Identification of the Soluble Starch Synthase Activities of Maize Endosperm

This study identified the complement of soluble starch synthases (SSs) present in developing maize (Zea mays) endosperm. The product of the du1 gene, DU1, was shown to be one of the two major soluble SSs. The C-terminal 450 residues of DU1 comprise eight sequence blocks conserved in 28 known or predicted glucan synthases. This region of DU1 was expressed in Escherichia coli and shown to possess SS activity. DU1-specific antisera detected a soluble endosperm protein of more than 200 kD that was lacking in du1- mutants. These antisera eliminated 20% to 30% of the soluble SS activity from kernel extracts. Antiserum against the isozyme zSSI eliminated approximately 60% of the total soluble SS, and immunodepletion of du1- mutant extracts with this antiserum nearly eliminated SS activity. Two soluble SS activities were identified by electrophoretic fractionation, each of which correlated specifically with zSSI or DU1. Thus, DU1 and zSSI accounted for the great majority of soluble SS activity present in developing endosperm. The relative activity of the two isozymes did not change significantly during the starch biosynthetic period. DU1 and zSSI may be interdependent, because mutant extracts lacking DU1 exhibited a significant stimulation of the remaining SS activity.     

Reduced Soluble Proteins Associated with Maize Endosperm Protein Bodies

Endosperm protein bodies from developing maize were purifiedby discontinuous sucrose gradient centrifugation and the proteincontent analysed by sodium dodecyl sulphate polyacrylamide gelelectrophoresis (SDS-PAE). Major proteins detected were zeinpolypeptides plus a component with Mr 28 000 and a doublet aroundMr 58 000. These proteins were present only in the protein bodyfraction of the sucrose gradient. Treatment of protein bodieswith the reducing agent dithiothreitol (DTT) in aqueous bufferdissolved the components with Mr 28 000 and 58 000, plus minorones, but not zein. The reduced soluble proteins were separatedby DEAE-Sephacel chromatography into three fractions: two ofthese contained the component with Mr 28 000, and the thirdthe components around Mr 58 000 plus minor ones. Proteins fromthe three fractions had characteristic amino acid compositions,markedly different from those of zein polypeptides. Chymotrypticdigestion experiments performed on protein bodies under variousconditions, and two-dimensional electrophoresis of proteinsfrom protein bodies suggested that the major zein polypeptides,the protein with Mr 28 000 and the other reduced soluble proteinshave different native organizations.     

Endosperm Protein Quality and Kernel Modification in the Quality Protein Maize Inbred Lines

Twenty-two selected quality protein maize (QPM) lines, including 13 lines developed in India (DMRQPM series) and nine lines released by CIMMYT, Mexico (CML series), were evaluated for their endosperm protein content and quality, besides kernel modification in terms of vitreousness. Endosperm protein contents in 13QPMlines were on par or better than that of the normal maize ‘checks’ (Trishulata and Parkash). The QPM endosperm proteins showed significantly higher % tryptophan as well as EF-1α (a multifunctional protein with a positive and highly significant correlation with lysine content in the endosperm) contents, in comparison with the normal maize genotypes. Evaluation of kernel modification revealed considerable scope for accumulation of endosperm modifiers in some of the QPM lines. Positive and highly significant correlation was revealed between tryptophan and EF-1α contents in the endosperm proteins, whereas the correlations between the quality parameters with kernel modification in the QPM genotypes were found to be non-significant. The study led to the identification of some promising QPM lines, such as DMRQPM-37, DMRQPM-44, CML176, CML142 and CML149, which could be effectively deployed in the QPM breeding programmes.     

The Starch-Debranching Enzymes Isoamylase and Pullulanase Are Both Involved in Amylopectin Biosynthesis in Rice Endosperm

The activities of the two types of starch debranching enzymes, isoamylase and pullulanase, were greatly reduced in endosperms of allelic sugary-1 mutants of rice (Oryza sativa), with the decrease more pronounced for isoamylase than for pullulanase. However, the decrease in isoamylase activity was not related to the magnitude of the sugary phenotype (the proportion of the phytoglycogen region of the endosperm), as observed with pullulanase. In the moderately mutated line EM-5, the pullulanase activity was markedly lower in the phytoglycogen region than in the starch region, and isoamylase activity was extremely low or completely lost in the whole endosperm tissue. These results suggest that both debranching enzymes are involved in amylopectin biosynthesis in rice endosperm. We presume that isoamylase plays a predominant role in amylopectin synthesis, but pullulanase is also essential or can compensate for the role of isoamylase in the construction of the amylopectin multiple-cluster structure. It is highly possible that isoamylase was modified in some sugary-1 mutants such as EM-273 and EM-5, since it was present in significant and trace amounts, respectively, in these mutants but was apparently inactive. The results show that the Sugary-1 gene encodes the isoamylase gene of the rice genome.     

A Nonaqueous Procedure for Isolating Starch Granules with Associated Metabolites from Maize (Zea mays L.) Endosperm

A nonaqueous procedure using glycerol and 3-chloro-1,2-propanediol was developed for the isolation from maize of starch granules with associated metabolites. In this procedure, immature endosperm tissue was quickly frozen at −156 C, freeze-dried, homogenized in cold glycerol, filtered through Miracloth, and centrifuged through a higher density medium of 3-chloro-1,2-propanediol. The procedure was used to isolate starch granules from the endosperm of normal and the mutant amylose-extender dull waxy. Starch and water-soluble polysaccharide recovery was high with low cytoplasmic (RNA) and nuclear (DNA) contamination.     

玉米籽粒胚乳细胞增殖及其与淀粉充实的关系

用纤维素酶解离胚乳、滤膜法统计玉米胚乳细胞的数目,进一步借助Logistic方程模拟胚乳细胞增殖动态的结果表明,整个灌浆期间胚乳细胞增殖呈现“慢-快-慢”的变化趋势。授粉15d后,不同类型胚乳的细胞数目依序为普通玉米〉糯玉米〉甜玉米〉爆裂玉米;胚乳细胞数目主要取决于细胞的增殖速率,并与淀粉充实和粒重关系密切。胚乳发育前期以胚乳细胞增殖为主,后期以淀粉积累为主。     

Deficiency of Starch Synthase IIIa and IVb Alters Starch Granule Morphology from Polyhedral to Spherical in Rice Endosperm

Starch granule morphology differs markedly among plant species. However, the mechanisms controlling starch granule morphology have not been elucidated. Rice (Oryza sativa) endosperm produces characteristic compound-type granules containing dozens of polyhedral starch granules within an amyloplast. Some other cereal species produce simple-type granules, in which only one starch granule is present per amyloplast. A double mutant rice deficient in the starch synthase (SS) genes SSIIIa and SSIVb (ss3a ss4b) produced spherical starch granules, whereas the parental single mutants produced polyhedral starch granules similar to the wild type. The ss3a ss4b amyloplasts contained compound-type starch granules during early developmental stages, and spherical granules were separated from each other during subsequent amyloplast development and seed dehydration. Analysis of glucan chain length distribution identified overlapping roles for SSIIIa and SSIVb in amylopectin chain synthesis, with a degree of polymerization of 42 or greater. Confocal fluorescence microscopy and immunoelectron microscopy of wild-type developing rice seeds revealed that the majority of SSIVb was localized between starch granules. Therefore, we propose that SSIIIa and SSIVb have crucial roles in determining starch granule morphology and in maintaining the amyloplast envelope structure. We present a model of spherical starch granule production.Starch is the most important carbohydrate storage material and contains the Glc polymers amylose and amylopectin. At least four classes of enzymes, ADP-Glc pyrophosphorylase (AGPase), starch synthase (SS), starch branching enzyme (BE), and starch debranching enzyme (DBE), are necessary for efficient starch biosynthesis in storage tissues.SSs (EC 2.4.1.21) play a central role in starch synthesis during α-glucan elongation by adding Glc residues from ADP-Glc to the nonreducing ends via α-1,4-glucosidic linkages. Rice (Oryza sativa) contains 11 SS genes that are grouped into six classes, SSI to SSV and granule-bound starch synthase (GBSS; Supplemental Fig. S1; Hirose and Terao, 2004; Ohdan et al., 2005). Every class contains multiple isozymes, except for SSI and SSV; SSI, SSIIa, SSIIIa, and GBSSI are highly expressed in developing rice endosperm (Hirose and Terao, 2004; Ohdan et al., 2005). SSI elongates short amylopectin chains with degree of polymerization (DP) from 6 or 7 to DP 8 to 12 (Fujita et al., 2006). SSIIa elongates amylopectin from DP 6 to 12 to DP 13 to 24 (Umemoto et al., 2002; Nakamura et al., 2005), and SSIIIa elongates long amylopectin chains with DP 33 or greater (Fujita et al., 2007). GBSSI synthesizes amylose and extra-long amylopectin chains (Sano, 1984; Takeda et al., 1987; Hizukuri, 1995). The functions of other SS isozymes, such as SSIIb, SSIIc, SSIIIb, SSIVa, SSIVb, SSV, and GBSSII, remain largely unknown due to the lack of respective mutant lines. It is not clear how SS isozymes contribute to starch granule formation.Rice endosperm amyloplasts produce characteristic compound-type starch granules, which consist of dozens of polyhedral, sharp-edged granules (Matsushima et al., 2010). Compound-type starch granules are the most common type in endosperm of Poaceae species (Tateoka, 1962; Grass Phylogeny Working Group, 2001; Prasad et al., 2011; Matsushima et al., 2013). Simple-type starch granules (one starch granule per amyloplast) are produced in some species of the Bambusoideae, Pooideae, Micrairoideae, Chloridoideae, and Panicoideae subfamilies. The taxonomic relationships in the Poaceae do not enable an accurate prediction of granule morphology (Tateoka 1962; Shapter et al., 2008; Matsushima et al., 2013).Two studies that changed starch granule shape from simple type to compound type have been reported (Suh et al., 2004; Myers et al., 2011). A hull-less cultivar of cv Betzes barley (Hordeum vulgare), cv Nubet, contains simple-type and bimodal starch granules, which are typical of wild-type barley. Chemical mutagenesis of cv Nubet produced a mutant called franubet, which contains compound-type starch granules (Suh et al., 2004). In the maize monogalactosyldiacylglycerol synthase-deficient mutant opaque5, simple-type granules are replaced by compound-type granules separated by a membranous structure (Myers et al., 2011). The molecular mechanisms that control starch granule morphology in cereal endosperm are largely unknown, although an alteration in membrane lipid synthesis may be involved (Myers et al., 2011).A structural model for the compound-type amyloplast is shown Figure 1. The amyloplast envelope contains an outer envelope membrane (OEM), inner envelope membrane (IEM), and intermembrane space (IMS). Each starch granule is enclosed by an IEM, and granules are separated by a septum-like structure (SLS; Yun and Kawagoe, 2010). In this model, the IMS and SLS are directly connected, and fluorescent proteins such as GFP and Cherry can move freely between the two (Fig. 1; Kawagoe, 2013). The chloroplast envelope membrane contains little protein compared with the thylakoid membrane (Heber and Heldt, 1981). The endosperm amyloplast envelope membrane contains even less protein. Low protein content could be a major reason why the amyloplast envelope in rice endosperm is difficult to observe using high-resolution electron microscopy. In transgenic rice, a fluorescent protein fused to an IEM protein, the ADP-Glc transporter BRITTLE1, visualized the amyloplast IEM (Yun and Kawagoe, 2010). Fluorescent proteins fused to the chloroplast OEM protein OEP7 visualized the amyloplast OEM in endosperm (Kawagoe, 2013). These studies revealed that the outermost membranes of rice amyloplasts are OEM and contain intraamyloplast compartments. Starch is synthesized within the amyloplast compartments and is ultimately formed as compound-type granules that are individually wrapped in IEM (Yun and Kawagoe, 2010; Kawagoe, 2013).Open in a separate windowFigure 1.Structural model of the wild-type amyloplast in developing rice endosperm. The OEM is in black, the IEM is in magenta, the IMS is in green, and the SLS is in blue. G, Starch granules.Confocal microscopy analyses of the rice IEM protein, BRITTLE1, revealed that an SLS, or cross wall, divides starch granules in the amyloplast (Yun and Kawagoe, 2010). A model for the synthesis of compound-type starch granules consisting of polyhedral, sharp-edged granules proposed that the SLS functions as a mold that casts growing granules into a characteristic shape (Yun and Kawagoe, 2010; Kawagoe, 2013). The model postulates a central role for the SLS in producing characteristic compound-type granules, although neither the SLS components nor the enzymes affecting its properties have been characterized.Arabidopsis (Arabidopsis thaliana) SS genes are grouped into six classes. Leaf transitory starch biosynthesis has been investigated in single mutants of SSI, SSII, SSIII, and SSIV and in various double and triple SS mutants (Ral et al., 2004; Delvallé et al., 2005; Zhang et al., 2005, 2008; Szydlowski et al., 2009, 2011). Starch granules in leaf chloroplasts are reduced in number but enlarged in the ssIV mutant (Roldán et al., 2007; Crumpton-Taylor et al., 2013) and in the ssIV double and triple mutants (Szydlowski et al., 2009). Immature ssIV leaves have no starch granules but accumulate the starch synthase substrate ADP-Glc at high concentrations. Starch granules are flattened and discoid in wild-type leaves but are rounded in mature leaves of ssIV, suggesting that SSIV is essential for coordinating granule formation with chloroplast division during leaf expansion (Crumpton-Taylor et al., 2013). The ssIII ssIV double mutant does not accumulate measurable amounts of starch in the leaves, despite the presence of SSI and SSII activity (Szydlowski et al., 2009), implying that Arabidopsis SSIII and SSIV are involved in the initiation of starch granule formation and that either SSIII or SSIV is sufficient. Overexpression of AtSSIV increases the starch level in Arabidopsis leaves and potato (Solanum tuberosum) tubers (Gámez-Arjona et al., 2011). In transgenic plants, the AtSSIV-GFP fusion protein is enriched in specific regions at the edge of granules in Arabidopsis chloroplasts and potato tuber amyloplasts. In rice, SSIVa and SSIVb are expressed in the endosperm and other organs at an early developmental stage (Hirose and Terao, 2004; Ohdan et al., 2005).In this study, two rice allelic SSIVb-deficient mutant lines (ss4b) were generated by insertion of the retrotransposon Tos17 and crossed with the SSIIIa null mutant (ss3a). Surprisingly, the ss3a ss4b endosperm produced spherical starch granules that were separated from each other within amyloplasts, whereas the single mutants produced compound-type polyhedral starch granules. The SSIVb and GBSSI enzymes were localized to distinct compartments in developing amyloplasts. We discuss the changes in rice starch structure due to the deficiency of both SSIIIa and SSIVb, the alteration in starch granule morphology, and possible unconventional functions of SSIIIa and SSIVb. We also present a model of how spherical granules are produced in ss3a ss4b rice endosperm.     

Alterations in Fibronectin Type III Domain Containing 1 Protein Gene Are Associated with Hypertension

Multiple quantitative trait loci (QTLs) for blood pressure (BP) have been detected in rat models of human polygenic hypertension. Great challenges confronting us include molecular identifications of individual QTLs. We first defined the chromosome region harboring C1QTL1 to a segment of 1.9 megabases that carries 9 genes. Among them, we identified the gene encoding the fibronectin type III domain containing 1 protein (Fndc1)/activator of G protein signaling 8 (Ags8) to be the strongest candidate for C1QTL1, since numerous non-synonymous mutations are found. Moreover, the 5’ Fndc1/Ags8 putative promoter contains numerous mutations that can account for its differential expression in kidneys and the heart, prominent organs in modulating BP, although the Fndc1/Ags8 protein was not detectable in these organs under our experimental conditions. This work has provided the premier evidence that Fndc1/Ags8 is a novel and strongest candidate gene for C1QTL1 without completely excluding other 8 genes in the C1QTL1-residing interval. If proven true by future in vivo function studies such as single-gene Fndc1/Ags8 congenics, transgenesis or targeted-gene modifications, it might represent a part of the BP genetic architecture that operates in the upstream position distant from the end-phase physiology of BP control, since it activates a Gbetagamma component in a signaling pathway. Its functional role could validate the concept that a QTL in itself can influence BP ‘indirectly’ by regulating other genes downstream in a pathway. The elucidation of the mechanisms initiated by Fndc/Ags8 variations will reveal novel insights into the BP modulation via a regulatory hierarchy.     

Formation and Deposition of Amylose in the Potato Tuber Starch Granule Are Affected by the Reduction of Granule-Bound Starch Synthase Gene Expression

The synthesis of amylose in amyloplasts is catalyzed by granule-bound starch synthase (GBSS). GBSS gene expression was inhibited via antisense RNA in Agrobacterium rhizogenes-transformed potato plants. Analysis of starch production and starch granule composition in transgenic tubers revealed that reduction of GBSS activity always resulted in a reduction of the production of amylose. Field experiments, performed over a 2-year period, showed that stable inhibition of GBSS gene expression can be obtained. Microscopic evaluation of iodine-stained starch granules was shown to be a sensitive system for qualitative and quantitative examination of amylose formation in starch granules of transgenic potato tubers. In plants showing inhibition of GBSS gene expression, the reduced amylose content in tuber starch was not a consequence of a lower amylose content throughout the entire starch granule. Starch granules of transgenic tubers were found to contain amylose at a percentage similar to wild-type starch in a core of varying size at the hilum of each granule. This indicated that reduced GBSS gene expression results in amylose formation in a restricted zone of the granules. The size of this zone is suggested to be dependent on the GBSS protein level. During development of the granules, the available GBSS protein is thought to become limiting, resulting in the formation of starch that lacks amylose. RNA gel blot analysis of tuber tissue showed that inhibition of GBSS gene expression resulted in a reduced GBSS mRNA level but did not affect the expression level of other starch synthesizing enzymes. Antisense RNA could only be detected in leaf tissue of the transgenic plants.     

多胞质玉米胚乳淀粉粒性状的扫描电镜观察

１１种多胞质系玉米胚乳淀粉粒的扫描电镜观察表明：不同的细胞质对细胞核有不同程度的互作,３种甜质胞质玉米的胚乳淀粉粒多呈球形,排列紧密,存在一定的共性;４种雄性不育胞质玉米的胚乳淀粉粒多呈不规则形,除（Ｔ）Ｍｏ１７外,排列疏松。这１１种玉米胚乳淀粉粒的平均直径为９．７８μｍ￣１４．６９μｍ,通过玉米胚乳淀粉粒形态特征的观察,在玉米淀粉性状和玉米籽粒的商品价值关系上进行一定程度探索,为玉米的进一步发展     

糯玉米胚乳淀粉粒粒度分布形成的酶学机理

以7个糯玉米品种为材料,测定其籽粒发育过程中淀粉粒粒度分布及淀粉合成相关酶活性的变化,分析两者之间的关系。结果表明,随着籽粒发育,糯玉米淀粉粒平均粒径逐渐增大,可溶性淀粉合成酶(SSS)和淀粉分支酶(SBE)活性呈单峰曲线变化。籽粒发育前期,小淀粉粒(≤7.4μm)所占体积较大;随着籽粒发育,小淀粉粒所占体积减少,大淀粉粒(>7.4μm)所占体积增多;籽粒发育后期,大淀粉粒所占体积较大。相关分析表明, SSS和SBE活性与大淀粉粒体积增大速率和平均粒径增大速率均呈显著或极显著正相关。因此, SSS和SBE是影响糯玉米胚乳淀粉粒粒度分布形成的主要酶, SSS和SBE活性越高,淀粉粒平均粒径越大,大淀粉粒所占体积越多。     

The Maize Invertase-Deficient miniature-1 Seed Mutation Is Associated with Aberrant Pedicel and Endosperm Development

Genetic evidence is presented to show that the developmental stability of maternal cells in the pedicel at the base of maize seeds is determined by the genotype of the developing endosperm. An early degeneration and withdrawal of maternal cells from the endosperm of homozygous miniature (mn mn) seed mutants were arrested if mn plants were pollinated by the wild-type Mn pollen. Similarly, the stability of the wild-type, Mn mn, maternal cells was also dependent on whether or not these cells were associated with the normal (Mn) or the mutant (mn) endosperm on the same ear. Biochemical and cellular analyses indicated that developing mn kernels have extremely low (<0.5% of the wild type) to undetectable levels of both soluble and wall-bound invertase activities. Extracts from endosperm with a single copy of the Mn gene showed a significant increase in both forms of invertases, and we suggest it is the causal basis of the wild-type seed phenotype. Collectively, these data provide evidence that invertase-mediated maintenance of a physiological gradient of photosynthate between pedicel and endosperm constitutes the rate-limiting step in structural stability of maternal cells as well as normal development of endosperm and seed.     

Regulation of Maize Leaf Sucrose-Phosphate Synthase by Protein Phosphorylation

Studies were conducted to determine the potential for regulationof maize leaf sucrose-phosphate synthase (SPS) by protein phosphorylation.Highly activated enzyme, in desalted crude leaf extracts preparedfrom illuminated leaves, was inactivated in vitro in a time-and ATP-de-pendent manner. Partial purification of SPS by polyethyleneglycol fractionation and Mono Q chromatography yielded enzymethat was not ATP-inactivated, possibly due to elimination ofcontaminating protein kinase. We used the partially purifiedSPS as substrate to identify an endogenous protein kinase. Theprotein kinase catalyzed the time- and ATP-dependent inacti-vationof SPS, and the apparent K_m for Mg-ATP was estimated to be approximately10µM. The partially purified maize SPS protein was phosphorylatedin vitro using [y-³²P]ATP and either the endogenous proteinkinase or the catalytic subunit of cAMP-dependent protein kinase.The incorporation of radiolabel was closely paralleled by inactivationof the enzyme. These results provide the first evidence forregulation of maize leaf SPS by protein phosphorylation, whichwe postulate is the mechanism of light-dark regulation in vivo. (Received October 23, 1990; Accepted January 7, 1991)     

Measurement of Metabolites Associated with Nonaqueously Isolated Starch Granules from Immature Zea mays L. Endosperm

Starch granules with associated metabolites were isolated from immature Zea mays L. endosperm by a nonaqueous procedure using glycerol and 3-chloro-1,2-propanediol. The soluble extract of the granule preparation contained varying amounts of neutral sugars, inorganic phosphate, hexose and triose phosphates, organic acids, adenosine and uridine nucleotides, sugar nucleotides, and amino acids. Based on the metabolites present and on information about translocators in chloroplast membranes, which function in transferring metabolites from the chloroplast stroma into the cytoplasm, it is suggested that sucrose is degraded in the cytoplasm, via glycolysis, to triose phosphates which cross the amyloplast membrane by means of a phosphate translocator. It is further postulated that hexose phosphates and sugars are produced from the triose phosphates in the amyloplast stroma by gluconeogenesis with starch being formed from glucose 1-phosphate via pyrophosphorylase and starch synthase enzymes. The glucose 1-phosphate to inorganic phosphate ratio in the granule preparation was such that starch synthesis by phosphorylase is highly unlikely in maize endosperm.     

Amyloplast Membrane Protein SUBSTANDARD STARCH GRAIN6 Controls Starch Grain Size in Rice Endosperm

Starch is a biologically and commercially important polymer of glucose. Starch is organized into starch grains (SGs) inside amyloplasts. The SG size differs depending on the plant species and is one of the most important factors for industrial applications of starch. There is limited information on genetic factors regulating SG sizes. In this study, we report the rice (Oryza sativa) mutant substandard starch grain6 (ssg6), which develops enlarged SGs in endosperm. Enlarged SGs are observed starting at 3 d after flowering. During endosperm development, a number of smaller SGs appear and coexist with enlarged SGs in the same cells. The ssg6 mutation also affects SG morphologies in pollen. The SSG6 gene was identified by map-based cloning and microarray analysis. SSG6 encodes a protein homologous to aminotransferase. SSG6 differs from other rice homologs in that it has a transmembrane domain. SSG6-green fluorescent protein is localized in the amyloplast membrane surrounding SGs in rice endosperm, pollen, and pericarp. The results of this study suggest that SSG6 is a novel protein that controls SG size. SSG6 will be a useful molecular tool for future starch breeding and applications.Starch is an important plant-derived Glc polymer that is widely used as food and in manufacturing applications involving nonfood products. Starch is water insoluble and osmotically inactive. These properties make starch a suitable molecule for long-term carbohydrate storage in seeds and tubers. Higher plant cells contain terminally differentiated plastids called amyloplasts, which is the organelle involved in starch synthesis and storage in endosperm and tubers (Sakamoto et al., 2008). Starch is organized as transparent grains (starch grains [SGs]) in amyloplasts (Buléon et al., 1998; Hancock and Tarbet, 2000). SGs are easily visualized using iodine solution and can be clearly observed with a normal light microscope (Matsushima et al., 2010).Cereal endosperm accumulates high levels of starch, which fill most of the amyloplast intracellular space. Therefore, the amyloplast volume is considered as approximately equivalent to the SG volume (Matsushima et al., 2014). Rice (Oryza sativa) endosperm SGs are normally 10 to 20 μm in diameter (Matsushima et al., 2010). Each amyloplast contains a single SG that is organized from the assembly of several dozen smaller starch granules. Each starch granule is a sharp-edged polyhedron with a typical diameter of 3 to 8 μm. This type of SG is called a compound SG (Tateoka, 1962). For compound SGs, starch granules are assembled but not fused to form a single SG, which is easily separated by conventional purification procedures. By contrast, a simple SG contains a single starch granule (Tateoka, 1962). In this case, both terms are used equally. Simple SGs are produced in several important crops such as maize (Zea mays), sorghum (Sorghum bicolor), barley (Hordeum vulgare), and wheat (Triticum aestivum; Tateoka, 1962; Matsushima et al., 2010, 2013). SG sizes in cereal endosperm are diverse. Maize and sorghum SGs have a uniform size distribution of approximately 10 μm in diameter (Jane et al., 1994; Matsushima et al., 2010; Ai et al., 2011). In barley and wheat, SGs of two discrete size classes (approximately 15−25 μm and less than 10 μm) coexist in the same cells (Evers, 1973; French, 1984; Jane et al., 1994; Matsushima et al., 2010, 2013).The size of starch granules is one of the most important characteristics for industrial applications (Lindeboom et al., 2004). Small starch granules can replace fat in food applications, because aqueous dispersions of small starch granules show fat-mimetic properties (Malinski et al., 2003). The larger starch granules of maize and cassava (Manihot esculenta) improve the final starch yield after wet-milling purification (Gutiérrez et al., 2002). In the case of simple SGs, the size of SGs is equal to the size of starch granules. Therefore, manipulation of the sizes of SGs and starch granules is a molecular target for bioengineering programs. SG size can be reduced in transgenic plants and genetic mutants by down-regulating several starch synthetic enzymes (Gutiérrez et al., 2002; Bustos et al., 2004; Ji et al., 2004; Stahl et al., 2004; Matsushima et al., 2010; Fujita, 2014). By contrast, our understanding of the genetic tools, biosynthetic enzymes, and plant materials that can be utilized to enlarge SGs is limited.Recent work identified a rice mutant that develops enlarged SGs; this mutant has been named substandard starch grain4 (ssg4; Matsushima et al., 2014). The enlarged SGs are observed in starch-accumulating tissues of ssg4, including endosperm, pollen, root caps, and young pericarp. Chloroplasts in young ssg4 leaves also are enlarged. SSG4 encodes an amyloplast-localized large protein with a domain of unknown function (DUF490). SSG4 homologs are conserved from bacteria to higher plants; however, the exact molecular function of SSG4 is unknown.In this study, we report the identification of another rice mutant (ssg6) that develops enlarged SGs in endosperm. We characterize the ssg6 mutation and identify the responsible gene. SSG6 encodes a protein homologous to aminotransferase. SSG6 is localized at the amyloplast membrane and is a novel factor that influences SG size. We also determined that ssg4 and ssg6 mutations act synergistically in pollens. SSG6 will be a useful molecular target for future starch breeding and starch biotechnology programs.