Starch Granules In Endosperm Research Articles

Variation in Granule Bound Starch Synthase I (GBSSI) loci amongst Australian wild cereal relatives (Poaceae)

A complex cascade of enzymes is responsible for the development of starch granules in grain endosperm. Granule Bound Starch Synthase I (GBSSI), encoded by the Waxy gene, is a key enzyme of starch synthesis and determines the accumulation of amylose in the starch granules. The complete genomic GBSSI sequence was ascertained for eight Australian cereal wild relatives (CWR) to determine diversity within the gene. A phylogeny derived from the coding sequence of the entire Waxy gene was compared to established phylogenetic relationships. Starch granule morphology observed in conjunction with this phylogeny suggests that small polygonal starch granules arranged as compound granules are the ancestral state, evolving subsequently to bimodal starch granules and to larger simple granules. Genomic sequence length varied within the species from 2800 to 3572 bp. Most variation occurred within the intron sequences, the largest insertion showing strong homology to a retrotransposon. One wild species was determined to have a deletion in the 3′-end of exon 1 resulting in a putatively non-functional allele. Alignment of the amino acid sequence showed strong homology throughout the central fragments of the gene but broad variation in the transit peptides. All putative functional alleles maintained the reported active sites for glycogen synthesis, though with variations in other highly conserved areas of the gene. These variations within the wild relatives of cultivated cereals may provide novel sources of genetic diversity for future cereal improvement programs.

Proteome and phosphoproteome analysis of starch granule-associated proteins from normal maize and mutants affected in starch biosynthesis.

In addition to the exclusively granule-bound starch synthase GBSSI, starch granules also bind significant proportions of other starch biosynthetic enzymes, particularly starch synthases (SS) SSI and SSIIa, and starch branching enzyme (BE) BEIIb. Whether this association is a functional aspect of starch biosynthesis, or results from non-specific entrapment during amylopectin crystallization, is not known. This study utilized genetic, immunological, and proteomic approaches to investigate comprehensively the proteome and phosphoproteome of Zea mays endosperm starch granules. SSIII, BEI, BEIIa, and starch phosphorylase were identified as internal granule-associated proteins in maize endosperm, along with the previously identified proteins GBSS, SSI, SSIIa, and BEIIb. Genetic analyses revealed three instances in which granule association of one protein is affected by the absence of another biosynthetic enzyme. First, eliminating SSIIa caused reduced granule association of SSI and BEIIb, without affecting GBSS abundance. Second, eliminating SSIII caused the appearance of two distinct electrophoretic mobility forms of BEIIb, whereas only a single migration form of BEIIb was observed in wild type or any other mutant granules examined. Third, eliminating BEIIb caused significant increases in the abundance of BEI, BEIIa, SSIII, and starch phosphorylase in the granule, without affecting SSI or SSIIa. Analysis of the granule phosphoproteome with a phosphorylation-specific dye indicated that GBSS, BEIIb, and starch phosphorylase are all phosphorylated as they occur in the granule. These results suggest the possibility that starch metabolic enzymes located in granules are regulated by post-translational modification and/or protein–protein interactions.

Roles of multiple surface sites, long substrate binding clefts, and carbohydrate binding modules in the action of amylolytic enzymes on polysaccharide substrates

Germinating barley seeds contain multiple forms of α-amylase, which are subject to both differential gene expression and differential degradation as part of the repertoire of starch-degrading enzymes. The α-amylases are endo-acting and possess a long substrate binding cleft with a characteristic subsite binding energy profile around the catalytic site. Furthermore, several amylolytic enzymes that facilitate attack on the natural substrate, i.e. the endosperm starch granules, have secondary sugar binding sites either situated on the surface of the protein domain or structural unit that contains the catalytic site or belonging to a separate starch binding domain. The role of surface sites in the function of barley α-amylase 1 has been investigated by using mutational analysis in conjunction with carbohydrate binding analyses and crystallography. The ability to bind starch depends on the surface sites and varies for starch granules of different genotypes and botanical origin. The surface sites, moreover, are candidates for being involved in degradation of polysaccharides by a multiple attack mechanism. Future studies of the molecular nature of the multivalent enzyme-substrate interactions will address surface sites in both barley α-amylase 1 and in the related isozyme 2.

Effect of endosperm starch granule size distribution on milling yield in hard wheat

Increased flour yield in hard wheat is associated with increased endosperm rheology index, calculated from strength and stiffness as measured by the SKCS. A study of the fractured endosperm of hard wheat varieties grouped according to similar rheology index values was performed using environmental scanning electron microscopy (ESEM). Differing microstructures and fracture patterns were observed between each group. Specifically, the group representing high rheology index had a greater concentration of small starch granules in prismatic cells. Samples of diverse wheat germplasm were grown at two sites and subjected to laboratory milling. Starch granule size distribution (SGSD) analysis using a laser diffraction method was undertaken on a subset of samples in triplicate representing a range in flour yield. The results supported an hypothesis for a significant influence of SGSD on flour yield of hard wheat varieties. In addition, a significant part ( R 2>0.40 ( p<0.05) at two sites) of the association appeared to be under genetic control. Results indicate a more even gradation of distributions involving an increase in the sample volume % of small granule (types B and C) and decrease in type A granules. This was associated with increased rheology index values and higher flour yield. The ratio of type A:C starch granules accounted for up to 58% ( p<0.05) of the variation in flour yield in the samples studied. Thus, rheological parameters measured using a rapid SKCS screening method can be linked to the genetic regulation of SGSD with implications for the improvement of commercial processing performance of hard wheat.

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.

Proso millet (Panicum miliaceumL.): An Evaluation of the Microstructural Changes in the Endosperm during the Malting Process by Using Scanning-Electron and Confocal Laser Microscopy

Scanning electron microscopy (SEM) and confocal scanning laser microscopy (CLSM) were used to investigate the micro-structural changes in the endosperm during the malting process. SEM was a suitable tool to characterise the microstructural constitution of starch granules in proso millet and the changes occurring during malting. An early visible degradation (after 24 h) of starch granules located in the floury endosperm, which is close to the embryo, could be observed. Due to this degradation, using confocal scanning laser microscopy, a less dense packaging of this part of the endosperm was observed. Degradation of starch granules in the vitreous endosperm was obvious at a distinct later stage of malting (78 h) and a preferred attack of small granules (>2.5 μm) was detected. Changes in protein structure could not be detected by either SEM or CLSM.

Physicochemical properties of endosperm and pericarp starches during maize development

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

Localization of a bacterial protein in starch granules of transgenic maize kernels.

The B subunit of Escherichia coli heat labile enterotoxin (LT-B) is a potent oral immunogen with potential for use as a vaccine, a carrier molecule to deliver antigens to gut-associated lymphoid tissues, and possibly an adjuvant to make coadministered vaccines more effective. LT-B produced in plants was shown to be functional and immunogenic in animals and humans. In this work, we show that maize-derived LT-B is strongly associated with starch granules in endosperm. Using immunogold labeling/electron microscopy, cell fractionation, and protein analysis techniques, we observed that LT-B protein could be detected both internally and externally in starch granules. This strong association confers an effective copurification of the antigen with the starch fraction of maize kernels, thermostability desirable in maize processing, and resistance to peptic degradation in simulated gastric fluid digests, an important attribute for an orally delivered antigen.

Mapping of a gene responsible for the difference in amylopectin structure between japonica-type and indica-type rice varieties.

The present investigation revealed that the alk and gel(t) genes, which cause the differences between a japonica rice variety Nipponbare and an indica rice variety Kasalath in terms of the disintegration of endosperm starch granules in alkali solution and their gelatinisation in a 4 M urea solution, respectively, cosegregated in backcross inbred lines derived from a cross between the two varieties. The segregation pattern of the profile for amylopectin chain-length, which was distinguished by enrichment in short chains of DP<==11 and depletion in intermediate-size chains of 12<==DP<==24 in japonica as compared with indica, was exactly the same as those of the above physico-chemical properties of starch granules, and the gene was designated as acl(t). Gene-mapping analysis showed that the starch synthase IIa ( SSIIa) gene is located at the alk locus on chromosome 6 in the rice genome. These results lead us to the possibility that different alleles of the SSIIa gene are responsible for differences in amylopectin structure between the two varieties, in that SSIIa plays a distinct role in the elongation of short chains within clusters (A+B(1) chains) of amylopectin. It is proposed that the activity of SSIIa in japonica rice is reduced in amount or functional capacity relative to the activity of this enzyme in indica rice. This, in turn, would explain why starch from japonica rice has a lower gelatinisation temperature than starch from indica rice and is more susceptible to disintegration in alkali or urea. The evidence for this hypothesis is that the alk(t), gel(t), acl(t) and SSIIa genes all map to the same locus.

Genetic Analysis of the Size of Endosperm Starch Granules in a Mapped Segregating Wheat Population

Le but de cet article est de localiser les regions du genome responsables du controle de la distribution de la taille des granules d'amidon de l'albumen de ble et de determiner l'effet de la taille sur la durete du grain dans une lignee recombinante.

Frequent Absence of GBSS 1 B Isoprotein in Endosperm Starch of Canadian Wheat Cultivars

Granule bound starch synthase 1 (GBSS 1) is an enzyme involved in amylose synthesis, and it is encoded at the waxy loci in wheat. Mutations have been reported for three GBSS1 loci in wheat. Mutations at wheat GBSS 1 loci result in the absence of GBSS 1 isoproteins with concomitant reduction in the amylose concentrations of endosperm starch. A recent survey of Canadian wheat cultivars detected the frequent absence of GBSS1 B isoprotein in endosperm starch granules. This was likely a result of many Canadian spring wheat cultivars having common parents, which have a mutation at the GBSS1 B locus.

Starch-branching enzymes preferentially associated with A-type starch granules in wheat endosperm.

Two starch granule-bound proteins (SGP), SGP-140 and SGP-145, were preferentially associated with A-type starch granules (>10 microm) in developing and mature wheat (Triticum aestivum) kernels. Immunoblotting and N-terminal sequencing suggested that the two proteins were different variants of SBEIc, a 152-kD isoform of wheat starch-branching enzyme. Both SGP-140 and SGP-145 were localized to the endosperm starch granules but were not found in the endosperm soluble fraction or pericarp starch granules younger than 15 d post anthesis (DPA). Small-size starch granules (<10 microm) initiated before 15 DPA incorporated SGP-140 and SGP-145 throughout endosperm development and grew into full-size A-type starch granules (>10 microm). In contrast, small-size starch granules harvested after 15 DPA contained only low amounts of SGP-140 and SGP-145 and developed mainly into B-type starch granules (<10 microm). Polypeptides of similar mass and immunologically related to SGP-140 and/or SGP-145 were also preferentially incorporated into A-type starch granules of barley (Hordeum vulgare), rye (Secale cereale), and triticale (x Triticosecale Wittmack) endosperm, which like wheat endosperm have a bimodal starch granule size distribution.

B‐type granule containing protrusions and interconnections between amyloplasts in developing wheat endosperm revealed by transmission electron microscopy and GFP expression

Starch granules in mature wheat endosperm show a bimodal size distribution. The formation of small starch granules in wheat endosperm cells was studied by transmission electron microscopy (TEM) and confocal laser scanning microscopy (CLSM) after expression and targeting of fluorescent protein into amyloplasts. Both techniques demonstrated the presence of protrusions emanating from A-type granules-containing amyloplasts and the presence of B-type starch granules in these evaginations. Moreover, CLSM recordings demonstrated the interconnection of the amyloplasts by these protrusions, suggesting a possible role of these protrusions in interplastid communication.

Characterization of a granule-bound starch synthase isoform found in the pericarp of wheat.

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

Characterization of the effect of heat stress on maternal and embryonic tissues of maize (Zea mays L.) kernels using scanning electron microscopy (SEM)

Heat stress during the early formative stages of maize (Zea mays L.) kernel development is detrimental to subsequent growth and grain yield. During this period, even a brief exposure to temperature above the optimum (25°C) can result in significant yield losses. The mechanisms involved in this response are not well understood, but appear to be associated with a thermally induced shift in hormone balance (cytokinin/ABA), which results in a reduction in kernel sink capacity (i.e. number of endosperm cells and starch granules formed) and the disruption of sugar metabolism and starch biosynthesis. However, the effect of heat stress on the morphology and ultra-structure of kernel component tissues (pedicel, pericarp, endosperm, and embryo) has not been studied. Therefore, the objective of our current study was to characterize heat stress induced changes in the pericarp (maternal) and the embryo (embryonic) tissues using SEM.

Location of α-amylase/subtilisin inhibitor during kernel development and germination

The location of an endogenous α-amylase/subtilisin inhibitor in developing and germinating barley (Hordeum vulgare, cv. Bonanza) was determined using immunohistochemical techniques. The inhibitor was found within protein bodies of cells containing starch granules in the starchy endosperm and embryo of developing caryopses. It could be detected as early as 2 weeks postanthesis in both organs. Upon germination, inhibitor was released from protein bodies, resulting in increased detection of the protein in regions of the starchy endosperm in which storage mobilization was occurring. Antibodies to α-amylase revealed large quantities of this protein in the same regions. Key words: α-amylase, α-amylase/subtilisin inhibitor, barley, germination, kernel development, starch.

The Major Proteins of Wheat Endosperm Starch Granules

Wheat starch contains two classes of associated proteins: proteins which are embedded within the granule and loosely associated surface proteins. The characterisation of the major proteins that are embedded in the granule are described. Gel electrophoresis on the basis of size resolved these proteins into five bands of molecular weights 60, 75, 85, 100 and 105 kDa. These polypeptides were demonstrated to be within the granule by their resistance to proteinase K digestion when granules were ungelatinised. The N-terminal sequences of these polypeptides are reported. The most prominent polypeptide is the 60 kDa granule-bound starch synthase. The N-terminal sequence obtained from the 75 kDa polypeptide shows homology to rice soluble starch synthase. The 85 kDa band was resolved into at least two types of polypeptides, one of which reacted with polyclonal antiserum to the maize branching enzyme IIb. The 100 and 105 kDa polypeptides were located only in the granule and are related, on the basis of N-terminal sequence similarity and cross-reactivity to monoclonal antibodies. SDS-PAGE and monoclonal antibody cross-reactivity experiments suggest that the 100 and 105 kDa polypeptides are absent from starch granules from all other species examined, including other cereals. It is speculated that all the major granule proteins are involved in starch biosynthesis.

Relationship Between Gelatinizing Temperature and Enthalpy of Starch from Gramineous Crops by Differential Scanning Calorimetry

AbstractIn this report endosperm starch granules from gramineous crops such as rice, barley, wheat, foxtail millet and proso millet were analyzed by Differential Scanning Calorimetry (DSC) and the relationship between gelatinized temperature and enthalpy were discussed. Peak temperatures (Tp) for gelatinization in these gramineous crops as a whole ranged from 57 to 80°C and enthalpy were distributed from 6.4 to 15.6 J/g. Crops having waxy phenotype, namely, rice, barley, foxtail millet and proso millet show a correlationship between Tp and enthalpy. A crop having no waxy phenotype such as wheat however shows no relationship between these parameters.

Molecular identification and comparison of the starch synthase bound to starch granules between endosperm and leaf blades in rice plants

Normal (nonglutinous) rice plants (Oryza sativa and O. glaberrima) contain more than 18% amylose in endosperm starch, while waxy (glutinous) plants lack it in this starch. In contrast, leaf starch contained more than 3.6% amylose even in waxy plants. SDS-PAGE analysis of proteins bound to endosperm starch granules in the normal plants revealed a single band with a Mr of 60 kd, whereas waxy plants did not exhibit a similar band. The activity of starch synthase (NDP-glucose-starch glucosyltransferase) was completely inhibited by antibody against the 60-kd protein. Thus, we conclude that the 60-kd protein is the waxy protein encoded by the Wx allele, which also plays a role in the synthesis of nonglutinous starch in endosperm tissue. In leaf blades, the proteins bound to starch granules separated into five bands with Mr's of 53.6 to 64.9 kd on SDS-PAGE. Analysis of these proteins by immunoblotting using antiserum against Wx protein and inhibition of starch synthase activity by the synthase antibody revealed that none of these proteins was homologous to Wx protein. We suggest that the synthesis of amylose in leaf blades is brought about by a protein encoded by a gene(s) different from the Wx gene expressed in the endosperm.

Comparative Levels of Waxy Gene Product of Endosperm Starch Granules of Different Rice Ecotypes

AbstractStarches of 40 rice varieties from different ecosystems and 2 amylose extender mutants were analyzed for their predominant waxy gene allele (Wxa or Wxb) through the amount of Wx gene product (60‐kdal polypeptide) by SDS‐polyacrylamide gel electrophoresis. Nonwaxy rices carrying the Wxa and Wxb alleles were clearly differentiated by two distinct regression equations correlating AC and the amount of Wx gene product. Indica, samba, and tropical upland rices hat the Wxa allele and contained about three times as much Wx gene protein at the same AC as japonica and long‐grain US rices carrying the Wxb allele. Indica‐japonica, bulu, and basmati‐type rices showed selection for either Wxa or Wxb. Identification of the predominant Wx allele provides an alternative method for classifying nonwaxy rice grains into indica or japonica type, and complements the embryo isozyme classification. Results of the two methods are generally consistent.