Knockout of a starch synthase gene <Emphasis Type="Italic">OsSSIIIa</Emphasis>/<Emphasis Type="Italic">Flo5</Emphasis> causes white-core floury endosperm in rice (<Emphasis Type="Italic">Oryza sativa</Emphasis> L.)

To elucidate the role of SSIIIa during starch synthesis in rice (Oryza sativa L.) endosperm, we characterized null mutants of this gene, generated by T-DNA insertions. Scanning electron microscope (SEM) analysis revealed that the starch granules in these mutants are smaller and rounder compared with the wild type controls, and that the mutant endosperm is characterized by a loosely packed central portion exhibiting a floury-like phenotype. Hence, the OsSSIIIa (Oryza sativa SSIIIa) mutations are referred to as white-core floury endosperm 5-1 (flo5-1) and flo5-2. Based upon their X-ray diffraction patterns, the crystallinity of the starch in the flo5 mutant endosperm is decreased compared with wild type. Through determination of the chain-length distribution of the mutant endosperm starch, we found that flo5-1 and flo5-2 mutants have reduced the content of long chains with degree of polymerization (DP) 30 or greater compared with the controls. This suggests that OsSSIIIa/Flo5 plays an important role in generating relatively long chains in rice endosperm. In addition, DP 6 to 8 and DP 16 to 20 appeared to be reduced in endosperm starch of flo5-1 and flo5-2, whereas DP 9 to 15 and DP 22 to 29 were increased in these mutants. By the use of differential scanning calorimetry (DSC), the gelatinization temperatures of endosperm starch were found to be 1–5°C lower than those of the control. We propose a distinct role for OsSSIIIa/Flo5 and the coordinated action of other SS isoforms during starch synthesis in the seed endosperm of rice.     

GBSS‐BINDING PROTEIN,encoding a CBM48 domain‐containing protein,affects rice quality and yield

The percentage of amylose in the endosperm of rice (Oryza sativa) largely determines grain cooking and eating qualities. Granule‐bound starch synthase I (GBSSI) and GBSSII are responsible for amylose biosynthesis in the endosperm and leaf, respectively. Here, we identified OsGBP, a rice GBSS‐binding protein that interacted with GBSSI and GBSSII in vitro and in vivo. The total starch and amylose contents in osgbp mutants were significantly lower than those of wild type in leaves and grains, resulting in reduced grain weight and quality. The carbohydrate‐binding module 48 (CBM48) domain present in the C‐terminus of OsGBP is crucial for OsGBP binding to starch. In the osgbp mutant, the extent of GBSSI and GBSSII binding to starch in the leaf and endosperm was significantly lower than wild type. Our data suggest that OsGBP plays an important role in leaf and endosperm starch biosynthesis by mediating the binding of GBSS proteins to developing starch granules. This elucidation of the function of OsGBP enhances our understanding of the molecular basis of starch biosynthesis in rice and contributes information that can be potentially used for the genetic improvement of yield and grain quality.     

Starch characteristics in cultures of normal and mutant maize endosperm

Summary Vigorously growing suspension cultures of normal, amylose-extender (ae) and waxy (wx) maize endosperm were established from near isogenic lines of maize inbred A636. The recovery of the ability to produce vigorous cultures of ae and wx endosperm by backcrossing demonstrate the genetic control of endosperm growth in vitro. Phenotypic expression of the endosperm mutants in culture was studied by examining the properties of starch accumulated in endosperm cultures and starch from developing and mature kernels of the same genotype. After 9 months in culture, the amylose contents of the starch in normal callus tissue and normal endosperm tissue were not significantly different, 28.2% and 31.7%, respectively. Starch granules from normal cultures and endosperm stained blue-black with iodine and were round to polygonal in shape. The starches of wx endosperm and callus cultures contained no amylose, and wx starch granules stained brown-orange with iodine. Although, wx starch granules were primarily round, a few granules with jagged edges were observed in starch samples isolated from cultures and kernels. The percent amylose in starch from ae callus was significantly lower than the amylose content of starch from ae endosperm tissue, 39.9% and 67.7%, respectively. Starch granules from ae endosperm and cultures were smaller than normal and wx starch granules. Irregular starch granules which are typical of ae endosperm were present in ae callus tissue, but were less frequently observed. We conclude that specific endosperm mutant phenotypes are expressed in vitro.Supported in part by the United States Department of Agriculture Competitive Grant 85-CRCR-1-1740. Contribution No. 94, Department of Horticulture. The Pennsylvania State University. Authorized for publication as paper No. 7373 in the journal series of the Pennsylvania Agricultural Experiment Station     

Starch biosynthesis in rice endosperm requires the presence of either starch synthase I or IIIa

Starch synthase (SS) I and IIIa are the first and second largest components of total soluble SS activity, respectively, in developing japonica rice (Oryza sativa L.) endosperm. To elucidate the distinct and overlapping functions of these enzymes, double mutants were created by crossing the ss1 null mutant with the ss3a null mutant. In the F(2) generation, two opaque seed types were found to have either the ss1ss1/SS3ass3a or the SS1ss1/ss3ass3a genotype. Phenotypic analyses revealed lower SS activity in the endosperm of these lines than in those of the parent mutant lines since these seeds had different copies of SSI and SSIIIa genes in a heterozygous state. The endosperm of the two types of opaque seeds contained the unique starch with modified fine structure, round-shaped starch granules, high amylose content, and specific physicochemical properties. The seed weight was ～90% of that of the wild type. The amount of granule-bound starch synthase I (GBSSI) and the activity of ADP-glucose pyrophosphorylase (AGPase) were higher than in the wild type and parent mutant lines. The double-recessive homozygous mutant prepared from both ss1 and ss3a null mutants was considered sterile, while the mutant produced by the leaky ss1 mutant×ss3a null mutant cross was fertile. This present study strongly suggests that at least SSI or SSIIIa is required for starch biosynthesis in rice endosperm.     

Starch granule initiation and growth are altered in barley mutants that lack isoamylase activity

Two mutant lines of barley, Risø 17 and Notch‐2, were found to accumulate phytoglycogen in the grain. Like the sugary mutants of maize and rice, these phytoglycogen‐accumulating mutants of barley lack isoamylase activity in the developing endosperm. The mutants were shown to be allelic, and to have lesions in the isoamylase gene, isa1 that account for the absence of this enzyme. As well as causing a reduction in endosperm starch content, the mutations have a profound effect on the structure, number and timing of initiation of starch granules. There are no normal A‐type or B‐type granules in the mutants. The mutants have a greater number of starch granules per plastid than the wild‐type and, particularly in Risø 17, this leads to the appearance of compound starch granules. These results suggest that, as well as suppressing phytoglycogen synthesis, isoamylase in the wild‐type endosperm plays a role in determining the number, and hence the form, of starch granules.     

Molecular identification and comparison of the starch synthase bound to starch granules between endosperm and leaf blades in rice plants

Normal (nonglutinous) rice plants (Oryza sativa andO. glaberrima) contain more than 18% amylose in endosperm starch, whilewaxy (glutinous) plants lack it in this starch. In contrast, leaf starch contained more than 3.6% amylose even inwaxy plants. SDS-PAGE analysis of proteins bound to endosperm starch granules in the normal plants revealed a single band with aMr of 60 kd, whereaswaxy plants did not exhibit a similar band. The activity of starch synthase (NDP-glucose-starch glucosyltransferase) was completely inhibited by antibody against the 60-kd protein. Thus, we conclude that the 60-kd protein is thewaxy protein encoded by theWx allele, which also plays a role in the synthesis of nonglutinous starch in endosperm tissue. In leaf blades, the proteins bound to starch granules separated into five bands withMr's of 53.6 to 64.9 kd on SDS-PAGE. Analysis of these proteins by immunoblotting using antiserum againstWx protein and inhibition of starch synthase activity by the synthase antibody revealed that none of these proteins was homologous toWx protein. We suggest that the synthesis of amylose in leaf blades is brought about by a protein encoded by a gene(s) different from theWx gene expressed in the endosperm.     

Fine mapping and candidate gene analysis of the floury endosperm gene, FLO(a), in rice

In addition to its role as an energy source for plants, animals and humans, starch is also an environmentally friendly alternative to fossil fuels. In rice, the eating and cooking quality of the grain is determined by its starch properties. The floury endosperm of rice has been explored as an agronomical trait in breeding and genetics studies. In the present study, we characterized a floury endosperm mutant, flo(a), derived from treatment of Oryza sativa ssp. japonica cultivar Hwacheong with MNU. The innermost endosperm of the flo(a) mutant exhibited floury characteristics while the outer layer of the endosperm appeared normal. Starch granules in the flo(a) mutant formed a loosely-packed crystalline structure and X-ray diffraction revealed that the overall crystallinity of the starch was decreased compared to wild-type. The FLO(a) gene was isolated via a map-based cloning approach and predicted to encode the tetratricopeptide repeat domaincontaining protein, OsTPR. Three mutant alleles contain a nucleotide substitution that generated one stop codon or one splice site, respectively, which presumably disrupts the interaction of the functionally conserved TPR motifs. Taken together, our map-based cloning approach pinpointed an OsTPR as a strong candidate of FLO(a), and the proteins that contain TPR motifs might play a significant role in rice starch biosynthetic pathways.     

Differential regulation of waxy gene expression in rice endosperm

Summary In order to examine the effects of different alleles on the gene expression at the waxy locus, the Wx gene product which controls the synthesis of amylose was isolated from endosperm starch of rice plants and analysed by electrophoretic techniques. The major protein bound to starch granules was absent in most of waxy strains and increased with the number of Wx alleles in triploid endosperms, suggesting that the major protein is the Wx gene product. In addition to wx alleles which result in the absence or drastic reduction of the Wx gene product and amylose, differentiation of Wx alleles seemed to have occurred among nonwaxy rice strains. At least two Wx alleles with different efficiencies in the production of the major protein as well as amylose were detected. These alleles are discussed in relation to regulation of the gene expression.     

The function of the Waxy locus in starch synthesis in maize endosperm

The soluble adenosine diphosphate glucose-starch glucosyltransferase of maize (Zea mays L.) endosperm uses adenosine diphosphate glucose as a sole substrate, but the starch granule-bound nucleoside diphosphate glucose-starch glucosyltransferase utilizes both adenosine diphosphate glucose and uridine diphosphate glucose. The soluble glucosyltransferase can be bound to added amylose or to maize starch granules that contain amylose. However, binding of the soluble enzyme to the starch granules does not change its substrate specificity to that of the natural starch granule-bound glucosyltransferase. Furthermore, the soluble glucosyltransferase bound to starch granules can be removed by repeated washing without a change in specificity. The bound glucosyltransferase can be released by mechanical disruption of starch granules, and the released enzyme behaves in a manner similar to that of the bound enzyme in several respects. These observations suggest that the soluble and bound glucosyltransferases are different enzymes. The starch granule-bound glucosyltransferase activity is linearly proportional to the number of Wx alleles present in the endosperm. This is compatible with the hypothesis that the Wx allele is a structural gene coding for the bound glucosyltransferase, which is important for the normal synthesis of amylose.Journal Paper No. 4818 of the Purdue University Agricultural Experiment Station.     

Free sugar fraction of the amylose-related mutants of maize

The free sugar fraction of normal and amylose-related mutants of maize has been studied. The mutant waxy, characterized by a starch deprived of amylose, does not differ from the normal maize so far as free sugars are concerned. We report, however, the presence of maltose in waxy extracts, a disaccharide otherwise supposed to be absent in this genotype. Three high-amylose mutants (amylose extender, dull, and sugary-2) can be differentiated on the basis of the content of free sugars: dull and sugary-2 enhance amylose synthesis without inducing the presence of starch amylolytic products, while amylose extender accumulates a large quantity of maltose and maltooligosaccharides with a degree of polymerization between 3 and 8. In developing endosperm of amylose extender an abnormal amylolytic activity may be responsible for the observed abnormalities in free sugars and starch characteristics.     

Antisense inhibition of isoamylase alters the structure of amylopectin and the physicochemical properties of starch in rice endosperm

This is the first report on regulation of the isoamylase1 gene to modify the structure of amylopectin and properties of starch by using antisense technology in plants. The reduction of isoamylase1 protein by about 94% in rice endosperm changed amylopectin into a water-insoluble modified amylopectin and a water-soluble polyglucan (WSP). As compared with wild-type amylopectin, the modified amylopectin had more short chains with a degree of polymerization of 5-12, while their molecular sizes were similar. The WSP, which structurally resembled the phytoglycogen in isoamylase-deficient sugary-1 mutants, accounted for about 16% of the total alpha-polyglucans in antisense endosperm, and it was distributed throughout the whole endosperm unlike in sugary-1 mutant. The reduction of isoamylase activity markedly lowered the gelatinization temperature from 54 to 43 degrees C and the viscosity, and modified X-ray diffraction pattern and the granule morphology of the starch. The activity of pullulanase, the other type of starch debranching enzyme, in the antisense endosperm was similar to that in wild-type, whereas it is deficient in sugary-1 mutants. These results indicate that the isoamylase1 is essential for amylopectin biosynthesis in rice endosperm, and that alteration of the isoamylase activity is an effective means to modify the physicochemical properties and granular structure of starch.     

Developmental Changes in the Structure of Endosperm Starch of Rice (Oryza sativa L.)

The developmental changes in the structure and properties of endosperm starches were investigated using the near-isogenic lines for wx alleles of rice. The amylose content in nonwaxy starch was increased during the development of rice grains. Because the accumulation of amylose in endosperm stopped earlier than that of amylopectin during development, the percentages of amylose reached a maximum at the 17th day after flowering in nonwaxy endosperm. Since the distributions of the unit-chain length of amylopectin in waxy and nonwaxy starches were unchanged with the development of the grains, these amylopectins would be synthesized in a similar manner through development. The structure and properties of endosperm starches were reconfirmed to be conspicuously affected by the temperature at the early developmental stages of the grain-filling period, namely, they appeared to be characterized by the temperature at which the starch was accumulated in the endosperm.     

Characterization of starch produced by suspension cell cultures of Indica rice (Oryza sativa L.)

Suspension cultures of rice (Oryza sativa L.), initiated from seed, produced significant amounts of starch. Starch accumulated in the cultured cells throughout the growth phase and reached a maximum of 7% of the cell dry weight at stationary phase. Starch was present in compound granules which were birefringent under polarized light. Suspension culture starch had a higher amylose content and a lower gelatinization temperature than rice grain starch. Additionally, starch branching enzyme, an enzyme involved in starch biosynthesis, was characterized by anion exchange chromatography in culture cells and endosperm. Culture cells had at least one major form of starch branching enzyme which differed from the multiple enzyme forms present in endosperm.     

Preparation and characterization of gelatinized granular starches from aqueous ethanol treatments

In order to modify the properties of native starch granules, the formation of gelatinized granular forms (GGS) from normal, waxy, and high amylose maize, as well as potato and tapioca starches was investigated by treating granules with aqueous ethanol at varying starch:water:ethanol ratios and then heating in a rotary evaporator to remove ethanol. The modified starches were characterized using bright field, polarized and electron microscopy. Short/long range molecular order and enthalpic transitions on heating were also studied using infrared spectroscopy, X-ray diffractometry and differential scanning calorimetry respectively. A diffuse birefringence pattern without Maltese cross was observed for most GGS samples. Treatment with aqueous ethanol resulted in starch-specific changes in the surface of granules, most noticeably swelling and disintegration in waxy maize, surface wrinkling in normal maize and tapioca, swelling and opening-up in potato starches, and swelling and bursting in high amylose maize. The ratio of ethanol to water at which original granular order was disrupted also varied with starch type. GGS had less short range molecular order than native granules as inferred by comparing 1047/1022 wave number ratio from infrared spectroscopy. Similarly, A- and B-type diffraction reflections were either reduced or completely lost with evolution of V-type patterns in GGS.     

Activity of starch synthase and the amylose content in rice endosperm

The content of amylose in endosperm of non-waxy japonica rice (Oryza sativa cv Akitakomachi) was increased by lowering the growth temperature from 25° to 15° during the ripening period. The activities of sucrose synthase, ADPglucose pyrophosphorylase, starch branching enzyme (Q-enzyme) and soluble starch synthase in endosperm developed at 15° were lower than or similar to those at 25°, when compared on a endosperm basis at the similar ripening stage. In contrast, the activity of starch granule-bound starch synthase, which is considered to be indispensable for amylose synthesis, was higher by 3–3.5-fold in the endosperm developed at the low temperature than that at the high ambient temperature. The results suggest that the low temperature specifically accelerates the expression of the bound starch synthase gene (waxy gene) in rice endosperm, which resulted in elevated amylose biosynthesis in the endosperm when developed at lower temperatures.     

Roles of isoamylase and ADP-glucose pyrophosphorylase in starch granule synthesis in rice endosperm

Amyloplast-targeted green fluorescent protein (GFP) was used to monitor amyloplast division and starch granule synthesis in the developing endosperm of transgenic rice. Two classical starch mutants, sugary and shrunken, contain reduced activities of isoamylase1 (ISA1) and cytosolic ADP-glucose pyrophosphorylase, respectively. Dividing amyloplasts in the wild-type and shrunken endosperms contained starch granules, whereas those in sugary endosperm did not contain detectable granules, suggesting that ISA1 plays a role in granule synthesis at the initiation step. The transition from phytoglycogen to sugary-amylopectin was gradual in the boundary region between the inner and outer endosperms of sugary. These results suggest that the synthesis of sugary-amylopectin and phytoglycogen involved a stochastic process and that ISA1 activity plays a critical role in the stochastic process in starch synthesis in rice endosperm. The reduction of cytosolic ADP-glucose pyrophosphorylase activity in shrunken endosperm did not inhibit granule initiation but severely restrained the subsequent enlargement of granules. The shrunken endosperm often developed pleomorphic amyloplasts containing a large number of underdeveloped granules or a large cluster of small grains of amyloplasts, each containing a simple-type starch granule. Although constriction-type divisions of amyloplasts were much more frequent, budding-type divisions were also found in the shrunken endosperm. We show that monitoring GFP in developing amyloplasts was an effective means of evaluating the roles of enzymes involved in starch granule synthesis in the rice endosperm.     

Physicochemical properties of giant embryo rice Seonong 17 and Keunnunjami

This study was carried out to determine the physicochemical properties of giant embryo rice “Seonong 17” and “Keunnunjami” in comparison with the normal embryo rice. Scanning electron microscopy revealed that Seonong 17 and Keunnunjami have larger embryo and that starch granules from Keunnunjami were more tightly packed with smaller air spaces between granules. Seonong 17 exhibited the lowest amylose content. Keunnunjami showed the highest protein content, pasting temperature, peak and breakdown viscosities, and gelatinization temperature and enthalpy. Both giant embryo rice samples contained significantly higher amounts of essential amino acids and unsaturated fatty acids than the normal rice. Proteomic analysis using two-dimensional gel electrophoresis revealed differences in the protein profile of Seonong 17 and Keunnunjami. The results could serve as baseline information in evaluating the quality of these two giant embryo rice cultivars and provide a better understanding of their potential uses and food industry applications.     

FLOURY ENDOSPERM16 encoding a NAD‐dependent cytosolic malate dehydrogenase plays an important role in starch synthesis and seed development in rice

Starch is the most important form of energy storage in cereal crops. Many key enzymes involved in starch biosynthesis have been identified. However, the molecular mechanisms underlying the regulation of starch biosynthesis are largely unknown. In this study, we isolated a novel floury endosperm rice (Oryza sativa) mutant flo16 with defective starch grain (SG) formation. The amylose content and amylopectin structure were both altered in the flo16 mutant. Map‐based cloning and complementation tests demonstrated that FLO16 encodes a NAD‐dependent cytosolic malate dehydrogenase (CMDH). The ATP contents were decreased in the mutant, resulting in significant reductions in the activity of starch synthesis‐related enzymes. Our results indicated that FLO16 plays a critical role in redox homeostasis that is important for compound SG formation and subsequent starch biosynthesis in rice endosperm. Overexpression of FLO16 significantly improved grain weight, suggesting a possible application of FLO16 in rice breeding. These findings provide a novel insight into the regulation of starch synthesis and seed development in rice.