The starch granule associated proteomes of commercially purified starch reference materials from rice and maize

Commercially available reference materials are integral components of many experimental protocols, as it is critical to compare one's results to those derived from well-characterized standards. Most reference materials are well defined, with all their components being cataloged. However, certain reference materials, such as commercially prepared starch samples, can have undefined components, potentially limiting their usefulness as standards. The proteome of commercially prepared starch has not been documented, and to that end, we initiated a mass spectrometry-based survey of the proteins associated with starch granules in commercially prepared rice and maize starch samples. We performed direct trypsin treatments of starch samples and sequenced both the water-soluble peptides liberated into the aqueous supernatant and the peptides released from the starch granule surface by isopropanol solvent washing. We discovered that the majority of proteins, in both rice and maize samples, were involved in either carbohydrate metabolism or storage. We also documented proteins that are markers for seed maturity and for starch mobilization.     

On the role of chloroplastic phosphoglucomutase in the regulation of starch turnover

When spinach (Spinacia oleracea L.) leaf disks were incubated in 10% polyethylene glycol to induce water stress, the ratio of glucose-1-phosphate to glucose-6-phosphate increased. This increase indicated an imbalance in the phosphoglucomutase (EC 2.7.5.1) reaction, which was earlier observed to be close to equilibrium, and was accompanied by higher fructose-1,6-bisphosphate and ribulose-1,5-bisphosphate concentrations. Because starch degradation was assumed to be the source of the glucose-1-phosphate accumulation, the kinetic properties of plastidic phosphoglucomutase were analysed. It was found that physiological concentrations of both sugar bisphosphates inhibited phosphoglucomutase by about 50%. From this observation it was concluded that under conditions in which fructose-1,6-bisphosphate and ribulose-1,5-bisphosphate accumulated, an inhibition of phosphoglucomutase activity restricted the carbon exchange between the Calvin cycle and starch turnover. Received: 23 March 1998 / Accepted: 26 August 1998     

Development of SNP-based CAPS and dCAPS markers in eight different genes involved in starch biosynthesis in rice

Single nucleotide polymorphisms (SNPs), which are inexhaustible, highly stable, and simply detectable sequence polymorphisms, can lead to phenotypic variations by affecting protein composition changes. Here, we report development of 25 new cleaved amplified polymorphic sequence or derived cleaved amplified polymorphic sequence markers that have discrete band sizes in relation to the SNP genotypes in eight putative gene regions. The average frequency of DNA polymorphisms was 1 per 175 bp (SNPs, 1 per 217 bp; In/dels, 1 per 906 bp). In primary statistical analysis of each marker on 55 diverse rice accessions, including different ecotypes, the mean value of the major allele frequency was 0.658 (0.509–0.927). The average polymorphism information content was 0.326 (0.126–0.375). The mean value of the inbreeding coefficient (f) was 0.950 and was positive (heterozygote deficiency) at all loci, corresponding to the inbreeding system in rice. In cluster analysis, all rice accessions clustered mainly into three groups according to the ecotypes. The association analysis showed that the SNP of Granule-bound starch synthase I and ADP-glucose pyrophosphorylase small subunit (ADPase-S) genes were highly associated with apparent amylose content variation than the others. These new SNP markers may be useful in genotyping rice germplasm, in marker-assisted selection for improving starch quality and content, and in linkage as well as association studies. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Temperature induced changes in the starch components and biosynthetic enzymes of two rice varieties

The effects of temperature on starch and amylose accumulation, fine structure of amylopectin and activities of some enzymes related to starch synthesis in developing rice endosperms was examined. Two early indica rice varieties were used, differing in amylose concentration (AC, %), namely Jia 935 (low AC) and Jia 353 (high AC). The results showed that the effects of high temperature on AC and amylopectin fine structure were variety-dependent. High temperature caused a reduction in amylose concentration and an increase in the short chain (CL<22) proportion of amylopectin for Jia 935; while opposite was true for Jia 353. High temperature also reduced and increased the activity of granule-bound starch synthase (GBSS) in Jia 935 and in Jia 353, respectively. This suggests that a change in the ratio of amylose/starch due to temperature was attributable to a change in GBSS activity. Moreover, obvious differences between the two rice varieties were detected in the activities of sucrose synthase (SuSy), ADP-glucose pyrophosphorylase (ADPG-Ppase), soluble starch synthase (SSS), starch branching enzyme (SBE), starch de-branching enzyme (SDBE) and starch phosphorylase (SPase) to high temperature. Accumulation rate of amylose was significantly and positively correlated with GBSS for Jia 935, but not for Jia 353. Amylose accumulation was also significantly and positively correlated with the activities of SDBE, SBE, ADPG-Ppase and SuSy for both varieties. The results suggest that the ratio of amylose to starch in rice endosperm is not only related to GBSS, but also affected by the activities of SDBE, SBE, ADPG-Ppase and SuSy.     

Sporophytic control of anther development and male fertility by glucose-6-phosphate/phosphate translocator 1(OsGPT1) in rice

Coordination between the sporophytic tissue and the gametic pollen within anthers is tightly controlled to achieve the optimal pollen fitness. Glucose-6-phosphate/phosphate translocator(GPT) transports glucose-6-phosphate, a key precursor of starch and/or fatty acid biosynthesis, into plastids. Here, we report the functional characterization of Os GPT1 in the rice anther development and pollen fertility. Pollen grains from homozygous osgpt1 mutant plants fail to accumulate starch granules, resulting in pollen sterility. Genetic analyses reveal a sporophytic effect for this mutation. Os GPT1 is highly expressed in the tapetal layer of rice anther. Degeneration of the tapetum, an important process to provide cellular contents to support pollen development, is impeded in osgpt1 plants. In addition, defective intine and exine are observed in the pollen from osgpt1 plants. Expression levels of multiple genes that are important to tapetum degeneration or pollen wall formation are significantly decreased in osgpt1 anthers. Previously, we reported that At GPT1 plays a gametic function in the accumulation of lipid bodies in Arabidopsis pollen. This report highlights a sporophytic role of Os GPT1 in the tapetum degeneration and pollen development. The divergent functions of Os GPT1 and At GPT1 in pollen development might be a result of their independent evolution after monocots and dicots diverged.     

Particle size distribution of rice flour affecting the starch enzymatic hydrolysis and hydration properties

Rice flour is becoming very attractive as raw material, but there is lack of information about the influence of particle size on its functional properties and starch digestibility. This study evaluates the degree of dependence of the rice flour functional properties, mainly derived from starch behavior, with the particle size distribution. Hydration properties of flours and gels and starch enzymatic hydrolysis of individual fractions were assessed. Particle size heterogeneity on rice flour significantly affected functional properties and starch features, at room temperature and also after gelatinization; and the extent of that effect was grain type dependent. Particle size heterogeneity on rice flour induces different pattern in starch enzymatic hydrolysis, with the long grain having slower hydrolysis as indicated the rate constant (k). No correlation between starch digestibility and hydration properties or the protein content was observed. It seems that in intact granules interactions with other grain components must be taken into account. Overall, particle size fractionation of rice flour might be advisable for selecting specific physico-chemical properties.     

The structure of the hot-water soluble components in the starch granules of new Japanese rice cultivars

In this study, the structures of the hot-water (80°C) soluble starch fractions (HWS) of six new Japanese rice cultivars (Saikai 194, Saikai 198, Hokuriku 149, Suigen 258, Hoshiyutaka, and Saikai 184) were investigated following a previous study [Mizukami, H., Hizukuri, S. and Takeda, Y. Structures and pasting properties of starches from new characteristic rice cultivars, Oyo Toshitu Kagaku (J. Appl. Glycosci.) 43 (1996) 15–23]. The HWS were subfractionated into 1-butanol-precipitate (SAM) and supernatant (SAP) fractions. The yields of the SAM and SAP fractions were 0.3%–2.4% and 3.1%–4.1% by starch weight, respectively. The Hoshiyutaka and the Saikai 184 yielded both relatively large (2.4%) and small amounts (0.3%) of SAM. The SAM were small amylose molecules with a _n between 320 and 420 and a _w between 950 and 1850. The SAM from the Hoshiyutaka and the Saikai 184 were the larger molecules with _n 390 and 420, respectively, and having slightly more branches (6.0 and 8.1) than those from the others (1.5–4.5). The SAP were smaller molecules having a _n between 60 and 190 as compared to the SAM. The SAP was composed of small amylopectin molecules ( _n 280–790, CL 17–32, β-amylolysis limit (β-AL), 54%–68%) including very small amylose molecules ( _n 24–34) having an average number of branch linkage ( ) of between 0.4 and 0.5. Both the amount and the structures of hot-water-extractable rice starch fractions vary with cultivar, and may influence their cooking properties.     

Expression of genes encoding the tobacco chloroplast phosphate translocator is not light-regulated and is repressed by sucrose

A cDNA encoding the complete precursor of the phosphate translocator of the chloroplast inner envelope membrane has been isolated from a tobacco leaf (Nicotiana tabacum cv. Samsun) gt 11 library. The tobacco cDNA is 1546 by in length and encodes a precursor protein of 401 amino acid residues with a deduced molecular weight of 43705. A putative processing site between Ala-73 and Ala-74 of the precursor protein is suggested by comparison with the N-terminal sequences of the pea and spinach proteins. Removal of the transit peptide produces the mature protein of 328 amino acid residues with a molecular weight of 36038. Southern blot analysis suggests there is probably one copy of the phosphate translocator gene in the pea haploid genome and two copies in the tobacco haploid genome, one derived from each ancestral parental genome. Messenger RNAs essentially equivalent in size to the cDNAs (approx. 1.6 kb) were detected in extracts of all organs examined from tobacco and pea, including leaves, stems, sepals, petals, seed-pods, tendrils and roots. An immunochemically related protein of a similar size to the phosphate translocator was detected in the equivalent pea organs. The levels of both mRNA and protein in non-photosynthetic organs were lower than those in photosynthetic organs. Tobacco phosphate translocator mRNA was present at high levels in etiolated tissue and did not increase significantly after 24 h illumination. Germination and growth of tobacco seedlings in the presence of sucrose caused a 3.3-fold decrease in the level of the phoshate translocator mRNA.     

Molecular analysis of the gene encoding a rice starch branching enzyme

Summary The sequence of a rice gene encoding a starch branching enzyme (sbe1) shows extreme divergence from that of the rice gene, that is homologous to bacterial glycogen branching enzyme (sbe2). sbe1 is expressed abundantly and specifically in developing seeds and maximally in the middle stages of seed development. This expression pattern completely coincides with that of the waxy gene, which encodes a granule-bound starch synthase. Three G-box motifs and consensus promoter sequences are present in the 5 flanking region of sbe1. It encodes a putative transit peptide, which is required for transport into the amyloplast. A 2.2 kb intron (intron 2) precedes the border between the regions encoding the transit peptide and the mature protein, and contains a high G/C content with several repeated sequences in its 5 half. Although only a single copy of sbe1 is present in the rice genome, Southern analysis using intron 2 as a probe indicates the presence of several homologous sequences in the rice genome, suggesting that this large intron and also the transit peptide coding region may be acquired from another portion of the genome by duplication and insertion of the sequence into the gene.     

Validation of housekeeping genes as internal control for studying gene expression in rice by quantitative real-time PCR

For accurate and reliable gene expression results, normalization of real-time PCR data is required against a control gene, which displays highly uniform expression in living organisms during various phases of development and under different environmental conditions. We assessed the gene expression of 10 frequently used housekeeping genes, including 18S rRNA, 25S rRNA, UBC, UBQ5, UBQ10, ACT11, GAPDH, eEF-1alpha, eIF-4a, and beta-TUB, in a diverse set of 25 rice samples. Their expression varied considerably in different tissue samples analyzed. The expression of UBQ5 and eEF-1alpha was most stable across all the tissue samples examined. However, 18S and 25S rRNA exhibited most stable expression in plants grown under various environmental conditions. Also, a set of two genes was found to be better as control for normalization of the data. The expression of these genes (with more uniform expression) can be used for normalization of real-time PCR results for gene expression studies in a wide variety of samples in rice.     

Association of waxy gene single nucleotide polymorphisms with starch characteristics in rice (Oryza sativa L.)

The waxy gene, which encodes the granule bound starch synthase enzyme, is one of the key genes influencing starch synthesis in the rice endosperm. To investigate functional differences between GBSS alleles, we cloned and sequenced GBSS cDNA from a series of cultivars that differed substantially in apparent amylose content and starch viscosity characteristics. We found two single nucleotide polymorphisms in exons 6 and 10 that resulted in amino acid substitutions. These substitutions are associated with differences in apparent amylose content and viscosity characteristics. Subsequent sequencing of these regions from additional cultivars confirmed their association with particular rice quality characteristics. These point mutations could prove useful as molecular markers in the production of cultivars with superior eating, cooking and processing quality, and contribute to our understanding of the various structural and functional differences among granule bound starch synthase alleles.     

Architectural changes of heated mungbean, rice and cassava starch granules: Effects of hydrocolloids and protein-containing envelope

Architectural changes of starch granules induced by heat were demonstrated using light microscopy and confocal laser scanning microscopy. Heat treatment (80 °C, 30 min) on mungbean starch, cassava starch and rice flour suspensions resulted in the rearrangement of amylose and granule-associated proteins within the deformed granules. The presence of alginate and carrageenan influenced the RVA pasting characteristics of starch/flour-hydrocolloid mixed suspensions by maintaining the granular structure of amylose-rich swollen granules or inducing the aggregation of the swollen ones. Generally, the addition of hydrocolloid increased peak viscosity, lowered breakdown and reduced setback of the flour-hydrocolloid mixed paste. This study demonstrated that the heat treatment in excess water generated the protein-containing granule envelope encasing the mungbean and cassava starch content within the deformed granules.     

Analysis of digestion of rice planthopper by Pardosa pseudoannulata based on CO-I gene

In order to systematically study the predatory behavior and digestion regularity of spiders, real-time fluorescence quantification PCR technique was used to detect the number of CO-I genes in Pardosa pseudoannulata after it preyed on rice planthoppers in different temperatures within different periods. At 28 °C, 0, 1, 2, 4, 8, 16, and 24 h after P. pseudoannulata preyed on rich planthopper, DNA was extracted from cephalothorax and abdomen of P. pseudoannulata. Routine PCR and real-time fluorescence PCR techniques were employed for CO-I gene amplification. The results show that: The prey liquid was temporarily stored in the sucking stomach of the spider head within 2 h after prey, and gradually transferred to the midgut of the abdomen with the prolongation of time. After 4 h, CO-I gene residues of rice planthopper in the cephalothorax gradually decreased. The CO-I gene of rice planthopper was basically transferred to the abdomen after 16 h. During 0–1 h, food contained in abdominal midgut and other digestive organs was very small, CO-I gene detection was not obvious. Over time, food entered into the midgut from the sucking stomach for digestion. During 2–4 h, CO-I gene amount increased, at 2–4 h, detected CO-I gene residue reached the peak; but rapidly declined after 8, 16, and 24 h, even it is still detectable. The results at different temperatures reveal that: As the temperature increased from 26 °C to 32 °C, CO-I gene residues of rich planthopper in cephalothorax and abdomen of P. pseudoannulata gradually decreased, which indicated that the digestion rate increased with the increase of temperature with some range. However, when the temperature continued to increase to 34 °C, the digestion rate decreased.