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.     

The altered pattern of amylose accumulation in the endosperm of low-amylose barley cultivars is attributable to a single mutant allele of granule-bound starch synthase I with a deletion in the 5'-non-coding region

Reasons for the variable amylose content of endosperm starch from waxy cultivars of barley (Hordeum vulgare) were investigated. The mature grains of most such cultivars contain some amylose, although amounts are much lower than in wild-type cultivars. In these low-amylose cultivars, amylose synthesis starts relatively late in grain development. Starch granules in the outer cell layers of the endosperm contain more amylose than those in the center. This distribution corresponds to that of granule-bound starch synthase I (GBSSI), which is more severely reduced in amount in the center of the endosperm than in the outer cell layers, relative to wild-type cultivars. A second GBSSI in the barley plant, GBSSIb, is not detectable in the endosperm and cannot account for amylose synthesis in the low-amylose cultivars. The change in the expression of GBSSI in the endosperm of the low-amylose cultivars appears to be due to a 413-bp deletion of part of the promoter and 5'-untranslated region of the gene. Although these cultivars are of diverse geographical origin, all carry this same deletion, suggesting that the low-amylose cultivars have a common waxy ancestor. Records suggest a probable source in China, first recorded in the 16th century. Two further families of waxy cultivars have no detectable amylose in the endosperm starch. These amylose-free cultivars were selected in the 20th century from chemically mutagenized populations of wild-type barley. In both cases, 1-bp alterations in the GBSSI gene completely eliminate GBSSI activity.     

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.     

Physicochemical properties and development of wheat large and small starch granules during endosperm development

Wheat mature seeds have large, lenticular A-type starch granules, and small, spherical B-type and irregular C-type starch granules. During endosperm development, large amyloplasts came from proplastid, divided and increased in number through binary fission from 4 to 12 days after flowering (DAF). Large starch granules formed and developed in the large amyloplast. One large amyloplast had only one large starch granule. Small amyloplasts came from the protrusion of large amyloplast envelope, divided and increased in number through envelope protrusion after 12 DAF. B-type starch granules formed and developed in small amyloplast from 12 to 18 DAF, C-type starch granules formed and developed in small amyloplast after 18 DAF. Many B- and C-type starch granules might form and develop in one small amyloplast. The amyloplast envelopes were asynchronously degraded and starch granules released into cell matrix when amyloplasts were full of starch granules. Apparent amylose contents of large starch granules were higher than that of small starch granules, and increased with endosperm development. The swelling powers and crystallinity of large starch granule were lower than that of small starch granules, and decreased with endosperm development. Small starch granules displayed broader gelatinization temperature ranges than did large starch granules.     

Physicochemical properties of endosperm and pericarp starches during maize development

Endosperm starch and pericarp starch were isolated from maize (B73) kernels at different developmental stages. Starch granules, with small size (2–4 μm diameter), were first observed in the endosperm on 5 days after pollination (DAP). The size of endosperm-starch granules remained similar until 12DAP, but the number increased extensively. A substantial increase in granule size was observed from 14DAP (diameter 4–7 μm) to 30DAP (diameter10–23 μm). The size of starch granules on 30DAP is similar to that of the mature and dried endosperm-starch granules harvested on 45DAP. The starch content of the endosperm was little before 12DAP (less than 2%) and increased rapidly from 10.7% on 14DAP to 88.9% on 30DAP. The amylose content of the endosperm starch increased from 9.2% on 14DAP to 24.2% on 30DAP and 24.4% on 45DAP (mature and dried). The average amylopectin branch chain-length of the endosperm amylopectin increased from DP23.6 on 10DAP to DP26.9 on14DAP and then decreased to DP25.4 on 30DAP and DP24.9 on 45DAP. The onset gelatinization temperature of the endosperm starch increased from 61.3 °C on 8DAP to 69.0 °C on 14DAP and then decreased to 62.8 °C on 45DAP. The results indicated that the structure of endosperm starch was not synthesized consistently through the maturation of kernel. The pericarp starch, however, showed similar granule size, starch content, amylose content, amylopectin structure and thermal properties at different developmental stages of the kernel.     

Characterization of a Granule-Bound Starch Synthase Isoform Found in the Pericarp of Wheat

Waxy wheat (Triticum aestivum L.) lacks the waxy protein, which is also known as granule-bound starch synthase I (GBSSI). The starch granules of waxy wheat endosperm and pollen do not contain amylose and therefore stain red-brown with iodine. However, we observed that starch from pericarp tissue of waxy wheat stained blue-black and contained amylose. Significantly higher starch synthase activity was detected in pericarp starch granules than in endosperm starch granules. A granule-bound protein that differed from GBSSI in molecular mass and isoelectric point was detected in the pericarp starch granules but not in granules from endosperm. This protein was designated GBSSII. The N-terminal amino acid sequence of GBSSII, although not identical to wheat GBSSI, showed strong homology to waxy proteins or GBSSIs of cereals and potato, and contained the motif KTGGL, which is the putative substrate-binding site of GBSSI of plants and of glycogen synthase of Escherichia coli. GBSSII cross-reacted specifically with antisera raised against potato and maize GBSSI. This study indicates that GBSSI and GBSSII are expressed in a tissue-specific manner in different organs, with GBSSII having an important function in amylose synthesis in the pericarp.     

Starch biosynthesis of amylomaize during endosperm development

In an attempt to investigate starch biosynthesis of amylomaize, the endosperm starches from four genotypes with different doses of amylose-extender (ae) between 0 and 3 were isolated at 14, 18, 22, 28, and 36 days after pollination, and their properties were examined. Gene effects of ae on the endosperm starches were found in the apparent amylose content from measurements of iodine binding capacity and in the elution pattern from fractionation of starches debranched by isoamylase on Sephadex G-75. With increasing doses of ae, the lengths of two side-chain fractions of amylopectin in the original starch granule and the carbohydrate content of shorter side-chain fraction both increased. However, the length of each side chain fraction varied little between 14 and 36 days in all genotypes. In addition, the amylose content in ae homozygous at the 14-day stage was ca 24 % lower than the value of amylose and intermediate fractions in the original starch granule.     

Physicochemical properties and structure of large, medium and small granule starches in fractions of normal barley endosperm

Normal barley grain was milled to flour with a machine used to polish brewers' rice from the surface layer to the center. Large (18.4 microm, median size), medium (12.3 microm) and small (2.2 microm) granule starches were isolated from classified flours. Their physicochemical properties and fine structure were investigated. The percentage (w%) of large granules decreased from the surface layer to the center, while the amounts of medium and small granules increased. Although all the starch granules were an A-type crystal, the relative crystallinity varied from 22.0 to 27.4%. The DPn of the amyloses was around 1600 and similar for all the samples. But the amylose content of the starches varied from 21.9 to 26.4%. Also, the amylopectins showed differences in DPn (around 5700-7900) and chain-length distribution between granule size or fractions. The transition temperature ranges and the enthalpy values of the starch granules differed with granule size. The gelatinization properties showed no correlation with any of the parameters, except the enthalpy value and relative crystallinity (gamma = +0.73). The findings suggested that the structural characteristics of the starches in classified flours of normal barley differed essentially from those of waxy barley.     

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     

Waxy Chlamydomonas reinhardtii: monocellular algal mutants defective in amylose biosynthesis and granule-bound starch synthase activity accumulate a structurally modified amylopectin.

Amylose-defective mutants were selected after UV mutagenesis of Chlamydomonas reinhardtii cells. Two recessive nuclear alleles of the ST-2 gene led to the disappearance not only of amylose but also of a fraction of the amylopectin. Granule-bound starch synthase activities were markedly reduced in strains carrying either st-2-1 or st-2-2, as is the case for amylose-deficient (waxy) endosperm mutants of higher plants. The main 76-kDa protein associated with the starch granule was either missing or greatly diminished in both mutants, while st-2-1-carrying strains displayed a novel 56-kDa major protein. Methylation and nuclear magnetic resonance analysis of wild-type algal storage polysaccharide revealed a structure identical to that of higher-plant starch, while amylose-defective mutants retained a modified amylopectin fraction. We thus propose that the waxy gene product conditions not only the synthesis of amylose from endosperm storage tissue in higher-plant amyloplasts but also that of amylose and a fraction of amylopectin in all starch-accumulating plastids. The nature of the ST-2 (waxy) gene product with respect to the granule-bound starch synthase activities is discussed.     

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.     

Comparison of the waxy locus sequence from a non-waxy strain and two waxy mutants of spontaneous and artificial origins in barley

Molecular characterization of 3 alleles of the waxy gene from a non-waxy strain "Shikoku hadaka No. 84" (SH84), an indigenous waxy strain "Mochimugi D" (MMD), and an artificial waxy mutant strain "Shikoku hadaka No. 97" (SH97) of barley (Hordeum vulgare ssp. vulgare) was performed via a PCR direct sequencing strategy. The 3 haplotypes were analyzed in terms of single nucleotide polymorphisms, insertion/deletion mutations, and simple sequence repeat polymorphisms. In comparison with the barley non-waxy gene sequence deposited in the public DNA database, 110 polymorphic sites were found in the 5,190-bp sequenced region of the non-waxy strain SH84. A 418-bp deletion in the 5' non-coding sequence was identified in the indigenous waxy strain MMD. Except for the deletion in the promoter region, the spontaneous mutant wax allele and non-waxy allele were identical. Such highly conserved sequences provide evidence for the recent occurrence of a deletion event in the cultivated barley gene pool. Compared to the original variety SH84, induced waxy mutant SH97 had a base substitution of a C to T in the exon 5, which converting Gln-89 of the wild-type gene into a stop codon, suggesting the involvement of a nonsense-mediated mRNA decay. These results will be helpful for understanding the mechanism of the variable amylose content in waxy cultivars of cereal species.     

Effect of high temperature on fine structure of amylopectin in rice endosperm by reducing the activity of the starch branching enzyme

Rice (Oryza sativa L.) grain quality is affected by the environmental temperature it experiences. To investigate the physiological molecular mechanisms of the effect of high temperatures on rice grain, a non-waxy indica rice was grown under two temperature conditions, (29/35 degrees C) and (22/28 degrees C), during the ripening stage in two phytotrons. The activities and gene expression of key enzymes for the biosynthesis of amylose and amylopectin were examined. The activity and expression levels of soluble endosperm starch synthase I were higher at 29/35 degrees C than that at 22/28 degrees C. In contrast, the activities and expression levels of the rice branching enzyme1, the branching enzyme3 and the granule bound starch synthase of the endosperm were lower at 29/35 degrees C than those at 22/28 degrees C. These results suggest that the decreased activity of starch branching enzyme reduces the branching frequency of the branches of amylopectin, which results in the increased amount of long chains of amylopectin of endosperm in rice grain at high temperature.     

Genetic analysis of waxy locus in rice (Oryza sativa L.)

Summary Inheritance of waxy locus was studied in crosses of a waxy variety with four non-waxy parents having high-, intermediate-, low- or very low-amylose content. The analysis for amylose content was done on a single grain basis in parents, F₁, F₂, B₁F₁, and B₂F₁ seeds. The waxy parent lacking synthesis of amylose content was found to differ from the ones having high-, intermediate-, low- or very low-amylose content by one gene with major effect. Dosage effects for amylose content were observed to have great influence on segregation pattern and efficiency of selection. Selection efficiency for amylose content can be enhanced by selecting for endosperm appearance in early segregating generations.     

Factors affecting dry weight accumulation in developing barley endosperm

Some factors that may be concerned in determining final grain weight in barley ( Hordeum vulgare L. var. distichum ) have been investigated. Variation in endosperm fresh and dry weight, volume and starch content have been recorded at different stages of grain development between anthesis and harvest-ripeness for two barleys, cvs Kym and Golden Promise, differing in final grain weight. Results were recorded under both field and glasshouse conditions. The results suggest that the higher final dry weight of Kym, in comparison with Golden Promise, is a function of both rate and duration of grain filling. Only at later stages of endosperm development did the differences in volume become significant and the Kym endosperms continued to increased rapidly in volume for two to three days after endosperm volume had reached a maximum in Golden Promise. The rates of starch accumulation in both cultivars were very similar but starch deposition continued in Kym endosperms for four to five days after deposition in Golden Promise endosperms had slowed down.     

Quantification of total iodine in intact granular starches of different botanical origin exposed to iodine vapor at various water activities

Iodine has been used as an effective tool for studying both the structure and composition of dispersed starch and starch granules. In addition to being employed to assess relative amylose contents for starch samples, it has been used to look at the molecular mobility of the glucose polymers within intact starch granules based on exposure to iodine vapor equilibrated at different water activities. Starches of different botanical origin including corn, high amylose corn, waxy corn, potato, waxy potato, tapioca, wheat, rice, waxy rice, chick pea and mung bean were equilibrated to 0.33, 0.75, 0.97 water activities, exposed to iodine vapor and then absorbance spectra and LAB color were determined. In addition, a new iodine quantification method sensitive to <0.1% iodine (w/w) was employed to measure bound iodine within intact granular starch. Amylose content, particle size distribution of granules, and the density of the starch were also determined to explore whether high levels of long linear glucose chains and the surface area-to-volume ratio were important factors relating to the granular iodine binding. Results showed, in all cases, starches complexed more iodine as water content increased and waxy starches bound less iodine than their normal starch counterparts. However, much more bound iodine could be measured chemically with waxy starches than was expected based on colorimetric determination. Surface area appeared to be a factor as smaller rice and waxy rice starch granules complexed more iodine, while the larger potato and waxy potato granules complexed less than would be expected based on measured amylose contents. Corn, high amylose corn, and wheat, known to have starch granules with extensive surface pores, bound higher levels of iodine suggesting pores and channels may be an important factor giving iodine vapor greater access to bind within the granules. Exposing iodine vapor to moisture-equilibrated native starches is an effective tool to explore starch granule architecture.     

Amylose Content and Starch Granule Size in Rice Grains are Affected By Growing Season

Amylose content and starch granule size in grains influence rice quality, which differs between the early (ES) and late season (LS). The objective of this study was to determine the variation of amylose content and starch granule size between seasons and find the main reasons (e.g., temperature and solar radiation) for the observed variation. Field experiments with six rice varieties (three high and three low amylose content rice) planted in the ES and LS were conducted in 2016 and 2017, respectively. The mean temperatures during the filling stage were higher in ES, however, the daily temperatures at 7-10 days after flowering (DAF) in 2016, and at 5-10, 13-14 DAF in 2017 were higher in LS. The results showed that amylose content in LS was lower than in ES with high amylose content rice varieties (HACV); the opposite trend occurred with low amylose content rice varieties (LACV). The mean starch granule diameter was higher in LS than ES in 2016, but the opposite result occurred in 2017 with all rice. Our results suggest that higher temperatures increased and decreased the amylose content in HACV and LACV, respectively. Temperatures at 5-15 DAF were important for the formation of starch granules: lower temperatures during 10-14 DAF increased the proportion of larger starch granules (d＞6.21 μm, some with d＞13.3 μm), and higher temperatures at 5-6 DAF increased the proportion of starch granules with diameter 4.24-6.21 μm.     

Enzymes of starch metabolism in the developing rice grain

The levels of starch, soluble sugars, protein, and enzymes involved in starch metabolism—α-amylase, β-amylase, phosphorylase, Q-enzyme, R-enzyme, and starch synthetase —were assayed in dehulled developing rice grains (Oryzasativa L., variety IR8). Phosphorylase, Q-enzyme, and R-enzyme had peak activities 10 days after flowering, whereas α- and β-amylases had maximal activities 14 days after flowering. Starch synthetase bound to the starch granule increased in activity up to 21 days after flowering. These enzymes (except the starch synthetases) were also detected by polyacrylamide gel electrophoresis. Their activity in grains at the midmilky stage (8-10 days after flowering) was determined in five pairs of lines with low and high amylose content from different crosses. The samples had similar levels of amylases, phosphorylase, R-enzyme, and Q-enzyme. The samples consistently differed in their levels of starch synthetase bound to the starch granule, which was proportional to amylose content. Granule-bound starch synthetase may be responsible for the integrity of amylose in the developing starch granule.