Starch Synthase IIa Research Articles

Effects of Homoeologous Wheat Starch Synthase IIa Genes on Starch Properties

Near-isogenic lines (NILs) of the eight haplotypes of starch synthase IIa (SSIIa) were used to analyze the effects of SSIIa gene dosage on branch chain length, gelatinization, pasting, retrogradation, and enzymatic hydrolysis of starches. Compared to wild-type, the amylopectin of lines missing one or more active SSIIa enzymes had increases in the proportion of short branch chains (DP6-10) and decreases in midlength chains (DP11-24), and the size of these differences depended on the dosage of active SSIIa enzymes. Of the three loci, SSIIa-A1 had the smallest contribution to amylopectin structure and SSIIa-B1 the largest. The different effects of the three SSIIa enzymes on starch properties were also seen in gelatinization, retrogradation, pasting, and enzymatic hydrolysis properties. Such differences in starch properties might be useful in influencing the texture and shelf life of food products.

Biomolecular Analyses of Starch and Starch Granule Proteins in the High-Amylose Rice Mutant Goami 2

Elevated proportions of amylose in cereals are commonly associated with either the loss of starch branching or starch synthase activity. Goami 2 is a high-amylose mutant of the temperate japonica rice variety Ilpumbyeo. Genotyping revealed that Goami 2 and Ilpumbyeo carry the same alleles for starch synthase IIa and granule-bound starch synthase I genes. Analyses of granule-bound proteins revealed that SSI and SSIIa accumulate inside the mature starch granules of Goami 2, which is similar to the amylose extender mutant IR36ae. However, unlike the amylose extender mutants, SBEIIb was still detectable inside the starch granules of Goami 2. Detection of SBEIIb after protein fractionation revealed that most of the SBEIIb in Goami 2 accumulates inside the starch granules, whereas most of it accumulates at the granule surface in Ilpumbyeo. Exhaustive mass spectrometric characterisations of granule-bound proteins failed to detect any peptide sequence mutation or major post-translational modifications in Goami 2. Moreover, the signal peptide was found to be cleaved normally from the precursor protein, and there is no apparent N-linked glycosylation. Finally, no difference was found in the SBEIIb structural gene sequence of Goami 2 compared with Ilpumbyeo. In contrast, a G-to-A mutation was detected in the SBEIIb gene of IR36ae located at the splice site between exon and intron 11, which could potentially introduce a premature stop codon and produce a truncated form of SBEIIb. It is suggested that the mutation responsible for producing high amylose in Goami 2 is not due to a defect in SBEIIb gene as was observed in IR36ae, even though it produces a phenotype analogous to the amylose extender mutation. Understanding the molecular genetic basis of this mutation will be important in identifying novel targets for increasing amylose and resistant starch contents in rice and other cereals.

Association Mapping for Grain Quality in a Diverse Sorghum Collection

Knowledge of the genetic bases of grain quality traits will complement plant breeding efforts to improve the end‐use value of sorghum [Sorghum bicolor (L.) Moench]. Candidate gene association mapping was used on a diverse panel of 300 sorghum accessions to assess marker–trait associations for 10 grain quality traits measured using the single kernel characterization system (SKCS) and near‐infrared reflectance spectroscopy (NIRS). The analysis of the accessions through 1290 genomewide single nucleotide polymorphisms (SNPs) separated the panel into five subpopulations that corresponded to three major sorghum races (durra, kafir, and caudatum), one intermediate race (guinea‐caudatum), and one working group (zerazera‐caudatum). These subpopulations differed in kernel hardness, acid detergent fiber, and total digestible nutrients. After model testing, association analysis between 333 SNPs in candidate genes and/or loci and grain quality traits resulted in eight significant marker–trait associations. A SNP in starch synthase IIa (SSIIa) gene was associated with kernel hardness (KH) with a likelihood ratio‐based R2 (RLR2) value of 0.08, a SNP in starch synthase (SSIIb) gene was associated with starch content with an RLR2 value of 0.10, and a SNP in loci pSB1120 was associated with starch content with an RLR2 value of 0.09.

Elongated phytoglycogen chain length in transgenic rice endosperm expressing active starch synthase IIa affects the altered solubility and crystallinity of the storage α-glucan

The relationship between the solubility, crystallinity, and length of the unit chains of plant storage α-glucan was investigated by manipulating the chain length of α-glucans accumulated in a rice mutant. Transgenic lines were produced by introducing a cDNA for starch synthase IIa (SSIIa) from an indica cultivar (SSIIa I, coding for active SSIIa) into an isoamylase1 (ISA1)-deficient mutant (isa1) that was derived from a japonica cultivar (bearing inactive SSIIa proteins). The water-soluble fraction accounted for >95% of the total α-glucan in the isa1 mutant, whereas it was only 35–70% in the transgenic SSIIa I /isa1 lines. Thus, the α-glucans from the SSIIa I /isa1 lines were fractionated into soluble and insoluble fractions prior to the following characterizations. X-ray diffraction analysis revealed a weak B-type crystallinity for the α-glucans of the insoluble fraction, while no crystallinity was confirmed for α-glucans in isa1. Concerning the degree of polymerization (DP) ≤30, the chain lengths of these α-glucans differed significantly in the order of SSIIa I /isa1 insoluble > SSIIa I /isa1 soluble > α-glucans in isa1. The amount of long chains with DP ≥33 was higher in the insoluble fraction α-glucans than in the other two α-glucans. No difference was observed in the chain length distributions of the β-amylase limit dextrins among these α-glucans. These results suggest that in the SSIIa I /isa1 transgenic lines, the unit chains of α-glucans were elongated by SSIIaI, whereas the expression of SSIIaI did not affect the branch positions. Thus, the observed insolubility and crystallinity of the insoluble fraction can be attributed to the elongated length of the outer chains due to SSIIaI.

Toward Understanding the Genetic and Molecular Bases of the Eating and Cooking Qualities of Rice

Apparent amylose content (AAC), gelatinization temperature (GT), gel consistency, and pasting viscosity are important predictors of the eating and cooking qualities of rice. Great progress has been made in our understanding of the genetic and molecular bases of rice eating and cooking qualities. The Wx gene encoding granule-bound starch synthase is the major gene controlling AAC, gel consistency, and pasting viscosity; it also regulates GT as a minor gene. Starch synthase IIa (SSIIa) is the major gene responsible for GT and amylopectin branch chain length distribution; it also has minor effects on AAC, gel consistency, and pasting viscosity. The functional nucleotide polymorphisms in Wx and SSIIa and their associations with physicochemical properties have been well characterized. Molecular breeding using these functional markers of Wx and SSIIa has resulted in the development of new high-quality rice cultivars (breeding lines). A multienzyme complex model is proposed to explain different AAC-GT combinatio...

Properties of a novel type of starch found in the double mutant “sweet wheat”

The double mutant “sweet wheat” (SW), which produces substantial amounts of sugars in immature seeds, is missing two starch synthases, namely granule-bound starch synthase I (GBSSI) and starch synthase IIa (SSIIa). The lack of these two enzymes causes major changes in the attributes of SW seed, starch, and starch granules. SW seeds appear normal during early stages of development, but become shrunken when seeds begin to mature and dry. However, even in immature seed, starch granules are small and misshapen, and high levels of maltose are present throughout seed development. The crystallinity of SW starch is altered in that a major peak typical of the cereal A-type diffraction pattern is absent, and the gelatinization temperature of SW starch is considerably lower than that of wild-type starch. Amylopectin from SW seed has a substantially lower molecular weight than that from wild-type seed, and a low molecular weight peak with a bimodal distribution is found only in SW starch. This peak contains linear malto-oligosaccharides as well as short, branched glucans. SW starch has an increased proportion of branches with DP<10, and chains with DP 2 and 3 are particularly increased. These changes suggest that sweet wheat starch is being modified in an atypical manner by isoamylases and/or β-amylases.

Sequence diversity, haplotype analysis, association mapping and functional marker development in the waxy and starch synthase IIa genes for grain-yield-related traits in hexaploid wheat (Triticum aestivum L.)

Development of high-yielding cereal crops could meet increasing global demands for food, feed and bio-fuels. Wheat is one of the world’s most important cereal crops. The biosynthesis of starch is the major determinant of yield in wheat. Two starch biosynthesis genes, the waxy (Wx) genes and the starch synthase IIa (SSIIa) genes, were amplified and sequenced in 92 diverse wheat genotypes using genome-specific primers. Nucleotide diversity, haplotype analysis and association mapping were performed. The first exon (5′-UTR) and the first intron of the three homoeologous Wx genes were isolated using expressed sequence tag sequences. The Wx genes contained 12 exons separated by 11 introns. SNP (single nucleotide polymorphism) frequency ranged from 1 SNP/3,648 bp for Wx-D1 to 1 SNP/135 bp for SSIIa-A1, with an average of 1 SNP/230 bp. The average SNP frequencies in exon and intron regions were 1 SNP/322 bp and 1 SNP/228 bp, respectively. Thirty, 23 and 5 SNPs were identified and formed five, six and five haplotypes for SSIIa-A1, SSIIa-B1 and SSIIa-D1, respectively. However, no association was found between these SNPs and seven yield-related traits. Twenty-two, 15 and 1 SNPs were detected and formed nine, five and two haplotypes for Wx-A1, Wx-B1 and Wx-D1, respectively. Three unique nucleotides C+A+T at SNP5, SNP6 and SNP12 formed Wx-B1-H3, which was significantly associated with increased grain weight, thousand kernel weight, and total starch content in three spring wheat genotypes and five winter wheat genotypes. Cost-effective and co-dominant SNP markers were developed using temperature-switch (TS)-PCR and are being used for marker-assisted selection of doubled haploid lines with enhanced grain yield and starch content in winter wheat breeding programs.

Lack of Low Frequency Variants Masks Patterns of Non-Neutral Evolution following Domestication

Detecting artificial selection in the genome of domesticated species can not only shed light on human history but can also be beneficial to future breeding strategies. Evidence for selection has been documented in domesticated species including maize and rice, but few studies have to date detected signals of artificial selection in the Sorghum bicolor genome. Based on evidence that domesticated S. bicolor and its wild relatives show significant differences in endosperm structure and quality, we sequenced three candidate seed storage protein (kafirin) loci and three candidate starch biosynthesis loci to test whether these genes show non-neutral evolution resulting from the domestication process. We found strong evidence of non-neutral selection at the starch synthase IIa gene, while both starch branching enzyme I and the beta kafirin gene showed weaker evidence of non-neutral selection. We argue that the power to detect consistent signals of non-neutral selection in our dataset is confounded by the absence of low frequency variants at four of the six candidate genes. A future challenge in the detection of positive selection associated with domestication in sorghum is to develop models that can accommodate for skewed frequency spectrums.

High Levels of Sugars and Fructan in Mature Seed of Sweet Wheat Lacking GBSSI and SSIIa Enzymes

Sweet wheat (SW), which lacks functional granule-bound starch synthase I (GBSSI) and starch synthase IIa (SSIIa), accumulates high levels of free sugars in immature seeds. Here, we examined the effects of the lack of these two enzymes on mature kernel composition. Whole grain flour of SW had higher levels of sugars, particularly maltose, slightly higher ash and protein content, approximately two to three times higher lipid levels, and about twice as much total dietary fiber as parental or wild-type lines. Considerably higher levels of low-molecular-weight soluble dietary fiber (LMW-SDF), largely consisting of fructan, were also detected in SW. Although there were no differences in total amino acid levels, the free amino acid content of SW was approximately 4-fold higher than that of wild type, and the levels of certain free amino acids such as proline were particularly high. Thus, we were able to clearly demonstrate that the lack of GBSSI and SSIIa caused dramatic changes in mature seed composition in SW. These compositional changes suggest that SW flour may provide health benefits when used as a food ingredient.

Association between nonsynonymous mutations of starch synthase IIa and starch quality in rice (Oryza sativa)

Starch quality is one of the most important agronomic traits in Asian rice, Oryza sativa. Starch synthase IIa (SsIIa) is a major candidate gene for starch quality variation. Within SsIIa, three nonsynonymous mutations in exon 8 have been shown to affect enzyme activity when expressed in Escherichia coli. To search for the variation in SsIIa that is responsible for starch quality variation in rice, we sequenced the SsIIa exon 8 region and measured starch quality as starch disintegration in alkali for 289 accessions of cultivated rice and 57 accessions of its wild ancestor, Oryza rufipogon. A general linear model and nested clade analysis were used to identify the associations between the three nonsynonymous single nucleotide polymorphisms (SNPs) and starch quality. Among the three nonsynonymous SNPs, we found strong evidence of association at one nucleotide site ('SNP 3'), corresponding to a Leu/Phe replacement at codon 781. A second SNP, corresponding to a Val/Met replacement at codon 737, could potentially show an association with increased sample sizes. Variation in SsIIa enzyme activity is associated with the cohesiveness of rice grains when cooked, and our findings are consistent with selection for more cohesive grains during the domestication of tropical japonica rice.

Development of new markers to genotype the functional SNPs of SSIIa, a gene responsible for gelatinization temperature of rice starch

Gelatinization temperature (GT) is an important quality predictor that determines the cooking quality of rice. GT is genetically controlled by the starch synthase IIa ( SSIIa) gene. Two functional single nucleotide polymorphisms (SNPs) inside the SSIIa have already been found to be responsible for the variation of GT. One of these, GC/TT SNP at 4329/4330 bp, could be genotyped by four primers in a single PCR ( Bao et al., 2006a), but another one, G/A SNP at 4198 bp, has not been detected by a PCR-based marker. Here, we developed cleaved amplified polymorphic sequence (CAPS) and derived cleaved amplified polymorphic sequence (dCAPS) markers to detect these SNPs. A dCAPS marker that the PCR products were cleaved by the BseR I restriction endonuclease was designed to detect GC/TT SNP. Both CAPS and dCAPS markers were designed to detect G/A SNP using the restriction endonuclease Nla III and Tsp45 I, respectively. All the markers developed were co-dominant. It was known that the A allele of G/A SNP was rare among rice germplasm, but it was still in use by rice breeders. 11 rice accessions including landrace and breeding lines with A allele of G/A SNP were detected. The F 2 individuals from two crosses were used to analyze the co-segregation between the SNP alleles and the GT. The segregation ratio of two SNPs did not conform to the expected Mendelian ratio of 1:2:1, but the SNPs were co-segregated with GT. The markers developed in the present study would be useful in molecular breeding for the improvement of the quality of rice grain.

Characterization of Small Heat Shock Proteins Associated with Maize Tolerance to Combined Drought and Heat Stress

To investigate how the mechanisms of small heat shock proteins (sHSPs) in regulating maize leaves respond to the combination of drought and heat stress, leaf protein patterns were monitored using a proteomic approach in maize plants exposed to combined drought and heat stress. Two-dimensional electrophoresis (2-DE) was used to identify combined drought- and heat-responsive protein spots in maize leaves. After Coomassie brilliant blue staining, approximately 450 protein spots were reproducibly detected on each gel, wherein 7 protein spots were expressed only under heat and combined drought and heat stress but were almost undetected under control and drought. Using MALDI-TOF mass spectrometry, a total of seven proteins were identified, including cytochrome b6-f complex iron-sulfur subunit, sHSP17.4, sHSP17.2, sHSP26, guanine nucleotide-binding protein β-subunit-like protein, putative uncharacterized protein, and granule-bound starch synthase IIa. Moreover, the gene expression of three sHSPs was analyzed at the transcriptional level and indicated that all three sHSPs were expressed under several treatments although their expression levels were obviously more enhanced by heat and combined drought and heat stress than by control and drought. In investigations of the effect of abscisic acid (ABA) on the three sHSPs, pretreatment with 100 μM ABA enhanced substantially the expression of the three sHSPs at the protein level, but only slightly at the mRNA level. These results show that transcription levels are not completely concomitant with translation and suggest that ABA induces the post-transcriptional regulation of sHSP17.2, sHSP17.4, and sHSP26 expression, which can lead to a better understanding of the mechanisms of plant response to the combination of drought and heat stress.

Genetic diversity and population structure of a diverse set of rice germplasm for association mapping

Germplasm diversity is the mainstay for crop improvement and genetic dissection of complex traits. Understanding genetic diversity, population structure, and the level and distribution of linkage disequilibrium (LD) in target populations is of great importance and a prerequisite for association mapping. In this study, 100 genome-wide simple sequence repeat (SSR) markers were used to assess genetic diversity, population structure, and LD of 416 rice accessions including landraces, cultivars and breeding lines collected mostly in China. A model-based population structure analysis divided the rice materials into seven subpopulations. 63% of the SSR pairs in these accessions were in LD, which was mostly due to an overall population structure, since the number of locus pairs in LD was reduced sharply within each subpopulation, with the SSR pairs in LD ranging from 5.9 to 22.9%. Among those SSR pairs showing significant LD, the intrachromosomal LD had an average of 25-50 cM in different subpopulations. Analysis of the phenotypic diversity of 25 traits showed that the population structure accounted for an average of 22.4% of phenotypic variation. An example association mapping for starch quality traits using both the candidate gene mapping and genome-wide mapping strategies based on the estimated population structure was conducted. Candidate gene mapping confirmed that the Wx and starch synthase IIa (SSIIa) genes could be identified as strongly associated with apparent amylose content (AAC) and pasting temperature (PT), respectively. More importantly, we revealed that the Wx gene was also strongly associated with PT. In addition to the major genes, we found five and seven SSRs were associated with AAC and PT, respectively, some of which have not been detected in previous linkage mapping studies. The results suggested that the population may be useful for the genome-wide marker-trait association mapping. This new association population has the potential to identify quantitative trait loci (QTL) with small effects, which will aid in dissecting complex traits and in exploiting the rich diversity present in rice germplasm.

米を中心とする穀物胚乳澱粉の構造と物性に関する研究

1． 近年わが国で育成された米品種の胚乳澱粉の構造と物性 近年わが国で育成されたモチ米，低アミロース米，中アミロース米，高アミロース米の4タイプの米試料の胚乳澱粉の性質を調べた．ウルチ性澱粉の真のアミロース含量とRVAによるピーク粘度との間に高い負の相関関係が観察された．高アミロース米の中には，見かけのアミロース含量は約30%と同様であるが，アミロペクチンの超長鎖含量，RVAのピーク粘度，セットバックが異なる性質を示す澱粉をもつ試料米が存在していた．アミロペクチンの超長鎖含量とRVAによる糊液のセットバックとの間に高い正の相関関係が観察されたことから，アミロペクチンに存在する超長鎖は澱粉糊のセットバックに大きな影響を及ぼすと考えられる．アミロペクチンの短鎖領域の側鎖長分布と超長鎖含量の両方とも，米澱粉の糊化温度に大きな影響を及ぼす．75°Cの水中で米澱粉を加熱する際，アミロペクチンに存在する超長鎖は，澱粉の膨潤および溶解に対して，いずれも関与しない傾向がみられた．2． アジアとその他の諸外国の品種米の胚乳澱粉の構造と性質 アジアとその他の諸外国の75品種米から胚乳澱粉を調製した．インディカおよび中国インディカのウルチ性品種米の澱粉は，見かけのアミロース含量，アミロペクチンの超長鎖含量ともに高い値を示し，アミロペクチンの最も短い側鎖画分(6-12量体，Fr.A)は低い値を示した．Fr.A含量は米澱粉のDSCによる糊化ピーク温度と負の関係が観察された．また，米澱粉中のWxタンパク質量と超長鎖含量との間に高い正の相関関係が得られた．米の胚乳澱粉の分類方法の一つとして，アミロペクチンの超長鎖含量とFr.A含量の間の関係を示すことを提案した． 3． 澱粉合成酵素の変異が澱粉の性質に及ぼす影響 ジャポニカ米の日本晴を遺伝的背景にもち，インディカ米のカサラスの染色体断片を含む3種の準同質遺伝子系統(NIL(Wxa)，NIL(SSIK)，NIL(Alk))の米試料を用いて，3種の澱粉合成酵素GBSSI，SSI，SSIIaが澱粉の性質に及ぼす影響を調べた．Wxa遺伝子によって日本晴とカサラスとの間のアミロース含量の違いをほぼ説明することができた．またWxa遺伝子によって澱粉のピーク粘度，ブレークダウンは減少した．NIL(Alk)のアミロペクチンは，6-11量体の短い側鎖が少なく，13-23量体の中鎖の多いカサラスと非常によく似た鎖長分布を示した．またDSCによる熱測定により，NIL(Alk)の炊飯米の老化速度が速いことがわかった．

Melting the secrets of gelatinisation temperature in rice

Gelatinisation temperature (GT) is one of the key traits measured in programs for breeding rice (Oryza sativa L.). It is commonly estimated by the alkali spreading value (ASV), and less commonly by differential scanning calorimetry (DSC). Using a diverse set of germplasm, it was determined that DSC values associate poorly with ASV, are not correlated with amylose content but correlate with cooking time. Rice varieties are traditionally grouped into three classes of GT based on ASV: high, intermediate and low. However, the distribution of DSC values of 4000 samples shows only two classes: high and low. Large differences in the distributions of chain lengths synthesised by starch synthase IIa (SSIIa) support the two classes as the major grouping, two haplotypes associating with each peak. Each peak of DSC values spanned 10°C. The chain length distribution of the amylopectin molecules from varieties at the upper boundary of each peak showed significantly more chains that span both the crystalline and amorphous lamellae of a cluster than varieties at the other end of that distribution. Improved varieties, classified as intermediate GT by ASV, belong to both of the classes defined by DSC, implying that some enzyme, other than SSIIa is involved in intermediate GT.

Molecular marker assisted selection for improvement of the eating, cooking and sensory quality of rice ( Oryza sativa L.)

II-32B is a key maintainer line used for hybrid rice breeding in China. However, it is of poor quality for most Chinese consumers because of its high apparent amylose content (AAC), high gelatinization temperature (GT) and non-fragrance. It is well known that the AAC, GT and fragrance traits are largely controlled by the Wx, starch synthase IIa ( SSIIa), and fragrance ( fgr) genes, respectively. With the aid of functional markers, we improved the quality of II-32B by introgressing the Wx, SSIIa, and fgr genes from Yixiang B, a fragrant maintainer line that has low AAC and low GT. The microsatellite allele (CT) 17 of Wx, the contiguous single nucleotide polymorphism TT allele of SSIIa and the 8-bp deletion allele of fgr were transferred to II-32B by two backcrosses and three selfings. Molecular marker assisted selection was applied in the series to select for individuals carrying Wx-(CT) 17, SSIIa-TT, and fgr-deletion alleles. According to the marker genotypes, seventeen homozygous lines for Wx-(CT) 17, SSIIa-TT, and fgr gene markers were finally selected. The improved II-32B lines were fragrant with reduced AAC and GT.

Granule‐bound SSIIa Protein Content and its Relationship with Amylopectin Structure and Gelatinization Temperature of Rice Starch

AbstractStarch characteristics such as gelatinization properties determine the quality of various products of rice. The eating, cooking and processing qualities of rice are a function of the gelatinization of rice starch. The gelatinization temperature (GT) of rice is controlled by the starch synthase IIa (SSIIa) gene. In the present study, the amount of the starch‐associated SSIIa protein and its relationship with amylopectin structure and GT were investigated. The results indicated that the starch associated SSIIa protein content was positively correlated with GT and level of amylopectin chains with degree of polymerization 12‐24 (DP12‐24), and negatively correlated with the fraction of chains with DP6‐11 and short chain ratio (SCR), i.e. DP6‐11/DP12‐24 (P&lt;0.001). GT was also negatively correlated with DP6‐11 SCR, and positively correlated with DP12‐24. The SSIIa protein content and amylopectin structure showed significant difference between the two SSIIa single nucleotide polymorphisms (SNPs, i.e. guanine‐cytosine/thymine‐thymine (GC/TT)) groups. Rice with GC and TT SNPs had average SSIIa protein contents of 1.076 and 0.681, respectively. Rice with the GC SNPs displayed a lower amount of chains with DP6‐11 and more chains with DP 12‐24 than those with TT SNPs. A mechanism is postulated to explain how SSIIa could determine the distribution of amylopectin side‐chains, which affects the gelatinization temperature of rice starch.

QTL detection of rice grain quality traits by microsatellite markers using an indica rice (Oryza sativa L.) combination

The gelatinization temperature (GT), gel consistency (GC) and amylose content (AC) are the three major rice traits that are directly related to cooking and eating quality (Little et al. 1958). GT is a physical trait responsible for cooking time and the capacity to absorb water during the processes cooking, and the temperature at which starch irreversibly loses its crystalline order during cooking. The GC is responsible for softness, and the AC is responsible for texture and appearance in rice. Hence, regulating AC in rice has been a major concern of rice breeders. To facilitate the development of new varieties with high cooking and eating qualities, it is necessary to understand the genetic bases of such traits. Lanceras et al. (2000) found four QTLs for AC on chromosomes three, four, six and seven. These QTLs accounted for 80% of phenotypic variation observeded in AC. Two QTLs on chromosome six, and one on chromosome seven were detected for GC, which accounted for 57% of phenotypic variation. Umemoto et al. (2002) confirmed this and demonstrated that the alk locus encodes the enzyme soluble starch synthase IIa. Bao et al. (2002) and Lanceras et al. (2000) found the effect of the wx region on GC. However, He et al. (1999) and Bao et al. (2002) showed that GC is controlled by two QTLs with minor effects. Zhou et al. (2003) improved the eating and cooking quality of Zhenshan 97 by introgressing the waxy gene region from Minghui 63 (wx-MH), a restorer line that had medium AC, soft GC and high GT. Li et al. (2004) identified four QTLs for AC, three for GT and five for GC using backcross-inbred lines. The four QTLs for AC were located on chromosomes three, four, five and six. The QTL on chromosome six covered the wx gene region and mainly contributed to the variance between

Proteins from Multiple Metabolic Pathways Associate with Starch Biosynthetic Enzymes in High Molecular Weight Complexes: A Model for Regulation of Carbon Allocation in Maize Amyloplasts

Starch biosynthetic enzymes from maize (Zea mays) and wheat (Triticum aestivum) amyloplasts exist in cell extracts in high molecular weight complexes; however, the nature of those assemblies remains to be defined. This study tested the interdependence of the maize enzymes starch synthase IIa (SSIIa), SSIII, starch branching enzyme IIb (SBEIIb), and SBEIIa for assembly into multisubunit complexes. Mutations that eliminated any one of those proteins also prevented the others from assembling into a high molecular mass form of approximately 670 kD, so that SSIII, SSIIa, SBEIIa, and SBEIIb most likely all exist together in the same complex. SSIIa, SBEIIb, and SBEIIa, but not SSIII, were also interdependent for assembly into a complex of approximately 300 kD. SSIII, SSIIa, SBEIIa, and SBEIIb copurified through successive chromatography steps, and SBEIIa, SBEIIb, and SSIIa coimmunoprecipitated with SSIII in a phosphorylation-dependent manner. SBEIIa and SBEIIb also were retained on an affinity column bearing a specific conserved fragment of SSIII located outside of the SS catalytic domain. Additional proteins that copurified with SSIII in multiple biochemical methods included the two known isoforms of pyruvate orthophosphate dikinase (PPDK), large and small subunits of ADP-glucose pyrophosphorylase, and the sucrose synthase isoform SUS-SH1. PPDK and SUS-SH1 required SSIII, SSIIa, SBEIIa, and SBEIIb for assembly into the 670-kD complex. These complexes may function in global regulation of carbon partitioning between metabolic pathways in developing seeds.

Endonucleolytic mutation analysis by internal labeling (EMAIL)

Mismatch-specific endonucleases are efficient tools for the targeted scanning of populations for subtle DNA variations. Conventional protocols involve 5'-labeled amplicon substrates and the detection of digestion products by LIF electrophoresis. A shortcoming of such protocols, however, is the limited 5'-signal strength. Normally the sensitivity of fluorescent DNA analyzers is superior to that of intercalating dye/agarose systems, however, pooling capacities of the former and latter approaches to mismatch scanning are somewhat similar. Detection is further limited by significant background. We investigated the activity of CEL nucleases using amplicon substrates labeled both internally and at each 5'-terminus. The amplicons were generated from exon 8 of the rice starch synthase IIa encoding gene. Signal of both 5'-labels was significantly reduced by enzyme activity, while that of the internal label was largely unaffected. In addition, background resulting from internal labeling was a significant improvement on that associated with 5'-labeling. Sizing of the multilabeled substrates suggests that 5'-modification enhances exonucleolytic activity, resulting in the removal of the dye-labeled terminal nucleotides. We have developed an alternative approach to mismatch detection, in which amplicon labeling is achieved via the incorporation of fluorescently labeled deoxynucleotides, which we have named Endonucleolytic Mutation Analysis by Internal Labeling (EMAIL). The strength of the EMAIL assay was demonstrated by the reclassification of a rice line as being heterozygous for the starch gene. This cultivar was assigned as being homozygous by a previous resequencing study. EMAIL shows potential for the clear identification of multiple mutations amongst allelic pools.