Granule-bound Starch Synthase Research Articles

Genetic diversity of the wx flanking region in rice landraces in northern Laos.

A glutinous texture of endosperm is one of the important traits of rice (Oyza sativa L.). Northern Laos is known as a center of glutinous rice diversity. We genotyped INDEL, SSR and SNP markers in a sample of 297 rice landraces collected in northern Laos. These glutinous varieties were confirmed to share a loss-of-function mutation in Granule bound starch synthase I (Wx). INDEL markers revealed a high frequency of recombinant genotypes between indica and japonica. Principal component analysis using SSR genotypes of Wx flanking region revealed that glutinous indica landraces were scattered between non-glutinous indica and glutinous-japonica types. High ratios of heterozygosity were found especially in glutinous indica. Haplotype analysis using SNP markers around Wx locus revealed that glutinous indica landraces would have a few chromosome segments of glutinous japonica. Frequent recombinations were confirmed outside of this region in glutinous indica. This intricate genetic structure of landraces suggested that glutinous indica landraces in Laos were generated through repeated natural crossing with glutinous-japonica landraces and severe selection by local farmers.

Polymorphism of starch pathway genes in cassava.

The distribution and frequency of single nucleotide polymorphisms (SNPs) can help to understand changes associated with characteristics of interest. We aimed to evaluate nucleotide diversity in six genes involved in starch biosynthesis in cassava using a panel of 96 unrelated accessions. The genes were sequenced, aligned, and used to obtain values for nucleotide diversity (π), segregating sites (θ), Tajima's D test, and neighbor-joining (NJ) clustering. On average, one SNP per 147 and 171 bp was identified in exon and intron regions, respectively. Thirteen heterozygous loci were found. Three of seven SNPs in the exon region resulted in non-synonymous replacement or four synonymous substitutions. However, no associations were noted between SNPs and root dry-matter content. The parameter π ranged from 0.0001 (granule bound starch synthase I) to 0.0033 (α-amylase), averaging 0.0011, while θ ranged from 0.00014 (starch branching enzyme) to 0.00584 (starch synthase I), averaging 0.002353. The θ diversity value was typically double that of the π. Results of the D test did not suggest any evidence of deviance of neutrality in these genes. Among the evaluated accession, 82/96 were clustered using the NJ method but without a clear separation of the root dry-matter content, root pulp coloration, and classification of the cyanogenic compound content. High variation in genes of the starch biosynthetic pathway can be used to identify associations with the functional properties of starch for the use of polymorphisms for selection purposes.

An Insight into the Glycemic Index of Rice

Glycemic index of rice is a highly complex trait. Rice varieties possessing slowly digestible starch (high amylose) are potentially characterized to have low glycemic index and can be useful for management of type II diabetes. Understanding genetic mechanisms underlying starch biosynthesis and metabolism of cooked rice can pave the way for developing efficient breeding and selection strategy for combining high grain yield with low glycemic index. In this context, reverse genetics can prove useful. Available rice genome sequence information encoding key enzymes involved in biosynthesis of amylose component of starch can unravel novel alleles involving single nucleotide polymorphisms (SNPs). A multi-allelic waxygene (Wx) encoding Granule-Bound Starch Synthase I (GBSS I) enzyme is known to determine amylose content in rice endosperm. Potential molecular markers are now available to detect GBSS I alleles (SNPs) associated with five classes of amylose (waxy: 0–5%, very low: 5–12%, low: 12–20%, intermediate: 20–25%, and high:25–33%). These can be routinely used to assist breeding programme. Besides, the presence of intra-class variations in amylose content could be attributed to additional regulatory elements or environmental conditions.

Modification variability of the new cultivar of wheat-rye amphidiploid by the ecological, industrial-valuable and molecular-genetic features

Studied ecological, industrial-valuable and molecular-genetic features of the new cultivar of wheat-rye amphidiploid triticale. It is shown that a cultivar Vivate Nosivske derived method of individual selection from the hybrid combination ([Avhusto NE x 312] x K 9844), and highlighted on the adaptability and resistance to a range of adverse abiotic and biotic factors, lodging and germination of the grain in the ear, is ecological stable in performance under Polissia-Forest-steppe and Forest-steppe ecotopes, generates a high yield (more than 9 t/ha) and grain quality (16 % protein) with timely use of science-based elements of farming. It is determined that the starch grains contained in this class is high (19,8 %) amylopectin content compared with amylose as evidenced by the consistency of the dough, its falling number and viscosity, and suitability for the baking of bakery products. This feature cultivar due to the presence in the genotype recessive allele (b) gene Wx-A1, which is responsible for blocking the GBSS – granulebound starch synthase.

Gel-free/label-free proteomic analysis of developing rice grains under heat stress

High temperature markedly reduces the yields and quality of rice grains. To identify the mechanisms underlying heat stress-induced responses in rice grains, proteomic technique was used. Developing Khao Dawk Mali 105 rice grains at the milky, dough, and mature stages were treated at 40°C for 3days. Aromatic compounds were decreased in rice grains under heat stress. The protein abundance involved in glycolysis and tricarboxylic acid cycle, including glyceraldehyde 3-phosphate dehydrogenase and citrate synthase, was changed in milky and dough grains after heat treatment; however, none changes in mature grains. The abundance involved in amino acid metabolism was increased in dough grains, but decreased in milky grains. In addition, the abundance involved in starch and sucrose metabolism, such as starch synthase, ADP-glucose pyrophosphorylase, granule-bound starch synthase, and alpha amylase, was decreased in milky grains, but increased in dough grains. A number of redox homeostasis-related proteins, such as ascorbate peroxidase and peroxiredoxin, were increased in developing rice grains treated with heat stress. These results suggest that in response to heat stress, the abundance of numerous proteins involved in redox homeostasis and carbohydrate biosynthetic pathways may play a major role in the development of KDML105 rice grains. Biological significanceYield of Khao Dawk Mali 105 rice, which is an economical aromatic rice, was disrupted by environmental stress. Rice grains developed under heat stress caused loss of aroma compound. To identify the mechanism of heat response in rice grain, gel-free/label-free proteomic technique was used. The abundance of proteins involved in glycolysis and tricarboxylic acid cycle was disrupted by heat stress. High temperature limited starch biosynthesis; however, it enhanced sugar biosynthesis in developing rice grains. Redox homeostasis related proteins were disrupted by heat stress. These results suggest that proteins involved in redox homeostasis and carbohydrate pathway might play a major role in developing grains in Khao Dawk Mali 105 rice under heat stress.

Identification and Phylogenetic Analysis of a Novel Starch Synthase in Maize.

Starch is an important reserve of carbon and energy in plants, providing the majority of calories in the human diet and animal feed. Its synthesis is orchestrated by several key enzymes, and the amount and structure of starch, affecting crop yield and quality, are determined mainly by starch synthase (SS) activity. To date, five SS isoforms, including SSI-IV and Granule Bound Starch Synthase (GBSS) have been identified and their physiological functions have been well characterized. Here, we report the identification of a new SS isoform in maize, designated SSV. By searching sequenced genomes, SSV has been found in all green plants with conserved sequences and gene structures. Our phylogenetic analysis based on 780 base pairs has suggested that SSIV and SSV resulted from a gene duplication event, which may have occurred before the algae formation. An expression profile analysis of SSV in maize has indicated that ZmSSV is mainly transcribed in the kernel and ear leaf during the grain filling stage, which is partly similar to other SS isoforms. Therefore, it is likely that SSV may play an important role in starch biosynthesis. Subsequent analysis of SSV function may facilitate understanding the mechanism of starch granules formation, number and structure.

Validation of molecular markers for the analysis of genetic diversity of amylase content and gel consistency among representative rice varieties in Sri Lanka

Starch is the major storage compound in almost all cereals including rice (Oryza sativa L.) and is used as a primary source of energy for humans. Furthermore, it is the main determinant of the eating and cooking quality (ECQ) of rice as amylose content (AC) and gel consistency (GC) are two parameters that significantly contribute to the ECQ. These parameters are mainly controlled by a major gene designated as Waxy or Wx, other minor genes such as ADP-glucose pyrophosphorylase isomerase (AGPiso) and environmental factors. The Waxy gene that encodes for the granule bound starch synthase (GBSS) enzyme which synthesizes amylase (meaning is not clear). The AGPiso (AGPase) gene, codes the protein glucose-1-phosphate adenylytransferase which plays a role in starch synthesis by catalyzing the synthesizing of activated glycosyl donor, ADP-glucose. In this study, an attempt was taken to demonstrate the correlation of AC and GC to three simple sequence repeat/ short tandem repeat (SSR/STS) molecular markers, AGPiso (linked to AGPiso gene), GBSS1 and WX (linked to Waxy gene) using 31 representative Sri Lankan rice varieties. Among the tested rice varieties, the AC and GC varied from 14.8 to 27.7%; and 3.4 to 9.0 cm, respectively. All three markers gave multiple alleles (2-5 alleles), however, these markers did not show a significant association to the AC and GC of the tested rice varieties. Further analysis of the genetic variation of Suduru Samba and Kalu Heenati will be useful as they clustered separately from the rest of the tested varieties based on the polymorphism for markers AGPiso, GBSS1 and WX. Tropical Agricultural Research Vol. 26 (2): 317 – 328 (2015)

Changes in the activities of starch metabolism enzymes in rice grains in response to elevated CO2 concentration.

The global atmospheric CO(2) concentration is currently (2012) 393.1 μmol mol(-1), an increase of approximately 42 % over pre-industrial levels. In order to understand the responses of metabolic enzymes to elevated CO(2) concentrations, an experiment was conducted using the Free Air CO(2) Enrichment (FACE )system. Two conventional japonica rice varieties (Oryza sativa L. ssp. japonica) grown in North China, Songjing 9 and Daohuaxiang 2, were used in this study. The activities of ADPG pyrophosphorylase, soluble and granule-bound starch synthases, and soluble and granule-bound starch branching enzymes were measured in rice grains, and the effects of elevated CO(2) on the amylose and protein contents of the grains were analyzed. The results showed that elevated CO(2) levels significantly increased the activity of ADPG pyrophosphorylase at day 8, 24, and 40 after flower, with maximum increases of 56.67 % for Songjing 9 and 21.31 % for Daohuaxiang 2. Similarly, the activities of starch synthesis enzymes increased significantly from the day 24 after flower to the day 40 after flower, with maximum increases of 36.81 % for Songjing 9 and 66.67 % for Daohuaxiang 2 in soluble starch synthase (SSS), and 25.00 % for Songjing 9 and 36.44 % for Daohuaxiang 2 in granule-bound starch synthase (GBSS), respectively. The elevated CO(2) concentration significantly increased the activity of soluble starch branching enzyme (SSBE) at day 16, 32, and 40 after flower, and also significantly increased the activity of granule-bound starch branching enzyme (GBSBE) at day 8, 32, and 40 after flower. The elevated CO(2) concentration increased the peak values of enzyme activity, and the timing of the activity peaks for SSS and GBSBE were earlier in Songjing 9 than in Daohuaxiang 2. There were obvious differences in developmental stages between the two varieties of rice, which indicated that the elevated CO(2) concentration increased enzyme activity expression and starch synthesis, affecting the final contents of starch and protein in the rice grains. Our results will provide a foundation for understanding the physiological mechanisms of rice yield under elevated atmospheric CO(2) concentrations.

Systematic Analysis of Pericarp Starch Accumulation and Degradation during Wheat Caryopsis Development

Although wheat (Triticum aestivum L.) pericarp starch granule (PSG) has been well-studied, our knowledge of its features and mechanism of accumulation and degradation during pericarp growth is poor. In the present study, developing wheat caryopses were collected and starch granules were extracted from their pericarp to investigate the morphological and structural characteristics of PSGs using microscopy, X-ray diffraction and Fourier transform infrared spectroscopy techniques. Relative gene expression levels of ADP-glucose pyrophosphorylase (APGase), granule-bound starch synthase II (GBSS II), and α-amylase (AMY) were quantified by quantitative real-time polymerase chain reaction. PSGs presented as single or multiple starch granules and were synthesized both in the amyloplast and chloroplast in the pericarp. PSG degradation occurred in the mesocarp, beginning at 6 days after anthesis. Amylose contents in PSGs were lower and relative degrees of crystallinity were higher at later stages of development than at earlier stages. Short-range ordered structures in the external regions of PSGs showed no differences in the developing pericarp. When hydrolyzed by α-amylase, PSGs at various developmental stages showed high degrees of enzymolysis. Expression levels of AGPase, GBSS II, and AMY were closely related to starch synthesis and degradation. These results help elucidate the mechanisms of accumulation and degradation as well as the functions of PSG during wheat caryopsis development.

Whole genome sequencing and analysis of Swarna, a widely cultivated indica rice variety with low glycemic index.

Swarna is a popular cultivated indica rice variety with low glycemic index (GI) but its genetic basis is not known. The whole genome of Swarna was sequenced using Illumina’s paired-end technology, and the reads were mapped to the Nipponbare reference genome. Overall, 65,984 non-synonymous SNPs were identified in 20,350 genes, and in silico analysis predicted that 4,847 of them in 2,214 genes may have deleterious effect on protein functions. Polymorphisms were found in all the starch biosynthesis genes, except the gene for branching enzyme IIa. It was found that T/G SNP at position 246, ‘A’ at position 2,386, and ‘C’ at position 3,378 in the granule bound starch synthase I gene, and C/T SNP at position 1,188 in the glucose-6-phosphate translocator gene may contribute to the low GI phenotype in Swarna. All these variants were also found in the genome of another low GI indica rice variety from Columbia, Fedearroz 50. The whole genome analysis of Swarna helped to understand the genetic basis of GI in rice, which is a complex trait involving multiple factors.

Effect of waxy (Low Amylose) on Fungal Infection of Sorghum Grain.

Loss of function mutations in waxy, encoding granule bound starch synthase (GBSS) that synthesizes amylose, results in starch granules containing mostly amylopectin. Low amylose grain with altered starch properties has increased usability for feed, food, and grain-based ethanol. In sorghum, two classes of waxy (wx) alleles had been characterized for absence or presence of GBSS: wx(a) (GBSS(-)) and wx(b) (GBSS(+), with reduced activity). Field-grown grain of wild-type; waxy, GBSS(-); and waxy, GBSS(+) plant introduction accessions were screened for fungal infection. Overall, results showed that waxy grains were not more susceptible than wild-type. GBSS(-) and wild-type grain had similar infection levels. However, height was a factor with waxy, GBSS(+) lines: short accessions (wx(b) allele) were more susceptible than tall accessions (undescribed allele). In greenhouse experiments, grain from accessions and near-isogenic wx(a), wx(b), and wild-type lines were inoculated with Alternaria sp., Fusarium thapsinum, and Curvularia sorghina to analyze germination and seedling fitness. As a group, waxy lines were not more susceptible to these pathogens than wild-type, supporting field evaluations. After C. sorghina and F. thapsinum inoculations most waxy and wild-type lines had reduced emergence, survival, and seedling weights. These results are valuable for developing waxy hybrids with resistance to grain-infecting fungi.

Differential expressions among five Waxy alleles and their effects on the eating and cooking qualities in specialty rice cultivars

Eating and cooking qualities (ECQs) of rice are important attributes due to its major influence on consumer acceptability. To better understand the molecular mechanism of the variation in ECQs, we investigated and compared the expressions among different alleles of the Waxy (Wx) gene and its effect on ECQs in specialty rice cultivars. The results showed that the accumulation of amylose was positively and significantly correlated to the level of mature Wx mRNA and granule-bound starch synthase I (GBSS I) in developing rice grain at 12 days after flowering. The amount of GBSS I and its activity together are the main factors controlling amylose synthesis. Differences in ECQs among five Wx allele types were investigated in samples from 15 rice varieties. The apparent amylose content (AAC) and gel consistency (GC) were similar in each type of Wx allele. The AAC followed the order, Wxa type>Wxin type>Wxb type>Wxmg type>wx. Contrary to this, the GC showed an opposite trend compared to AAC. There was a wide variation in rapid visco analyzer (RVA) profile among five Wx allele types, while varieties sharing a specified Wx allele had basically the similar RVA profile, although there was a slight difference in some RVA parameters, peak, hot paste and cool paste viscosities.

Photoperiodic control of sugar release during the floral transition: What is the role of sugars in the florigenic signal?

Florigen is a mobile signal released by the leaves that reaching the shoot apical meristem (SAM), changes its developmental program from vegetative to reproductive. The protein FLOWERING LOCUS T (FT) constitutes an important element of the florigen, but other components such as sugars, have been also proposed to be part of this signal.1-5 We have studied the accumulation and composition of starch during the floral transition in Arabidopsis thaliana in order to understand the role of carbon mobilization in this process. In A. thaliana and Antirrhinum majus the gene coding for the Granule-Bound Starch Synthase (GBSS) is regulated by the circadian clock6,7 while in the green alga Chlamydomonas reinhardtii the homolog gene CrGBSS is controlled by photoperiod and circadian signals.8,9 In a recent paper10 we described the role of the central photoperiodic factor CONSTANS (CO) in the regulation of GBSS expression in Arabidopsis. This regulation is in the basis of the change in the balance between starch and free sugars observed during the floral transition. We propose that this regulation may contribute to the florigenic signal and to the increase in sugar transport required during the flowering process.

Overexpression of STARCH BRANCHING ENZYME II increases short-chain branching of amylopectin and alters the physicochemical properties of starch from potato tuber.

BackgroundStarch is biosynthesised by a complex of enzymes including various starch synthases and starch branching and debranching enzymes, amongst others. The role of all these enzymes has been investigated using gene silencing or genetic knockouts, but there are few examples of overexpression due to the problems of either cloning large genomic fragments or the toxicity of functional cDNAs to bacteria during cloning. The aim of this study was to investigate the function of potato STARCH BRANCHING ENZYME II (SBEII) using overexpression in potato tubers.ResultsA hybrid SBEII intragene consisting of potato cDNA containing a fragment of potato genomic DNA that included a single intron was used in order to prevent bacterial translation during cloning. A population of 20 transgenic potato plants exhibiting SBEII overexpression was generated. Compared with wild-type, starch from these tubers possessed an increased degree of amylopectin branching, with more short chains of degree of polymerisation (DP) 6–12 and particularly of DP6. Transgenic lines expressing a GRANULE-BOUND STARCH SYNTHASE (GBSS) RNAi construct were also generated for comparison and exhibited post-transcriptional gene silencing of GBSS and reduced amylose content in the starch. Both transgenic modifications did not affect granule morphology but reduced starch peak viscosity. In starch from SBEII-overexpressing lines, the increased ratio of short to long amylopectin branches facilitated gelatinisation, which occurred at a reduced temperature (by up to 3°C) or lower urea concentration. In contrast, silencing of GBSS increased the gelatinisation temperature by 4°C, and starch required a higher urea concentration for gelatinisation. In lines with a range of SBEII overexpression, the magnitude of the increase in SBEII activity, reduction in onset of gelatinisation temperature and increase in starch swollen pellet volume were highly correlated, consistent with reports that starch swelling is greatly dependent upon the amylopectin branching pattern.ConclusionThis work reports the first time that overexpression of SBEII has been achieved in a non-cereal plant. The data show that overexpression of SBEII using a simple single-intron hybrid intragene is an effective way to modify potato starch physicochemical properties, and indicate that an increased ratio of short to long amylopectin branches produces commercially beneficial changes in starch properties such as reduced gelatinisation temperature, reduced viscosity and increased swelling volume.Electronic supplementary materialThe online version of this article (doi:10.1186/s12896-015-0143-y) contains supplementary material, which is available to authorized users.

Registration of A/BN641 and RN642 waxy Grain Sorghum Genetic Stocks

Loss-of-function mutations in the granule-bound starch synthase gene result in an endosperm with a waxy appearance and a near absence of starch amylose. Three waxy grain sorghum [Sorghum bicolor (L.) Moench] lines, AN641 (Reg. No. GS-741, PI 672150), BN641 (Reg. No. GS-742, PI 672151), and RN642 (Reg. No. GS-743, PI 672152) were developed jointly by the USDA–ARS and the Agricultural Research Division, Institute of Agriculture and Natural Resources, University of Nebraska, and were released in June 2014. AN641 and BN641 have the waxyb (wxb) allele and are near-isogenic to ‘Wheatland’. RN642 has the waxya (wxa) allele and is near-isogenic to ‘Tx430’. Release of these lines with cytoplasmic male-sterile (A), maintainer (B), and fertility restorer (R) fertility reactions to A1 cytoplasm facilitates the production and evaluation of interallelic (wxb × wxa) waxy and heterowaxy [wxb × wild-type (WT) and WT × wxa] hybrids as a source of low-amylose starch for the ethanol and food industries.

Effect of high temperature on the expressions of genes encoding starch synthesis enzymes in developing rice endosperms

High temperature is the major environmental factor affecting grain starch properties of cooking rice cultivars. In this study, two non-waxy indica rice genotypes, cv. 9311 and its mutant with extremely high amylose phenotype (9311eha) were used to study the differential expressions of genes in starch synthesis and their responses to high temperature (HT). Significant increase in apparent amylose content and hot-water-soluble starch content in mutant 9311eha were genetically caused by a substitution from AGTTATA to AGGTATA at the leader intron 5′ splice site in Wx gene. This mutation resulted in different mRNA transcript levels, mRNA splicing efficiencies and protein levels of Wx between the two rice genotypes, which also lead to the genotype-dependent alteration in the temporal pattern of Wx transcription and granule-bound starch synthase (GBSS) activity in response to HT. However, changes in the activities of other starch synthesizing enzymes and their expressions of distinct isoform genes were not significant with the Wx gene mutation, thus only minor difference in the particle size of starch granule, chain-length distribution and gelatinization enthalpy were found between the two genotypes. The temporal-specific expression of multiple isoform genes responsive to different temperature regiments indicated that the reduction of GBSS transcript expression under HT was generally accompanied by the decreased expressions of SSSIIa, SSSIIIa and SBEIIb. Consequently, high temperature-ripened grains in 9311eha showed high proportion of intermediate and long B chains and somewhat lower level of short A chain compared to the wildtype. The temperature-dependent alteration of amylose content was not only attributed to the reduced expression of GBSS, but also associated with the complimentary effect of SSSIIa and SBEIIb.

Genetic variation of the granule-bound starch synthase I (GBSSI) genes in waxy and non-waxy accessions of Chenopodium berlandieri ssp. nuttalliae from Central Mexico

Huauzontle (Chenopodium berlandieri ssp. nuttalliae) is a locally important vegetable crop native to the highland valleys of Central Mexico and a potential source of genes for improving its Andean sister crop, quinoa (Chenopodium quinoa). A previous work involving two huauzontle lines identified one waxy genotype that lacked amylose due to mutations in granule-bound starch synthase I (GBSSI), major amylose-synthesis genes with two constituent subgenomes, A and B. We conducted this study to determine the extent of waxy genotypes and cryptic GBSSI mutations in 11 huauzontle accessions or landrace populations extending from Puebla in the southeast to Jalisco in the northwest. This represents one of the first published studies of genetic variation in C. berlandieri ssp. nuttalliae. Accessions were phenotyped for opaque versus translucent seed morphology and their seed starches were stained with Lugol's Stain. In addition, complete or partial GBSSI genes from their A and B genomes were polymerase chain reaction (PCR)-amplified, cloned and sequenced. Seven accessions were either wholly or partially non-waxy while six were either entirely or partially waxy. All waxy accessions carried the same putatively null alleles, designated gbssIa-tp (A-genome) and gbssIb-del (B-genome). The identification of publicly available genotypes carrying gbssIa-tp and their potential use in breeding waxy grain quinoa is discussed.

PROTEIN TARGETING TO STARCH is required for localising GRANULE-BOUND STARCH SYNTHASE to starch granules and for normal amylose synthesis in Arabidopsis.

The domestication of starch crops underpinned the development of human civilisation, yet we still do not fully understand how plants make starch. Starch is composed of glucose polymers that are branched (amylopectin) or linear (amylose). The amount of amylose strongly influences the physico-chemical behaviour of starchy foods during cooking and of starch mixtures in non-food manufacturing processes. The GRANULE-BOUND STARCH SYNTHASE (GBSS) is the glucosyltransferase specifically responsible for elongating amylose polymers and was the only protein known to be required for its biosynthesis. Here, we demonstrate that PROTEIN TARGETING TO STARCH (PTST) is also specifically required for amylose synthesis in Arabidopsis. PTST is a plastidial protein possessing an N-terminal coiled coil domain and a C-terminal carbohydrate binding module (CBM). We discovered that Arabidopsis ptst mutants synthesise amylose-free starch and are phenotypically similar to mutants lacking GBSS. Analysis of granule-bound proteins showed a dramatic reduction of GBSS protein in ptst mutant starch granules. Pull-down assays with recombinant proteins in vitro, as well as immunoprecipitation assays in planta, revealed that GBSS physically interacts with PTST via a coiled coil. Furthermore, we show that the CBM domain of PTST, which mediates its interaction with starch granules, is also required for correct GBSS localisation. Fluorescently tagged Arabidopsis GBSS, expressed either in tobacco or Arabidopsis leaves, required the presence of Arabidopsis PTST to localise to starch granules. Mutation of the CBM of PTST caused GBSS to remain in the plastid stroma. PTST fulfils a previously unknown function in targeting GBSS to starch. This sheds new light on the importance of targeting biosynthetic enzymes to sub-cellular sites where their action is required. Importantly, PTST represents a promising new gene target for the biotechnological modification of starch composition, as it is exclusively involved in amylose synthesis.

Identification of Bioactive Peptide Sequences from Amaranth (Amaranthus hypochondriacus) Seed Proteins and Their Potential Role in the Prevention of Chronic Diseases.

Amaranth (Amaranthus hypochondriacus) is a pseudocereal with higher protein concentration than most cereal grains. Enzymatic hydrolysis and food processing could produce biopeptides from amaranth proteins; however, there is limited information about the bioactivity of peptides from amaranth proteins. The objective of this comprehensive review was to determine bioactive peptide sequences in amaranth proteins that may prevent cardiovascular disease, cancer, and diabetes. Amaranth proteins, reported in UniProt database, were evaluated for potential bioactive peptide using BIOPEP database. The 15 main proteins present in amaranth seed are 11S globulin, 7S globulin, α-amylase inhibitor, trypsin inhibitor, antimicrobial proteins, nonspecific lipid-transfer-protein-1, superoxide dismutase, ring-zinc finger protein, prosystemin, amaranth albumin 1, glucose-1-phosphate adenyltransferase, glucosyltransferase, polyamine oxidase, granule-bound starch synthase 1,and acetolactate synthase. All proteins showed high occurrence frequencies of angiotensin-converting enzyme-inhibitor peptides (A = 0.161 to 0.362), as well as of dipeptidyl peptidase IV inhibitor (A = 0.003 to 0.087). Other proteins showed antioxidative (A = 0.012 to 0.063) and glucose uptake-stimulating activity (A = 0.023 to 0.042), and also antithrombotic (A = 0.002 to 0.031) and anticancer sequences (A = 0.001 to 0.042). The results of this study support the concept that amaranth grain could be part of a "healthy" diet and thereby prevent chronic human diseases.

AtCCR4a and AtCCR4b are Involved in Determining the Poly(A) Length of Granule-bound starch synthase 1 Transcript and Modulating Sucrose and Starch Metabolism in Arabidopsis thaliana.

Removing the poly(A) tail is the first and rate-limiting step of mRNA degradation and apparently an effective step not only for modulating mRNA stability but also for translation of many eukaryotic transcripts. Carbon catabolite repressor 4 (CCR4) has been identified as a major cytoplasmic deadenylase in Saccharomyces cerevisiae. The Arabidopsis thaliana homologs of the yeast CCR4, AtCCR4a and AtCCR4b, were identified by sequence-based analysis; however, their role and physiological significance in plants remain to be elucidated. In this study, we revealed that AtCCR4a and AtCCR4b are localized to cytoplasmic mRNA processing bodies, which are specific granules consisting of many enzymes involved in mRNA turnover. Double mutants of AtCCR4a and AtCCR4b exhibited tolerance to sucrose application but not to glucose. The levels of sucrose in the seedlings of the atccr4a/4b double mutants were reduced, whereas no difference was observed in glucose levels. Further, amylose levels were slightly but significantly increased in the atccr4a/4b double mutants. Consistent with this observation, we found that the transcript encoding granule-bound starch synthase 1 (GBSS1), which is responsible for amylose synthesis, is accumulated to a higher level in the atccr4a/4b double mutant plants than in the control plants. Moreover, we revealed that GBSS1 has a longer poly(A) tail in the double mutant than in the control plant, suggesting that AtCCR4a and AtCCR4b can influence the poly(A) length of transcripts related to starch metabolism. Our results collectively suggested that AtCCR4a and AtCCR4b are involved in sucrose and starch metabolism in A. thaliana.