/cyppdkZm1 functions are eliminated. The mutation of chOsPPDKB in M14 did not significantly affect the growth of the seedlings (Supplemental Figure S3), while loss-of-function mutant of chOsPPDKB homolog (pdk1/C 4 ppdkZm1) in maize is seedling lethal. One hypothesis is that other genes with a similar function in photosynthesis might offset the loss caused by mutation in the chOsPPDKB gene in M14. Unlike rice, there are no significant differences in amylose content and the fine structure of amylopectin in maize PPDK-deficient transgenic seeds [35], which may be due to more storage protein rather than starch in maize endosperm. Owing to that the expression of most genes in the starch synthesis pathway reduced in M14, the amylose content and amylopectin structure changed significantly ( Figures 3B,D and 7).
Three hypotheses for PPDK functions in endosperm were reported, (i) participation in the process of gluconeogenesis and provision of hexose for starch synthesis, (ii) providing pyruvate for the synthesis of lipids, and (iii) regulating the sucrose synthase by controlling the homoeostasis of PPi [16,35,36]. The M14 had a single nucleotide T to C substitution in the coding region of cyOsPPDKB, leading to the Leu-637 being replaced by Phe. The single amino acid substitution did not affect the expression of cyOsPPDKB in M14 endosperm, which was different from the Suweon 542 mutant [25]. This residue was just located in the PEP-utilizers domain (Supplemental Figure  S4A). Amino acid sequence analysis revealed that the Leu-637 residue was highly conserved in the PPDK among different plant species (Supplemental Figure S4B). PPDK catalyzes the interconversion of ATP, Pi, and pyruvate to AMP, PPi, and PEP. The pyruvate kinase (PK) catalyzes PEP to pyruvate, which is an irreversible reaction. Therefore, the Leu-637 mutation of OsPPDKB in M14 might affect the reaction, that pyruvate and Pi in the cytosol of developing endosperm is converted into PEP and PPi. The reaction of PEP to pyruvate is normal. In this case, the recycling of PEP into hexose pools 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.
Three hypotheses for PPDK functions in endosperm were reported, (i) participation in the process of gluconeogenesis and provision of hexose for starch synthesis, (ii) providing pyruvate for the synthesis of lipids, and (iii) regulating the sucrose synthase by controlling the homoeostasis of PPi [16,35,36]. The M14 had a single nucleotide T to C substitution in the coding region of cyOsPPDKB, leading to the Leu-637 being replaced by Phe. The single amino acid substitution did not affect the expression of cyOsPPDKB in M14 endosperm, which was different from the Suweon 542 mutant [25]. This residue was just located in the PEP-utilizers domain (Supplemental Figure S4A). Amino acid sequence analysis revealed that the Leu-637 residue was highly conserved in the PPDK among different plant species (Supplemental Figure S4B). PPDK catalyzes the interconversion of ATP, Pi, and pyruvate to AMP, PPi, and PEP. The pyruvate kinase (PK) catalyzes PEP to pyruvate, which is an irreversible reaction. Therefore, the Leu-637 mutation of OsPPDKB in M14 might affect the reaction, that pyruvate and Pi in the cytosol of developing endosperm is converted into PEP and PPi. The reaction of PEP to pyruvate is normal. In this case, the recycling of PEP into hexose pools may be reduced, while the concentration of PPi in the cytosol may be increased. The PEP participates in the gluconeogenesis and provides hexose for starch biosynthesis [37]. The PPi is utilized as an alternative energy donor for the conversion of imported sucrose into hexose by sucrose synthase that is important for the early process of starch biosynthesis [38]. The developing kernel is an oxygen-limited tissue and the ATP level gradually decreased during the kernel filling [39]. PPDK is induced in response to hypoxia or anoxic stress and can also act glycolytically, which releases part of the energy to form ATP [23,40]. The PPDK reaction may have important roles in a storage metabolism by converting AMP to ATP in the hypoxic developing kernels. Several amyloplast enzymes exist in a large protein complex, which includes PPDK, AGPase, SSIIa, SSIIIa, BEIIa, and BEIIb, and this complex is responsible for coordinate regulation of starch synthesis [41]. Indeed, the expression of the complex members AGPS1, AGPS2, AGPL1, AGPL3, AGPL4, SSIIa, SSIIIa, BEIIa, and BEIIb in M14 endosperm were significantly reduced ( Figure 7). Therefore, the mutation of PPDK might also affect the function of the large protein complex. may be reduced, while the concentration of PPi in the cytosol may be increased. The PEP participates in the gluconeogenesis and provides hexose for starch biosynthesis [37]. The PPi is utilized as an alternative energy donor for the conversion of imported sucrose into hexose by sucrose synthase that is important for the early process of starch biosynthesis [38]. The developing kernel is an oxygen-limited tissue and the ATP level gradually decreased during the kernel filling [39]. PPDK is induced in response to hypoxia or anoxic stress and can also act glycolytically, which releases part of the energy to form ATP [23,40]. The PPDK reaction may have important roles in a storage metabolism by converting AMP to ATP in the hypoxic developing kernels. Several amyloplast enzymes exist in a large protein complex, which includes PPDK, AGPase, SSIIa, SSIIIa, BEIIa, and BEIIb, and this complex is responsible for coordinate regulation of starch synthesis [41]. Indeed, the expression of the complex members AGPS1, AGPS2, AGPL1, AGPL3, AGPL4, SSIIa, SSIIIa, BEIIa, and BEIIb in M14 endosperm were significantly reduced ( Figure 7). Therefore, the mutation of PPDK might also affect the function of the large protein complex.

Plant Materials and Growth Conditions
The grains of japonica rice variety Kitaake were irradiated with 60 Co-γ ray at the dose of 300 Gy. Mature grains of M1 plant were harvested respectively and dehusked with a rice huller to screen endosperm mutants. A floury endosperm mutant M14 was identified. The M14 and an indica variety Dular were hybridized to produce F2 population for map-based cloning. The WT and M14 were planted at the experimental farm of Yangzhou University in May and harvested in September.

Microscopy
The samples were prepared following the method as described previously [13]. Briefly, mature seeds were transversely broken by two tweezers, coated with gold, and examined with a scanning electron microscope (SEM, S-4800, Hitachi, Tokyo, Japan). For the observation of compound starch granules, transverse sections of WT and M14 kernels at 9 DAF were fixed overnight in 2% (v/v) glutaraldehyde. The samples were dehydrated in an alcohol series, embedded in LR White resin, and sectioned using an ultrathin microtome (EM UC7, Leica, Wetzla, Germany). Semi-thin sections

Plant Materials and Growth Conditions
The grains of japonica rice variety Kitaake were irradiated with 60 Co-γ ray at the dose of 300 Gy. Mature grains of M1 plant were harvested respectively and dehusked with a rice huller to screen endosperm mutants. A floury endosperm mutant M14 was identified. The M14 and an indica variety Dular were hybridized to produce F 2 population for map-based cloning. The WT and M14 were planted at the experimental farm of Yangzhou University in May and harvested in September.

Microscopy
The samples were prepared following the method as described previously [13]. Briefly, mature seeds were transversely broken by two tweezers, coated with gold, and examined with a scanning electron microscope (SEM, S-4800, Hitachi, Tokyo, Japan). For the observation of compound starch granules, transverse sections of WT and M14 kernels at 9 DAF were fixed overnight in 2% (v/v) glutaraldehyde. The samples were dehydrated in an alcohol series, embedded in LR White resin, and sectioned using an ultrathin microtome (EM UC7, Leica, Wetzla, Germany). Semi-thin sections were dried at 40 • C, stained with I 2 -KI, and observed with a light microscope (BX53, Olympus, Tokyo, Japan).

Measurement of Starch Properties
Mature rice grains were air-dried and dehulled. Brown rice was further ground with a mortar and passed through a 0.15 mm sieve. The total starch content in the flour was determined with a total starch assay kit (Megazyme, Wicklow, Ireland). The amylose content was measured by using an iodine-potassium iodide colorimetric method as described previously [42]. The chain length distribution of amylopectin was analyzed using a fluorophore-assisted capillary electrophoresis (FACE) method according to the previous report [43]. The thermal properties and pasting properties of starch were measured by differential scanning calorimetry (DSC, 200-F3, Netzsch, Selb, Germany) and rapid visco analyzer (RVA, 3D, Newport Scientific, Narrabeen, Australia), respectively, following the method as described previously [44].

Map-Based Cloning of the OsPPDKB Gene
The samples with floury endosperm phenotype were selected from the F 2 population of M14 (japonica) and Dular (indica) for map-based cloning. One-hundred-and-seventy genome-wide InDel markers showing polymorphism were selected (Supplemental Table S2). The sequences of markers for fine mapping are shown in Supplemental Table S3.

Vector Construction and Complementation Test
The WT cyOsPPDKB cDNA sequence was cloned by PCR and inserted into the binary vector pCUbi1390 to generate the transformation cassette pUbi:cyOsPPDKB for complementation. The plasmid pUbi:cyOsPPDKB was introduced into calli generated from M14 by Agrobacterium strain EHA105. Sixteen independent positive transgenic lines were obtained.

RNA Extraction and qRT-PCR
Total RNA from various tissues of WT and M14 was extracted using the OminiPlant RNA Kit (CWBIO, Beijing, China). The first-strand cDNA was reverse transcribed by priming with oligo (dT18) based on HiScript 1st Strand cDNA Synthesis Kit (Vazyme, Nanjing, China). The qRT-PCR was performed on a CFX Connect real-time PCR system (Bio-Rad, Hercules, CA, USA) using AceQ qPCR SYBR Green Master Mix Kit (Vazyme, Nanjing, China). The PCR procedure was carried out using the following program: 95 • C for 10 min, then 40 cycles of 95 • C for 15 s, 60 • C for 1 min. The rice Actin1 was employed as an endogenous control. Quantitative gene expression was analyzed from three biological replicates by the 2 −∆∆Ct method, where ∆∆C t = (C t, Target gene − C t, Actin1 ) M14 − (C t, Target gene − C t, Actin1 ) WT [45]. The primers of OsPPDKB and Actin1 were listed in Supplemental Table  S3. The primers detecting starch biosynthesis have been reported elsewhere [12].

Subcellular Localization Analysis of OsPPDKB
The two transcripts of OsPPDKB (chOsPPDKB and cyOsPPDKB) were cloned in the pCAMBIA1305-GFP vector to create 35s::chOsPPDKB-GFP and 35s::cyOsPPDKB-GFP. The two binary vectors were transformed into Nicotiana benthamiana leaves following the method as described previously [11]. Laser scanning confocal microscope (LSM710, Zeiss, Oberkochen, Germany) was used to detect the GFP fluorescent signals.

Protein Extraction and Immunoblot Analysis
In order to separate the endosperm tissue, the developing kernels of WT and M14 at 9 DAF were harvested, and the embryo and pericarp were removed subsequently. Proteins were extracted following the method as described previously [43], and transferred electrophoretically to nitrocellulose (NC) membrane after SDS-PAGE. The NC membrane was incubated in blocking buffer adding antibodies and was detected with ECL Plus reagent. Antibodies against GBSSI, SSI, BEI, BEIIa and BEIIb were described previously [46].

Conclusions
A floury endosperm mutant M14 was isolated from Kitaake grains irradiated with 300 Gy 60 Co-γ ray. Map-based cloning analysis showed that the floury endosperm phenotype was controlled by cyOsPPDKB. Abundant single and abnormal starch granules were observed in M14 endosperm. Compared with WT, amylopectin short branch-chains with DP 6-12 increased, but intermediate branch-chains with DP 13-50 decreased in M14. The M14 starch had a significantly lower gelatinization temperature and peak, hot and final viscosities. The chloroplast-localized PPDK highly expressed in leaf and leaf sheath and the cytoplasm-localized PPDK exhibited a high expression in developing endosperm. The expression of starch synthesis-related genes was significantly altered in M14. The substitution of single amino acid in M14 affects the catalysis of PPDK on the reaction of Pyruvate to PEP, leading to the change of starch metabolism and structure through regulating the expression of starch synthesis-related enzymes in rice endosperm. This study contributed to an understanding of the role of PPDK in starch metabolism and structure and provided some information for quality breeding of rice.