Mutation of the maize sbe1a and ae genes alters morphology and physical behavior of wx-type endosperm starch granules

In maize, three isoforms of starch-branching enzyme, SBEI, SBEIIa, and SBEIIb, are encoded by the Sbe1a, Sbe2a, and Amylose extender (Ae) genes, respectively. The objective of this research was to explore the effects of null mutations in the Sbe1a and Ae genes alone and in combination in wx background on kernel characteristics and on the morphology and physical behavior of endosperm starch granules. Differences in kernel morphology and weight, starch accumulation, starch granule size and size distribution, starch microstructure, and thermal properties were observed between the ae wx and sbe1a ae wx plants but not between the sbe1a wx mutants when compared to wx. Starch from sbe1a ae wx plants exhibited a larger granule size with a wider gelatinization temperature range and a lower endotherm enthalpy than ae wx. Microscopy shows weaker iodine staining in sbe1a ae wx starch granules. X-ray diffraction revealed A-type crystallinity in wx and sbe1a wx starches and B-type in sbe1a ae wx and ae wx. This study suggests that, while the SBEIIb isoform plays a dominant role in maize endosperm starch synthesis, SBEI also plays a role, which is only observable in the presence of the ae mutation.     

Allelic variants of the amylose extender mutation of maize demonstrate phenotypic variation in starch structure resulting from modified protein-protein interactions

Amylose extender (ae(-)) starches characteristically have modified starch granule morphology resulting from amylopectin with reduced branch frequency and longer glucan chains in clusters, caused by the loss of activity of the major starch branching enzyme (SBE), which in maize endosperm is SBEIIb. A recent study with ae(-) maize lacking the SBEIIb protein (termed ae1.1 herein) showed that novel protein-protein interactions between enzymes of starch biosynthesis in the amyloplast could explain the starch phenotype of the ae1.1 mutant. The present study examined an allelic variant of the ae(-) mutation, ae1.2, which expresses a catalytically inactive form of SBEIIb. The catalytically inactive SBEIIb in ae1.2 lacks a 28 amino acid peptide (Val272-Pro299) and is unable to bind to amylopectin. Analysis of starch from ae1.2 revealed altered granule morphology and physicochemical characteristics distinct from those of the ae1.1 mutant as well as the wild-type, including altered apparent amylose content and gelatinization properties. Starch from ae1.2 had fewer intermediate length glucan chains (degree of polymerization 16-20) than ae1.1. Biochemical analysis of ae1.2 showed that there were differences in the organization and assembly of protein complexes of starch biosynthetic enzymes in comparison with ae1.1 (and wild-type) amyloplasts, which were also reflected in the composition of starch granule-bound proteins. The formation of stromal protein complexes in the wild-type and ae1.2 was strongly enhanced by ATP, and broken by phosphatase treatment, indicating a role for protein phosphorylation in their assembly. Labelling experiments with [γ-(32)P]ATP showed that the inactive form of SBEIIb in ae1.2 was phosphorylated, both in the monomeric form and in association with starch synthase isoforms. Although the inactive SBEIIb was unable to bind starch directly, it was strongly associated with the starch granule, reinforcing the conclusion that its presence in the granules is a result of physical association with other enzymes of starch synthesis. In addition, an Mn(2+)-based affinity ligand, specific for phosphoproteins, was used to show that the granule-bound forms of SBEIIb in the wild-type and ae1.2 were phosphorylated, as was the granule-bound form of SBEI found in ae1.2 starch. The data strongly support the hypothesis that the complement of heteromeric complexes of proteins involved in amylopectin synthesis contributes to the fine structure and architecture of the starch granule.     

Starch-branching enzyme I-deficient mutation specifically affects the structure and properties of starch in rice endosperm

We have isolated a starch mutant that was deficient in starch-branching enzyme I (BEI) from the endosperm mutant stocks of rice (Oryza sativa) induced by the treatment of fertilized egg cells with N-methyl-N-nitrosourea. The deficiency of BEI in this mutant was controlled by a single recessive gene, tentatively designated as starch-branching enzyme mutant 1 (sbe1). The mutant endosperm exhibited the normal phenotype and contained the same amount of starch as the wild type. However, the mutation apparently altered the fine structure of amylopectin. The mutant amylopectin was characterized by significant decrease in both long chains with degree of polymerization (DP) > or = 37 and short chains with DP 12 to 21, marked increase in short chains with DP < or = 10 (A chains), and slight increase in intermediate chains with DP 24 to 34, suggesting that BEI specifically synthesizes B1 and B2-3 chains. The endosperm starch from the sbe1 mutant had a lower onset concentration for urea gelatinization and a lower onset temperature for thermo-gelatinization compared with the wild type, indicating that the genetic modification of amylopectin fine structure is responsible for changes in physicochemical properties of sbe1 starch.     

Importance of isoforms of starch-branching enzyme in determining the structure of starch in pea leaves

The impact of a mutation at the r locus of peas ( Pisum sativum L.) on the structure of starch in the leaf has been studied. The mutation specifically eliminates the A class of isoform of starch-branching enzyme (SBE A) from the leaf, causing a 10-fold reduction in the total activity of the enzyme. Gel-permeation chromatography and thymol precipitation show that wild-type leaf starch consists of polymers with the general characteristics of amylose and amylopectin, although amylose is only a very minor component of the starch. High-performance anion exchange chromatography (HPAEC) of debranched amylopectin reveals that the distribution profile of branch lengths is strongly polymodal, and distinctly different from that of the amylopectin of storage starches. The mutation at the r locus results in the appearance of an amylopectin-like glucan of low molecular weight in the starch. The absorbance of the iodine complex of the amylopectin and analysis by HPAEC both indicate that the mutation causes an increase in the average branch length of the amylopectin but does not affect the polymodal nature of the distribution of branch lengths. The extent to which these effects of the mutation are specifically due to the loss of SBE A is discussed. It is suggested that differences in properties between isoforms of SBE are not the main factors that determine the polymodal distribution of branch lengths in amylopectin.     

Functional interactions between heterologously expressed starch-branching enzymes of maize and the glycogen synthases of Brewer's yeast

Starch-branching enzymes (SBEs) catalyze the formation of alpha(1-->6) glycoside bonds in glucan polymers, thus, affecting the structure of amylopectin and starch granules. Two distinct classes of SBE are generally conserved in higher plants, although the specific role(s) of each isoform in determination of starch structure is not clearly understood. This study used a heterologous in vivo system to isolate the function of each of the three known SBE isoforms of maize (Zea mays) away from the other plant enzymes involved in starch biosynthesis. The ascomycete Brewer's yeast (Saccharomyces cerevisiae) was employed as the host species. All possible combinations of maize SBEs were expressed in the absence of the endogenous glucan-branching enzyme. Each maize SBE was functional in yeast cells, although SBEI had a significant effect only if SBEIIa and SBEIIb also were present. SBEI by itself did not support glucan accumulation, whereas SBEIIa and SBEIIb both functioned along with the native glycogen synthases (GSs) to produce significant quantities of alpha-glucan polymers. SBEIIa was phenotypically dominant to SBEIIb in terms of glucan structure. The specific branching enzyme present had a significant effect on the molecular weight of the product. From these data we suggest that SBEs and GSs work in a cyclically interdependent fashion, such that SBE action is needed for optimal GS activity; and GS, in turn, influences the further effects of SBE. Also, SBEIIa and SBEIIb appear to act before SBEI during polymer assembly in this heterologous system.     

Starch-branching enzyme IIa is required for proper diurnal cycling of starch in leaves of maize

Starch-branching enzyme (SBE), a glucosyl transferase, is required for the highly regular pattern of α-1,6 bonds in the amylopectin component of starch. In the absence of SBEIIa, as shown previously in the sbe2a mutant of maize (Zea mays), leaf starch has drastically reduced branching and the leaves exhibit a severe senescence-like phenotype. Detailed characterization of the maize sbe2a mutant revealed that SBEIIa is the primary active branching enzyme in the leaf and that in its absence plant growth is affected. Both seedling and mature sbe2a mutant leaves do not properly degrade starch during the night, resulting in hyperaccumulation. In mature sbe2a leaves, starch hyperaccumulation is greatest in visibly senescing regions but also observed in green tissue and is correlated to a drastic reduction in photosynthesis within the leaf. Starch granules from sbe2a leaves observed via scanning electron microscopy and transmission electron microscopy analyses are larger, irregular, and amorphous as compared with the highly regular, discoid starch granules observed in wild-type leaves. This appears to trigger premature senescence, as shown by an increased expression of genes encoding proteins known to be involved in senescence and programmed cell death processes. Together, these results indicate that SBEIIa is required for the proper diurnal cycling of transitory starch within the leaf and suggest that SBEIIa is necessary in producing an amylopectin structure amenable to degradation by starch metabolism enzymes.     

Independent genetic control of maize starch-branching enzymes IIa and IIb. Isolation and characterization of a Sbe2a cDNA.

In maize (Zea mays L.) three isoforms of starch-branching enzyme (SBEI, SBEIIa, and SBEIIb) are involved in the synthesis of amylopectin, the branched component of starch. To isolate a cDNA encoding SBEIIa, degenerate oligonucleotides based on domains highly conserved in Sbe2 family members were used to amplify Sbe2-family cDNA from tissues lacking SBEIIb activity. The predicted amino acid sequence of Sbe2a cDNA matches the N-terminal sequence of SBEIIa protein purified from maize endosperm. The size of the mature protein deduced from the cDNA also matches that of SBEIIa. Features of the predicted protein are most similar to members of the SBEII family; however, it differs from maize SBEIIb in having a 49-amino acid N-terminal extension and a region of substantial sequence divergence. Sbe2a mRNA levels are 10-fold higher in embryonic than in endosperm tissue, and are much lower than Sbe2b in both tissues. Unlike Sbe2b, Sbe2a-hybridizing mRNA accumulates in leaf and other vegetative tissues, consistent with the known distribution of SBEIIa and SBEIIb activities.     

Impact of down-regulation of starch branching enzyme IIb in rice by artificial microRNA- and hairpin RNA-mediated RNA silencing

The inactivation of starch branching IIb (SBEIIb) in rice is traditionally associated with elevated apparent amylose content, increased peak gelatinization temperature, and a decreased proportion of short amylopectin branches. To elucidate further the structural and functional role of this enzyme, the phenotypic effects of down-regulating SBEIIb expression in rice endosperm were characterized by artificial microRNA (amiRNA) and hairpin RNA (hp-RNA) gene silencing. The results showed that RNA silencing of SBEIIb expression in rice grains did not affect the expression of other major isoforms of starch branching enzymes or starch synthases. Structural analyses of debranched starch showed that the doubling of apparent amylose content was not due to an increase in the relative proportion of amylose chains but instead was due to significantly elevated levels of long amylopectin and intermediate chains. Rices altered by the amiRNA technique produced a more extreme starch phenotype than those modified using the hp-RNA technique, with a greater increase in the proportion of long amylopectin and intermediate chains. The more pronounced starch structural modifications produced in the amiRNA lines led to more severe alterations in starch granule morphology and crystallinity as well as digestibility of freshly cooked grains. The potential role of attenuating SBEIIb expression in generating starch with elevated levels of resistant starch and lower glycaemic index is discussed.     

Granule-bound starch synthase I is responsible for biosynthesis of extra-long unit chains of amylopectin in rice

A rice Wx gene encoding a granule-bound starch synthase I (GBSSI) was introduced into the null-mutant waxy (wx) rice, and its effect on endosperm starches was examined. The apparent amylose content was increased from undetectable amounts for the non-transgenic wx cultivars to 21.6-22.2% of starch weight for the transgenic lines. The increase was in part due to a significant amount of extra-long unit chains (ELCs) of amylopectin (7.5-8.4% of amylopectin weight), that were absent in the non-transgenic wx cultivars. Thus, actual amylose content was calculated to be 14.9-16.0% for the transgenic lines. Only slight differences were found in chain-length distribution for the chains other than ELCs, indicating that the major effect of the Wx transgene on amylopectin structure was ELC formation. ELCs isolated from debranched amylopectin exhibited structures distinct from amylose. Structures of amylose from the transgenic lines were slightly different from those of cv. Labelle (Wx(a)) in terms of a higher degree of branching and size distribution. The amylose and ELC content of starches of the transgenic lines resulted in the elevation of pasting temperature, a 50% decrease in peak viscosity, a large decrease in breakdown and an increase in setback. As yet undetermined factors other than the GBSSI activity are thought to be involved in the control of formation and/or the amount of ELCs. Structural analysis of the Wx gene suggested that the presence of a tyrosine residue at position 224 of GBSSI correlates with the formation of large amounts of ELCs in cultivars carrying Wx(a).     

Starch branching enzyme IIb in wheat is expressed at low levels in the endosperm compared to other cereals and encoded at a non-syntenic locus

Studies of maize starch branching enzyme mutants suggest that the amylose extender high amylose starch phenotype is a consequence of the lack of expression of the predominant starch branching enzyme II isoform expressed in the endosperm, SBEIIb. However, in wheat, the ratio of SBEIIb and SBEIIa expression are inversely related to the expression levels observed in maize and rice. Analysis of RNA at 15 days post anthesis suggests that there are about 4-fold more RNA for SBE IIa than for SBE IIb. The genes for SBE IIa and SBE IIb from wheat are distinguished in the size of the first three exons, allowing isoform-specific antibodies to be produced. These antibodies were used to demonstrate that in the soluble fraction, the amount of SBE IIa protein is two to three fold higher than SBIIb, whereas in the starch granule, there is two to three fold more SBE IIb protein amount than SBE IIa. In a further difference to maize and rice, the genes for SBE IIa and SBE IIb are both located on the long arm of chromosome 2 in wheat, in a position not expected from rice–maize–wheat synteny.     

Scanning Electron Microscopy and Fermentation Studies on Selected Known Maize Starch Mutants Using STARGEN? Enzyme Blends

The conversion of maize (corn) kernels to bio-ethanol is an energy-intensive process involving many stages. One step typically required is the liquefaction of the ground kernel to enable enzyme hydrolysation of the starch to glucose. The enzyme blends STARGEN? (Genencor) are capable of hydrolysing starch granules without liquefaction, reducing energy inputs and increasing efficiency. Studies were conducted on maize starch mutants amylose extender 1 (ae1), dull 1 (du1) and waxy 1 (wx1) in the inbred line Oh43 to determine whether different maize starches affected hydrolysation rates by STARGEN? 001 and STARGEN? 002. All mutants contained similar proportions of starch in the kernel but varied in the amylose to amylopectin ratio. Ground maize kernels were incubated with STARGEN? 001 and viewed using scanning electron microscopy to examine the hydrolysis action of STARGEN? 001 on the starch granules. The ae1 mutant exhibited noticeably less enzymic hydrolysis action, on the granules visualised, than wx1 and background line Oh43. Kernels were batch-fermented with STARGEN? 001 and STARGEN? 002. The ae1 mutant exhibited a 50% lower ethanol yield compared to the wx1 mutant and background line. A final study compared hydrolysation rates of STARGEN? 001 and STARGEN? 002 on purified maize starch, amylopectin and amylose. Though almost twice the amylopectin was hydrolysed using STARGEN? 002 than STARGEN? 001 in this trial, fermentations using STARGEN? 002 resulted in lower ethanol yields than fermentations using STARGEN? 001. Both STARGEN? enzyme blends were more suitable for the fermentation of high amylopectin maize starches than high amylose starches.     

Protein phosphorylation in amyloplasts regulates starch branching enzyme activity and protein-protein interactions

Protein phosphorylation in amyloplasts and chloroplasts of Triticum aestivum (wheat) was investigated after the incubation of intact plastids with gamma-(32)P-ATP. Among the soluble phosphoproteins detected in plastids, three forms of starch branching enzyme (SBE) were phosphorylated in amyloplasts (SBEI, SBEIIa, and SBEIIb), and both forms of SBE in chloroplasts (SBEI and SBEIIa) were shown to be phosphorylated after sequencing of the immunoprecipitated (32)P-labeled phosphoproteins using quadrupole-orthogonal acceleration time of flight mass spectrometry. Phosphoamino acid analysis of the phosphorylated SBE forms indicated that the proteins are all phosphorylated on Ser residues. Analysis of starch granule-associated phosphoproteins after incubation of intact amyloplasts with gamma-(32)P-ATP indicated that the granule-associated forms of SBEII and two granule-associated forms of starch synthase (SS) are phosphorylated, including SSIIa. Measurement of SBE activity in amyloplasts and chloroplasts showed that phosphorylation activated SBEIIa (and SBEIIb in amyloplasts), whereas dephosphorylation using alkaline phosphatase reduced the catalytic activity of both enzymes. Phosphorylation and dephosphorylation had no effect on the measurable activity of SBEI in amyloplasts and chloroplasts, and the activities of both granule-bound forms of SBEII in amyloplasts were unaffected by dephosphorylation. Immunoprecipitation experiments using peptide-specific anti-SBE antibodies showed that SBEIIb and starch phosphorylase each coimmunoprecipitated with SBEI in a phosphorylation-dependent manner, suggesting that these enzymes may form protein complexes within the amyloplast in vivo. Conversely, dephosphorylation of immunoprecipitated protein complex led to its disassembly. This article reports direct evidence that enzymes of starch metabolism (amylopectin synthesis) are regulated by protein phosphorylation and indicate a wider role for protein phosphorylation and protein-protein interactions in the control of starch anabolism and catabolism.     

控制水稻胚乳淀粉合成代谢若干关键酶基因对花后高温的响应表达

以稻米品质温度敏感型的早籼稻品种嘉早935为材料,利用人工气候箱控温试验和实时荧光定量PCR技术,探讨了不同灌浆温度(日均温分别为22和32 ℃)处理下胚乳淀粉分支酶(SBE)、淀粉去分支酶(DBE)和淀粉合酶(SS)的10个同工型基因(sbe1、sbe3、sbe4、pul、isa1、isa2、isa3、Wx、sss1和sss2a)的相对表达量差异及动态变化特征.结果表明: 淀粉合成相关功能基因对水稻灌浆期高温胁迫的响应表达方式存在明显差异,而且因同工型的类型而不同.在高温处理下,sbe1和sbe3的相对表达量显著下降,二者属于SBE类基因中对高温胁迫较敏感的主要同工型;DBE基因中,pul属于高表达的同工型,而且其对高温胁迫响应比isa1、isa2和isa3敏感;在Wx、sss1和sss2a中,sss2a的相对表达量显著低于sss1和Wx, 但sss2a和sss1对高温胁迫响应比Wx敏感,因此二者可能也是高温胁迫对胚乳淀粉结构进行调控的重要位点,尤其在水稻灌浆的中后期发挥重要作用.     

Biochemical and genetic analysis of the effects of amylose-extender mutation in rice endosperm

Biochemical analysis of amylose-extender (ae) mutant of rice (Oryza sativa) revealed that the mutation in the gene for starch-branching enzyme IIb (BEIIb) specifically altered the structure of amylopectin in the endosperm by reducing short chains with degree of polymerization of 17 or less, with the greatest decrease in chains with degree of polymerization of 8 to 12. The extent of such change was correlated with the gelatinization properties of the starch granules, as determined in terms of solubility in urea solution. The ae mutation caused a dramatic reduction in the activity of BEIIb. The activity of soluble starch synthase I (SSI) in the ae mutant was significantly lower than in the wild type, suggesting that the mutation had a pleiotropic effect on the SSI activity. In contrast, the activities of BEI, BEIIa, ADP-Glc pyrophosphorylase, isoamylase, isoamylase, pullulanase, and Suc synthase were not affected by the mutation. Therefore, it is stressed that the function of BEIIb cannot be complemented by BEIIa and BEI. These results strongly suggest that BEIIb plays a specific role in the transfer of short chains, which might then be extended by SS to form the A and B(1) chains of amylopectin cluster in rice endosperm.