The effect of waxy mutations on the granule-bound starch synthases of barley and maize endosperms

The effects of waxy mutations on starch-granule-bound starch synthases (EC 2.4.1.18) in the developing endosperm of barley (Hordeum vulgare L.) and maize (Zea mays L.) have been investigated. Three granule-bound starch synthases in barley endosperm were identified by use of antibodies to known starch synthases, by reconstitution and assay of individual proteins from sodium dodecyl sulphate-polyacrylamide gels of granule-bound proteins, and by partial purification of proteins released by enzymic digestion of starch. These are proteins of 60, 77 and 90 kDa. Use of antibodies to known starch synthases and partial purification of proteins released by enzymic digestion of starch indicated that there may be at least four granule-bound starch synthases in maize endosperm: proteins of 59, 74, 77 and 83 kDa. Mutations at the waxy loci of both species affected only the 60- (barley) and 59-(maize) kDa isoforms. No evidence was found that other putative isoforms are altered in abundance or activity by the mutations. The contribution of our results to understanding of the starch synthase activity of intact starch granules and the mechanism of amylose synthesis is discussed.We are very grateful to Dr. Roger Ellis (SCRI, Dundee, Scotland) for the gift of barley seeds, and to Drs Roger Ellis, Alan Schulman and Cathie Martin for helpful advice and comments during the course of this work.     

Nutritional Interdependence Among Rumen Bacteria, Bacteroides amylophilus, Megasphaera elsdenii, and Ruminococcus albus

Nutritional interdependence among three representatives of rumen bacteria, Bacteroides amylophilus, Megasphaera elsdenii, and Ruminococcus albus, was studied with a basal medium consisting of minerals, vitamins, cysteine hydrochloride, and NH₄⁺. B. amylophilus grew well in the basal medium supplemented with starch and produced branched-chain amino acids after growth ceased. When cocultured with B. amylophilus in the basal medium supplemented with starch and glucose, amino acid-dependent M. elsdenii produced an appreciable amount of branched-chain fatty acids, which are essential growth factors for cellulolytic R. albus. A small addition of starch (0.1 to 0.3%) to the basal medium containing glucose and cellobiose brought about successive growth of the three species in the order of B. amylophilus, M. elsdenii, and R. albus, and successive growth was substantiated by the formation of branched-chain amino acids and fatty acids in the culture. Supplementation with 0.5% starch, however, failed to support the growth of R. albus. On the basis of these results, the effects of supplementary starch or branched-chain fatty acids on cellulose digestion in the rumen was discussed.     

Effects of vanillin, quillaja saponin, and essential oils on in vitro fermentation and protein-degrading microorganisms of the rumen

This study investigated the effects of vanillin on methanogenesis and rumen fermentation, and the responses of ruminal protein-degrading bacteria to vanillin (at concentrations of 0, 0.76 and 1.52 g/L), essential oils (clove oil, 1 g/L; origanum oil, 0.50 g/L, and peppermint oil, 1 g/L), and quillaja saponin (at concentration of 0 and 6 g/L) in vitro. Methane production, degradabilities of feed substrate, and ammonia concentration decreased linearly with increasing doses of vanillin. Concentration of total volatile fatty acids also decreased, whereas proportion of butyrate tended to increase linearly with increasing doses of vanillin. Protozoa population decreased, but abundances of Ruminococcus flavefaciens, Prevotella bryantii, Butyrivibrio fibrisolvens, Prevotella ruminicola, Clostridium aminophilum, and Ruminobacter amylophilus increased with increasing doses of vanillin. Origanum and clove oils resulted in lower ammonia concentrations compared to control and peppermint oil. All the tested essential oils decreased abundances of protozoa, Selenomonas ruminantium, R. amylophilus, P. ruminicola and P. bryantii, with the largest decrease resulted from origanum oil followed by clove oil and peppermint oil. The abundances of Megasphaera elsdenii, C. aminophilum, and Clostridium sticklandii were deceased by origanum oil while that of B. fibrisolvens was lowered by both origanum and clove oils. Saponin decreased ammonia concentration and protozoal population, but increased the abundances of S. ruminantium, R. amylophilus, P. ruminicola, and P. bryantii, though the magnitude was small (less than one log unit). The results suggest that reduction of ammonia production by vanillin and saponin may not be caused by direct inhibition of major known proteolytic bacteria, and essential oils can have different inhibitory effects on different proteolytic bacteria, resulting in varying reduction in ammonia production.     

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.     

In Vitro Utilization of Amylopectin and High-Amylose Maize (Amylomaize) Starch Granules by Human Colonic Bacteria

It has been well established that a certain amount of ingested starch can escape digestion in the human small intestine and consequently enters the large intestine, where it may serve as a carbon source for bacterial fermentation. Thirty-eight types of human colonic bacteria were screened for their capacity to utilize soluble starch, gelatinized amylopectin maize starch, and high-amylose maize starch granules by measuring the clear zones on starch agar plates. The six cultures which produced clear zones on amylopectin maize starch- containing plates were selected for further studies for utilization of amylopectin maize starch and high-amylose maize starch granules A (amylose; Sigma) and B (Culture Pro 958N). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to detect bacterial starch-degrading enzymes. It was demonstrated that Bifidobacterium spp., Bacteroides spp., Fusobacterium spp., and strains of Eubacterium, Clostridium, Streptococcus, and Propionibacterium could hydrolyze the gelatinized amylopectin maize starch, while only Bifidobacterium spp. and Clostridium butyricum could efficiently utilize high-amylose maize starch granules. In fact, C. butyricum and Bifidobacterium spp. had higher specific growth rates in the autoclaved medium containing high-amylose maize starch granules and hydrolyzed 80 and 40% of the amylose, respectively. Starch-degrading enzymes were cell bound on Bifidobacterium and Bacteroides cells and were extracellular for C. butyricum. Active staining for starch-degrading enzymes on SDS-PAGE gels showed that the Bifidobacterium cells produced several starch-degrading enzymes with high relative molecular (M_r) weights (>160,000), medium-sized relative molecular weights (>66,000), and low relative molecular weights (<66,000). It was concluded that Bifidobacterium spp. and C. butyricum degraded and utilized granules of amylomaize starch.     

Purification of a debranching enzyme (R-enzyme) from malted barley,and the role of the enzyme in the digestion of starch granules during the germination of barley seeds

A debranching enzyme (R-enzyme or pullulan-6-glucanohydrolase, EC3.2.1.41), free from contaminating carbohydrases and homogeneous by poly(acrylamide) disc-gel electrophoresis, has been purified from malted barley. A partially purified preparation of this enzyme (3.1 units/mg of protein) accelerated the rate of digestion of barley-starch granules by the action of purified alpha and beta amylases to the same extent as was effected by the dialyzed, crude extract from malted barley. Contrary to expectation, the debranching enzyme, purified to homogeneity (10 units/mg of protein), had very little accelerating effect. These results indicate that a factor or factors, which may be maltase or α-d-glucosidase and were lost during the purification of the debranching enzyme, may play a role in the digestion of starch granules by the dialyzed, crude extract from malted barley in vitro and by enzymes in the endosperm of germinating barley seeds in vivo. The debranching enzymes, including barley-malt R-enzyme, Aerobacter pullulanase, and Pseudomonas isoamylase, did not digest starch granules to a detecble extent.     

Surface Localization of Zein Storage Proteins in Starch Granules from Maize Endosperm : Proteolytic Removal by Thermolysin and in Vitro Cross-Linking of Granule-Associated Polypeptides

Starch granules from maize (Zea mays) contain a characteristic group of polypeptides that are tightly associated with the starch matrix (C. Mu-Forster, R. Huang, J.R. Powers, R.W. Harriman, M. Knight, G.W. Singletary, P.L. Keeling, B.P. Wasserman [1996] Plant Physiol 111: 821–829). Zeins comprise about 50% of the granule-associated proteins, and in this study their spatial distribution within the starch granule was determined. Proteolysis of starch granules at subgelatinization temperatures using the thermophilic protease thermolysin led to selective removal of the zeins, whereas granule-associated proteins of 32 kD or above, including the waxy protein, starch synthase I, and starch-branching enzyme IIb, remained refractory to proteolysis. Granule-associated proteins from maize are therefore composed of two distinct classes, the surface-localized zeins of 10 to 27 kD and the granule-intrinsic proteins of 32 kD or higher. The origin of surface-localized δ-zein was probed by comparing δ-zein levels of starch granules obtained from homogenized whole endosperm with granules isolated from amyloplasts. Starch granules from amyloplasts contained markedly lower levels of δ-zein relative to granules prepared from whole endosperm, thus indicating that δ-zein adheres to granule surfaces after disruption of the amyloplast envelope. Cross-linking experiments show that the zeins are deposited on the granule surface as aggregates. In contrast, the granule-intrinsic proteins are prone to covalent modification, but do not form intermolecular cross-links. We conclude that individual granule intrinsic proteins exist as monomers and are not deposited in the form of multimeric clusters within the starch matrix.It has long been known that starch granules contain bound polypeptides, with protein levels of isolated starch granules from maize (Zea mays) ranging from 0.3 to 1.0% based upon measurement of N₂ (May, 1987). A recent study by our laboratory demonstrates that isolated starch granules from maize contain several dozen strongly bound polypeptides (Mu-Forster et al., 1996). The granule-associated proteins include starch-biosynthetic enzymes such as the waxy protein, SSI, and SBEIIb. These polypeptides are not removed from intact starch granules by protease treatment or detergent washing; therefore, they are believed to bind to the starch and to become irreversibly entrapped within the starch matrix.Based upon staining intensities of polypeptides extracted from the starch granule (Mu-Forster et al., 1996), approximately one-half of the granule-associated proteins in maize consist of low-molecular-mass polypeptides ranging between 10 and 27 kD. These bands fall within the size range displayed by the zein storage proteins, however, the spatial distribution of these polypeptides within the starch granule is unknown. Zeins have been defined as alcohol-soluble proteins that occur principally in protein bodies of maize endosperm and that may or may not require reduction before extraction (Wilson, 1991). The association of zeins with starch granules during endosperm development would not be expected because zein genes do not contain transit peptides that would target these proteins through the amyloplast envelope into the amyloplast stroma.The objective of this study was to establish the topology of granule-associated zeins in starch granules from maize endosperm. To accomplish this, it was necessary to distinguish between surface-localized and internalized polypeptides. Our working hypothesis defines polypeptides localized at the starch granule surface as those that are susceptible to hydrolysis upon treatment of intact granules with exogenous proteases. Conversely, internal granule proteins are defined as those that (a) become susceptible to proteolysis only following thermal disruption of the starch matrix, and (b) resist extraction by 2% SDS at room temperatures (Denyer et al., 1993; Rahman et al., 1995; Mu-Forster et al., 1996).In this study we were able to distinguish between surface-localized and internalized granule-associated polypeptides in starch granules from maize endosperm by use of the thermophilic protease thermolysin. Thermolysin is well suited for this purpose because it is highly active at starch-gelatinization temperatures, and has also been shown to effectively hydrolyze hydrophobic proteins located at the surfaces of chloroplasts and other subcellular organelles (Cline et al., 1984; Xu and Chitnis, 1995). Upon extended incubation of intact starch granules with thermolysin at subgelatinization temperatures, we found that zeins were selectively removed from the starch granule surface. All other granule-associated polypeptides remained inaccessible to proteolytic attack or to extraction by 2% SDS, unless the starch matrix was first disrupted by gelatinization. Our results distinguish between the surface-localized and granule-intrinsic proteins of maize endosperm, and establish that zeins are localized at the starch-granule surface. In addition, cross-linking experiments were conducted to determine nearest-neighbor relationships among zein subunits localized at the granule surface and granule intrinsic polypeptides localized within the starch matrix.     

Starch granule initiation and growth are altered in barley mutants that lack isoamylase activity

Two mutant lines of barley, Risø 17 and Notch‐2, were found to accumulate phytoglycogen in the grain. Like the sugary mutants of maize and rice, these phytoglycogen‐accumulating mutants of barley lack isoamylase activity in the developing endosperm. The mutants were shown to be allelic, and to have lesions in the isoamylase gene, isa1 that account for the absence of this enzyme. As well as causing a reduction in endosperm starch content, the mutations have a profound effect on the structure, number and timing of initiation of starch granules. There are no normal A‐type or B‐type granules in the mutants. The mutants have a greater number of starch granules per plastid than the wild‐type and, particularly in Risø 17, this leads to the appearance of compound starch granules. These results suggest that, as well as suppressing phytoglycogen synthesis, isoamylase in the wild‐type endosperm plays a role in determining the number, and hence the form, of starch granules.     

High amylose mutants of rice,Oryza sativa L.

Summary Five mutant lines of rice with increased amylose content in starch granules were identified among floury endosperm mutants. The amylose contents of the mutants ranged from 29.4% to 35.4% and were about twice as high as that of the normal counterpart. Starch properties of the high amylose mutants were analyzed by column chromatography, X-ray diffractometry, photopastegraphy and scanning electron microscopy. The high amylose mutants produced longer unit chains of amylopectin than those of the normal counterpart as well as an increased amount of amylose. A X-ray diffractogram of starch in the mutant was characterized by a type B pattern, while that in the normal counterpart showed a type A pattern which is typical for starches of common cereals. The temperatures at the initiation of gelatinization of the mutants were much higher than that for the normal counterpart. The endosperm cells of the mutant were loosely packed with irregular round-shaped starch granules, whereas those of the normal counterpart were densely packed with polyhedral starch granules. Judging from the results obtained, it was concluded that starch properties of the high amylose mutants of rice were similar to those of the amylose-extender (ae) mutant of maize.     

RFLP-based mapping of three mutant loci in rye (Secale cereale L.) and their relation to homoeologous loci within the Gramineae

 Three mutant loci of rye determining absence of ligules (al), waxless plant (wa1) and waxy endosperm (Wx) characters were mapped in a single F₂ population, comprising 84 individual plants. The three loci could be clearly tagged in relation to 7 (al on chromosome 2R), 4 (wa1 on chromosome 7R) or 6 (Wx on chromosome 4R) RFLP markers. The mapping data are compared with existing data for homoeologous regions containing equivalent mutants of wheat, barley, rice and maize. It is shown that the loci analysed are highly conserved across the cereal species, including rye. Received: 14 March 1997 / Accepted: 21 March 1997     

Effects of rhizobial inoculation, cropping systems and growth stages on endophytic bacterial community of soybean roots

Interspecific interactions and soil nitrogen supply levels affect intercropping productivity. We hypothesized that interspecific competition can be alleviated by increasing N application rate and yield advantage can be obtained in competitive systems. A field experiment was conducted in Wuwei, Gansu province in 2007 and 2008 to study intercropping of faba bean/maize, wheat/maize, barley/maize and the corresponding monocultures of faba bean (Vicia faba L.), wheat (Triticum aestivum L.), barley (Hordeum vulgare L.) and maize (Zea mays L.) with N application rates of 0, 75, 150, 225 and 300 kg N ha^?1. Total land equivalent ratios (TLER) were 1.22 for faba bean/maize, 1.16 for wheat/maize, and 1.13 for barley/maize intercropping over the 2-year study period. Maize was overyielding when intercropped with faba bean, but underyielding when intercropped with wheat or barley according to partial land equivalent ratios (PLER) based on grain yields of individual crops in intercropping and sole cropping. There was an interspecific facilitation between intercropped faba bean and maize, and interspecific competition between maize and either wheat or barley. The underyielding of maize was higher when intercropped with barley than with wheat. Fertilizer N alleviated competitive interactions in intercrops with adequate fertilizer N at 225 kg ha^?1. Yield advantage of intercropping can be acquired with adequate nitrogen supply, even in an intensive competitive system such as barley/maize intercropping. This is important when using intercropping to develop intensive farming systems with high inputs and high outputs.     

Starch-synthesizing Enzymes in the Endosperm and Pollen of Maize

Two mutations, amylose-extender and waxy, which affect the proportion of amylose and amylopectin of starch synthesized in the endosperm of maize (Zea mays L.) seeds, are also expressed in the pollen. However, most mutations that affect starch synthesis in the maize endosperm are not expressed in the pollen. In an attempt to understand the nonconcordance between the endosperm and pollen, extracts of mature pollen grains were assayed for a number of the enzymes possibly implicated in starch synthesis in the endosperm. Sucrose synthetase (sucrose-UDP glucosyl transferase, EC 2.4.1.13) activity was not detectable in either mature or immature pollen grains of nonmutant maize, but both bound and soluble invertase (EC 3.2.1.26) exhibited much greater specific activity (per milligram protein) in pollen extracts than in 22-day-old endosperm extracts. Phosphorylase (EC 2.4.1.1) activity was also higher in pollen than in endosperm extracts. ADP-Glucose pyrophosphorylase (EC 2.7.7.27) activity was much lower in pollen than endosperm extracts, but mutations that drastically reduced ADP-glucose pyrophosphorylase activity in the endosperm (brittle-2 and shrunken-2) did not markedly affect enzymic activity in the pollen. Specific activities of other enzymes implicated in starch synthesis were similar in endosperm and pollen extracts.     

披碱草属及其近源属植物种子胚乳细胞多样性研究

披碱草属不仅是小麦、大麦等作物的重要基因库,也是优良牧草的重要组成部分。胚乳是麦类作物种子的重要组成部分,其重量占籽粒重量在90%以上。胚乳特性是一个相对稳定的遗传性状,可以将胚乳特性作为植物分类和系统关系的一个指标。胚乳细胞特征作为植物的一类微形态特征,在麦类植物系统分类与进化研究中具有一定的价值。该研究对小麦族的6个披碱草属(Elymus)物种、2个拟鹅观草属(Pseudoroegneria)物种、1个大麦属(Hordeum)物种和2个冰草属(Agropyron)物种,共4属11份材料的胚乳细胞特征进行解剖观察并测量相关指标。结果表明:(1)不同属、种的植物种子胚乳细胞之间存在丰富的多样性,属间差异大于属内种间差异;(2)同样具有P染色体组的Agropyron cristatum和A.mongolicum与同样具有St染色体组的Pseudoroegneria libanotica和P.spicata各自在细胞形状和大小上的差异很小;(3)不同物种的胚乳细胞在大小、形状和数量上均表现出差异,但不能很好地反映属以及基因组间的差异,研究结果为揭示披碱草属植物的系统关系提供了胚乳细胞方面的证据,同时也为利用该属植物改良麦类作物品质积累资料。     

Direct fermentation of various pure and crude starchy substrates to L(+) lactic acid using Lactobacillus amylophilus GV6

Lactobacillus amylophilus GV6 fermented a variety of pure and natural starches directly to L(+) lactic acid. Starch to lactic acid conversion efficiency was more than 90% by strain GV6 at low substrate concentrations with all starches. The strain GV6 produced high yields of lactic acid per g of substrate utilized with pure starches such as soluble starch, corn starch, and potato starch, yielding 92–96% at low substrate concentrations in 2 days and 78–89% at high substrate (10%) concentrations in 4–6 days. Strain GV6 also produced high yields of lactic acid per g of substrate utilized with crude starchy substrates such as wheat flour, sorghum flour, cassava flour, rice flour and barley flour yielding 90–93% at low substrate concentrations in 2 days and 80% or more at high substrate concentrations in 6–7 days. Lactic acid yields by L. amylophilus GV6 with pure starches were comparable when low cost crude starchy substrates were used. Lactic acid productivity by strain GV6 is higher than for any other previously reported strains of L. amylophilus.     

Programmed cell death during endosperm development

The endosperm of cereals functions as a storage tissue in which the majority of starch and seed storage proteins are synthesized. During its development, cereal endosperm initiates a cell death program that eventually affects the entire tissue with the exception of the outermost cells, which differentiate into the aleurone layer and remain living in the mature seed. To date, the cell death program has been described for maize and wheat endosperm, which exhibits common and unique elements for each species. The progression of endosperm programmed cell death (PCD) in both species is accompanied by an increase in nuclease activity and the internucleosomal degradation of nuclear DNA, hallmarks of apoptosis in animals. Moreover, ethylene and abscisic acid are key to mediating PCD in cereal endosperm. The progression of the cell death program in developing maize endosperm follows a highly organized pattern whereas in wheat endosperm, PCD initiates stochastically. Although the essential characteristics of cereal endosperm PCD are now known, the molecular mechanisms responsible for its execution remain to be identified.     

Effects of Ammonia and Amino Acids on the Growth and Proteolytic Activity of Three Species of Rumen Bacteria: Prevotella albensis, Butyrivibrio fibrisolvens, and Streptococcus bovis

The addition of increasing physiological concentrations of ammonia or amino acids had distinct effects on the growth and proteolytic activity of Streptococcus bovis JB1, Prevotella albensis, and Butyrivibrio fibrisolvens DSM3071. The growth of S. bovis and B. fibrisolvens was enhanced by NH₃ and AA, and that of P. albensis was reduced compared with a control with protein as the sole source of nitrogen. The proteolytic activity of S. bovis and P. albensis was reduced, but that of B. fibrisolvens was improved. NH₃ seemed to act mainly on the cell-associated fraction of the proteolytic activity, while the action of AA was not specific. In the rumen the proteolytic activity of S. bovis and P. albensis would be optimal at low concentrations of NH₃ or AA (<0.05 and <0.27 g/L respectively). In contrast, B. fibrisolvens would need higher concentrations (0.5 g/L of NH₃ or 2.7 g/L of AA). It can be assumed that these bacteria will grow in different ecological niches. Received: 5 October 1999 / Accepted: 30 December 1999     

Estimation of the in situ degradation of the washout fraction of starch by using a modified in situ protocol and in vitro measurements

The in situ degradation of the washout fraction of starch in six feed ingredients (i.e. barley, faba beans, maize, oats, peas and wheat) was studied by using a modified in situ protocol and in vitro measurements. In comparison with the washing machine method, the modified protocol comprises a milder rinsing method to reduce particulate loss during rinsing. The modified method markedly reduced the average washout fraction of starch in these products from 0.333 to 0.042 g/g. Applying the modified rinsing method, the fractional degradation rate (k _d) of starch in barley, oats and wheat decreased from on average 0.327 to 0.144 h⁻¹ whereas for faba beans, peas and maize no differences in k _d were observed compared with the traditional washing machine rinsing. For barley, maize and wheat, the difference in non-fermented starch in the residue between both rinsing methods during the first 4 h of incubation increased, which indicates secondary particle loss. The average effective degradation of starch decreased from 0.761 to 0.572 g/g when using the new rinsing method and to 0.494 g/g when applying a correction for particulate matter loss during incubation. The in vitro k _d of starch in the non-washout fraction did not differ from that in the total product. The calculated ratio between the k _d of starch in the washout and non-washout fraction was on average 1.59 and varied between 0.96 for oats and 2.39 for maize. The fractional rate of gas production was significantly different between the total product and the non-washout fraction. For all products, except oats, this rate of gas production was larger for the total product compared with the non-washout fraction whereas for oats the opposite was observed. The rate of increase in gas production was, especially for grains, strongly correlated with the in vitro k _d of starch. The results of the present study do not support the assumption used in several feed evaluation systems that the degradation of the washout fraction of starch in the rumen is much faster than that of the non-washout fraction.