Block-surface staining for differentiation of starch and cell walls in wheat endosperm.

A staining technique for differentiating starch granules and cell walls was developed for computer-assisted studies of starch granule distribution in cells of wheat (Triticum aestivum L.) caryopses. Blocks of embedded caryopses were sectioned, exposing the endosperm tissue, and stained with iodine potassium iodide (IKI) and Calcofluor White. Excessive tissue hydration during staining was avoided by using stains prepared in 80% ethanol and using short staining times. The IKI quenched background fluorescence which facilitated the use of higher concentrations of Calcofluor White. Cell wall definition was improved with the IKI-Calcofluor staining combination compared to Calcofluor alone. The high contrast between darkly stained starch granules and fluorescent cell walls permitted computer assisted analysis of data from selected hard and soft wheat varieties. The ratio of starch granule area to cell area was similar for both wheat classes. The starch granule sizes ranged from 2.1 microns 3 to 22,000 microns 3 with approximately 90% of the granules measuring less than 752 microns 3 (ca. 11 microns in diameter). Hard wheat samples had a greater number of small starch granules and a lower mean starch granule area compared to the soft wheat varieties tested. The starch size distribution curve was bimodal for both the hard and soft wheat varieties. Three-dimensional starch size distribution was measured for four cells near the central cheek region of a single caryopsis. The percentage of small granules was higher at the ends than at the mid-section of the cells.     

不同供水条件对小麦强、弱势籽粒中淀粉粒度分布的影响

以3个淀粉含量不同的冬小麦品种山农12、鲁麦21和济南17为材料,设灌溉和旱作2种栽培处理,对不同水分条件下小麦强、弱势籽粒中淀粉粒的体积、数目和表面积的分布特征进行了研究.结果表明,小麦强、弱势籽粒均含有A(＞9.8 μm)、B(2.0～9.8 μm)、C(＜2.0 μm)3种类型的淀粉粒,但不同类型淀粉粒的分布状况存在明显差异.在强势籽粒中,淀粉粒的体积和表面积分布均表现为三峰分布,而弱势籽粒中淀粉粒的体积和表面积分布则表现为双峰分布.与弱势粒相比较,强势粒中C型淀粉粒(＜2.0 μm)的体积百分比为7.25%～9.31%,表面积百分比为34.88%～41.51%,而弱势粒的体积和表面积百分比分别为5.33%～6.40%和26.31%～33.54%.强、弱势籽粒中＜0.6 μm和0.6～2.0 μm范围内的淀粉粒数目存在明显差异,强势粒为1.86%～6.13%和83.77%～87.77%,而弱势粒为25.72%～37.42%和52.77%～58.48%.与灌溉栽培相比较,旱作栽培条件下籽粒中B、C型淀粉粒体积和表面积百分比显著增加,而A型淀粉粒体积和表面积显著减少;弱势粒中＜0.6 μm的淀粉粒数目显著增加,强势籽粒中淀粉粒的数目无显著变化.与弱势粒相比较,强势粒中的蛋白质含量较高,C型淀粉粒的体积和表面积所占比例较大,而强势粒中的淀粉含量较低,且A、B型淀粉粒比例也较小.与灌溉栽培相比较,旱作栽培条件下强、弱势籽粒中B、C型淀粉粒体积和表面积百分比增加,蛋白质含量也显著增加,淀粉含量降低.表明水分亏缺能提高籽粒中B、C型淀粉粒体积和表面积百分比及蛋白质含量.     

Soft and Hard Textured Wheat Differ in Starch Properties as Indicated by Trimodal Distribution,Morphology, Thermal and Crystalline Properties

Starch and proteins are major components in the wheat endosperm that affect its end product quality. Between the two textural classes of wheat i.e. hard and soft, starch granules are loosely bound with the lipids and proteins in soft wheat due to higher expression of interfering grain softness proteins. It might have impact on starch granules properties. In this work for the first time the physiochemical and structural properties of different sized starch granules (A-, B- and C-granules) were studied to understand the differences in starches with respect to soft and hard wheat. A-, B- and C-type granules were separated with >95% purity. Average number and proportion of A-, B-, and C-type granules was 18%, 56%, 26% and 76%, 19%, 5% respectively. All had symmetrical birefringence pattern with varied intensity. All displayed typical A-type crystallites. A-type granules also showed V-type crystallinity that is indicative of starch complexes with lipids and proteins. Granules differing in gelatinization temperature (ΔH) and transition temperature (ΔT), showed different enthalpy changes during heating. Substitution analysis indicated differences in relative substitution pattern of different starch granules. Birefringence, percentage crystallinity, transmittance, gelatinization enthalpy and substitution decreased in order of A>B>C being higher in hard wheat than soft wheat. Amylose content decreased in order of A>B>C being higher in soft wheat than hard wheat. Reconstitution experiment showed that starch properties could be manipulated by changing the composition of starch granules. Addition of A-granules to total starch significantly affected its thermal properties. Effect of A-granule addition was higher than B- and C-granules. Transmittance of the starch granules paste showed that starch granules of hard wheat formed clear paste. These results suggested that in addition to differences in protein concentration, hard and soft wheat lines have differences in starch composition also.     

Detection of areas of wall differentiation in fungi using fluorescent staining

Fungal colonies were stained with a fluorescent brightner, Calcofluor White M2R New. When viewed using ultraviolet light certain hyphal regions showed intense fluorescence, whereas others showed a lower intensity fluorescence. The apical 10 μm of leadingBotrytis cinerea hyphae showed intense fluorescence. Developing septa and side branches also showed this intense localised fluorescence. Calcofluor staining of hyphae and their subsequent growth illustrated the phenomenon of tip extension.     

不同筋力小麦品种在不同生态环境下籽粒淀粉糊化特性分析

以不同筋力小麦品种 强筋、中筋、弱筋 在河南省5个纬度点 32°N～36°N 种植为材料,利用快速粘度分析仪 RVA 研究了不同筋力品种的淀粉糊化特性,结果表明:弱筋小麦品种的淀粉糊化特性和强筋、中筋小麦品种有明显的差异;弱筋小麦品种更易受环境条件的影响;两个中筋小麦品种的表现不一致,其中豫麦70的环境变异程度大于豫麦49的变异程度;弱筋小麦品种、强筋小麦品种以及中筋品种豫70的峰值粘度、低谷粘度、最终粘度均随着纬度从北到南呈逐渐下降的趋势;所有品种的淀粉糊化指标均在信阳点表现为最低.     

The Number and Sizes of Starch Granules in the Wheat Endosperm, and their Association with Grain Weight

The number and size of starch granules was measured in developingand mature endosperms of two wheat varieties, Chinese Springand Spica. Chromosomal effects on particular aspects of starchgranule size and number were detected in the analysis of disomicgenotypes derived from reciprocal monosomic hybrids betweenthe two parental varieties. Among these genotypes, a greaterweight per endosperm cell was associated with a greater volumeper A-type starch granule. It is suggested that the number percell and volume of these large starch granules are the majordeterminants of endosperm cell weight, and there exists separategenetic control of these parameters. It should therefore bepossible genetically to combine these attributes to achievedirected increases in mature grain weight. Triticum aestivum, grain weight, starch granules     

Regulation of Grain Weight by Supply of Assimilates and Starch Granule Development in Three Winter Wheat Varieties

On removing the top half of the ear (halving) on several datesafter anthesis, dry weight per grain increased in three winterwheat (Triticum aestivum L.) varieties, in two pot experiments;the increase was greater with early than with late halving.The variety Splendeur had a lower dry weight and water percentagein grains than either Hobbit or Maris Huntsman. The ratio ofthe green area integrated over the post-anthesis period to thenumber of grains per ear (green area per grain) was highestin Splendeur and lowest in Hobbit in the first experiment; inthe second, Splendeur gave a lower ratio than the other twovarieties, which showed similar values. The green area per grainwas greater the earlier the ear was halved. The number of A-typestarch granules per endosperm, but not the volume per A granule(modal volume) and the modal volume of B starch granules, butnot their number, increased to a greater extent with early thanwith late halving. In Splendeur the grains had fewer A starchgranules, although these were of greater modal volume than inHobbit and Maris Huntsman and a number of B starch granulessimilar to the other two varieties, but of smaller modal volume.Maris Huntsman had more A granules than Hobbit, but with smallermodal volume. Dry weight per grain increased linearly with thenumber of A starch granules per endosperm, which in turn increasedasymptotically with green area per grain. The regressions forthe three varieties differed significantly. The influence ofthe supply of assimilates and the capacity for starch granuleformation in the regulation of grain weight is discussed. Key words: Grain weight, starch granules, assimilate supply, variety, wheat     

Starch-bound 2S proteins and kernel texture in einkorn, <Emphasis Type="Italic">Triticum</Emphasis> <Emphasis Type="Italic">monococcum</Emphasis> ssp <Emphasis Type="Italic">monococcum</Emphasis>

The starch granule proteins from 113 einkorn wheat (Triticum monococcum ssp monococcum) accessions were analyzed by acidic, polyacrylamide gel electrophoresis (A-PAGE), and two-dimensional A-PAGE x SDS-PAGE. All accessions were confirmed to contain equal amounts of two polypeptide chains corresponding to puroindoline B (Pin-B), as well as a prominent component plus a faint band corresponding to puroindoline A (Pin-A). When compared with soft-textured common wheat, “monococcum” accessions showed an increase of 3.2- and 2.7-fold in Pin-A and Pin-B levels on the starch granules, respectively. In addition, all accessions contained a novel component of the 2S super-family of seed proteins named Einkorn Trypsin Inhibitor (ETI), which was found to be encoded as a pre-protein 148 residues long. Wild-type ETI encoded by allele Eti-A ^m 1a and “valine-type” ETI encoded by allele Eti-A ^m 1b, which occurred in 107 and six einkorn accessions, respectively, were found to accumulate on starch granules as a mature protein of 121 amino acids with a hydrophobic central domain. The einkorn accessions exhibited an average SKCS index as low as −2.05 ± 11.4, which is typical of extra-soft kernels. The total surface area of starch granules in “monococcum” wheat, as determined by visual assessments in counting chambers, was estimated at 764 mm²/mg of starch, and was about 1.5 times higher than that for common wheat. The results are discussed in relation to the identification of factors that cause the extra-soft texture of einkorn kernels.     

Kinetic Analysis of Glucoamylase-Catalyzed Hydrolysis of Starch Granules from Various Botanical Sources

The kinetics of glucoamylase-catalyzed hydrolysis of starch granules from six different botanical sources (rice, wheat, maize, cassava, sweet potato, and potato) was studied by the use of an electrochemical glucose sensor. A higher rate of hydrolysis was obtained as a smaller size of starch granules was used. The adsorbed amount of glucoamylase on the granule surface per unit area did not vary very much with the type of starch granules examined, while the catalytic constants of the adsorbed enzyme (k ₀) were determined to be 23.3±4.4, 14.8±6.0, 6.2±1.8, 7.1±4.1, 4.6±3.0, and 1.6±0.6 s^?1 for rice, wheat, maize, cassava, sweet potato, and potato respectively, showing that k ₀ was largely influenced by the type of starch granules. A comparison of the k ₀-values in relation to the crystalline structure of the starch granules suggested that k ₀ increases as the crystalline structure becomes dense.     

A comparison of the highly selective fluorescence staining of fungi in tissue sections with Uvitex 2B and Calcofluor White M2R

Summary The selective fluorescence staining of two fungi,Candida albicans andBlastomyces dermatitides, with Uvitex 2B and Calcofluor White M2R was studied in deparaffinized and frozen sections of mouse kidney and lung. Both fluorochromes emitted maximally at about 430nm, independent of the mounting media (Kaiser's gelatin or Entellan). In addition to fungi, both fluorochromes also stained elastic fibres. The fluorescence intensity remained unchanged after storage of sections for more than 6 months in conventional slide boxes. the two fluorochromes showed the following differences: Calcofluor faded 1.25 times faster than Uvitex when illuminated with ultraviolet light. Calcofluor showed a greater affinity for tissues in general, and red cells and renal tubular casts in particular. Counterstaining of deparaffinized sections with Hemalum and Eosin reduced the fungi fluorescence and suppressed the general background fluorescence. However, it led to an intensification of Eosin staining and the fluorescence of red cells in Calcofluorstained sections but not in Uvitex-stained ones. Similarly, the background fluorescence in frozen sections was reduced by Evans Blue, although elastic fibres still fluoresced after staining with Calcofluor. The degree of staining selectivity, and thus the contrast produced within a histological specimen, was greater with Uvitex 2B than with Calcofluor White M2R.     

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.     

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

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

小麦籽粒发育及淀粉晶体特性对花后高温胁迫的响应

选用2个品质类型和成熟期不同的新疆主栽小麦品种‘新春11号’和‘新春39号’,分别进行花后灌浆早期高温(花后5~8d,32℃,T_1)和中期高温(花后15~18d,38℃,T_2)处理,分析花后高温对小麦籽粒发育及淀粉晶体的影响。结果显示:(1)T_1处理明显降低了两品种籽粒长度和粒重,而T_2处理显著影响籽粒宽度和厚度;高温处理虽然降低了籽粒灌浆速率,但两品种灌浆最大峰值出现时间均在花后18d。(2)T_1处理对小麦籽粒A型淀粉粒形态的影响较大,中熟品种‘新春11号’的A型淀粉粒表面在花后10d时可观察到微孔,在花后15~20d时其粒径明显小于同期对照,在花后20~25d时淀粉粒表面压痕增多且A、B型淀粉粒表面出现明显缢缩;而早熟品种‘新春39号’淀粉粒形态和粒径大小受花后高温的影响相对较小。(3)两品种在不同高温处理下,其淀粉粒晶体特性衍射峰出现的位置相同,但淀粉粒的尖峰强度不同,表明高温胁迫不影响淀粉粒的晶体类型,但可能改变了淀粉粒内部的层状结构。研究表明,花后早期高温不仅对小麦籽粒外部形态有较大的影响,同时也影响到籽粒内部淀粉粒的形态和晶体的特性。     

Effect of starch addition on the biological conversion and microbial community in a methanol-fed UASB reactor during long-term continuous operation

The effect of starch addition on the microbial composition and the biological conversion was investigated using two upflow anaerobic sludge bracket (UASB) reactors treating methanolic wastewater: one reactor was operated with starch addition, and another reactor was operated without starch addition. Approximately 300 days of operation were performed at 30 kg COD/m³/d, and then, the organic load of the reactors was gradually increased to 120 kg COD/m³/d. Successful operation was achieved at 30 kg COD/m³/d in both reactors; however, the methanol-fed reactor did not perform well at 120 kg COD/m³/d while the methanol-starch-fed reactor did. The granule analysis revealed the granule developed further only in the methanol-starch-fed reactor. The results of the microbial community analysis revealed more Methanosaeta cells were present in the methanol-starch-fed reactor, suggesting the degradation of starch produced acetate as an intermediate, which stimulated the growth of Methanosaeta cells responsible for the extension of granules.     

Structural development of aleurone and its function in common wheat

The wheat aleurone is formed from surface endosperm cells, and its developmental status reflects its biogenesis, structural characteristics, and physiological functions. In this report, wheat caryopses at different development stages were embedded in Spurr’s low-viscosity embedding medium for observation of the development of aleurone cells (ACs) by light microscopy, scanning electron microscopy, and fluorescence microscopy, respectively. According to their structures and physiological characterization, the ACs development process was divided into five stages: endosperm cellulization, spherosome formation, aleurone grain formation, filling material proliferation, and maturation. Furthermore, ACs in different parts of the caryopsis formed differently. ACs near the vascular bundle developed earlier and formed transfer cells, but other ACs formed slowly and did not form transfer cells. ACs on the caryopsis backside were a regular square shape; however, ACs in the caryopsis abdomen were mainly irregular. There were also differences in development between wheat varieties. ACs were rectangular in hard wheat but square in soft wheat. ACs were larger and showed a greater degree of filling in hard compared to soft wheat. The storage materials in ACs were different compared to inner endosperm cells (IECs). The concentrations of minerals such as sodium, magnesium, silicon, phosphorus and potassium were higher in ACs than in IECs. ACs contained many aleurone grains and spherosomes, which store lipids and mineral nutrients, respectively. The cell nucleus did not disappear and the cells were still alive during aleurone maturation. However, IECs were dead and mainly contained amyloplast and protein bodies, which store starch and protein, respectively. Overall, the above results characterized major structural features of aleurone and revealed that the wheat aleurone has mainly four functions.     

Effect of High Temperature During Grain-filling on the Structure of Developing and Malted Barley Grains

High temperatures (up to 35 °C) were applied to plants ofmalting barley,Hordeum vulgareL. (‘Schooner’) fora period of 5 d during grain-filling. Heat treatment had a profoundeffect on the structure of the mature barley grain. There wasevidence of degradation of endosperm storage products in heat-treatedgrain. Starch granule development was reduced in sub-aleuronecells following heat treatment and alterations to starch granuledistribution and growth were observed in the endosperms of thesegrains. Endosperm cell wall and crushed cell layer (CCL) developmentwere sensitive to high temperatures, with the reduced thicknessof the CCL and generally patchy Calcofluor fluorescence of endospermcell walls indicative of partial hydrolysis of ß-glucans.Increased growth of the embryo took place in heat-treated grainscompared with control grains. Endosperm texture was generallymore friable in heat-treated grains than in control grains,and these grains overmodified during malting, with considerabledegradation of starch in the form of extensive pitting of A-typestarch granules. Evidence is presented for developmental andgermination events occurring simultaneously within the developinggrain.Copyright 1998 Annals of Botany Company Barley,Hordeum vulgareL., starch granules, crushed cell layer, scutellum, embryo, fluorescence microscopy, scanning electron microscopy, confocal microscopy, malting quality.     

Kinetic analysis of glucoamylase-catalyzed hydrolysis of starch granules from various botanical sources

The kinetics of glucoamylase-catalyzed hydrolysis of starch granules from six different botanical sources (rice, wheat, maize, cassava, sweet potato, and potato) was studied by the use of an electrochemical glucose sensor. A higher rate of hydrolysis was obtained as a smaller size of starch granules was used. The adsorbed amount of glucoamylase on the granule surface per unit area did not vary very much with the type of starch granules examined, while the catalytic constants of the adsorbed enzyme (k(0)) were determined to be 23.3+/-4.4, 14.8+/-6.0, 6.2+/-1.8, 7.1+/-4.1, 4.6+/-3.0, and 1.6+/-0.6 s(-1) for rice, wheat, maize, cassava, sweet potato, and potato respectively, showing that k(0) was largely influenced by the type of starch granules. A comparison of the k(0)-values in relation to the crystalline structure of the starch granules suggested that k(0) increases as the crystalline structure becomes dense.     

Starch biosynthesis: experiments on how starch granules grow in vivo

Four varieties of starch granules from potato, wheat, maize, and rice were fractionated into homogeneous 10-μm-sized ranges. The size with the largest amount of granules was reacted with ADP-[¹⁴C]Glc, washed, and peeled into 7−9 layers, using a controlled peeling process, involving 90:10 volume proportions of Me₂SO-H₂O at 10 °C. All of the starches showed biosynthesis of starch throughout the granules. Starch synthase activities were determined for each of the layers. Three of the starches had a relatively large amount of synthase activity in the second layer, with only a small amount in the first layer. Potato starch had the largest amount of activity in the first layer. Starch synthase activity was found to alternate between higher and lower activities throughout all of the varieties of granules, showing that the synthesis was not uniform and also was not exclusively occurring at the surface of the starch granules, which had previously been hypothesized. From these results and our previous studies on the mechanism of starch chain elongation by the addition of d-glucose to the reducing end of a growing chain that is covalently attached to the active site of starch synthase, a hypothesis is proposed for how starch granules grow in vivo.     

Genetic elimination of a starch granule protein, SGP-1, of wheat generates an altered starch with apparent high amylose

A starch granule protein, SGP-1, is a starch synthase bound to starch granules in wheat endosperm. A wheat lacking SGP-1 was produced by crossing three variants each deficient in one of three SGP-1 classes, namely SGP-A1, -B1 or -D1. This deficient wheat (SGP–1 null wheat) showed some alterations in endosperm starch, meaning that SGP-1 is involved in starch synthesis. Electrophoretic experiments revealed that the levels of two starch granule proteins, SGP-2 and -3, decreased considerably in the SGP-1 null wheat though that of the waxy protein (granule-bound starch syn- thase I) did not. The A-type starch granules were deformed. Apparent high amylose level (30.8–37.4%) was indicated by colorimetric measurement, amperometric titration, and the concanavalin A method. The altered structure of amylopectin was detected by both high- performance size-exclusion chromatography and high-performance anion exchange chromatography. Levels of amylopectin chains with degrees of polymerization (DP) 6–10 increased, while DP 11–25 chains decreased. A low starch crystallinity was shown by both X-ray diffraction and differential scanning calorimetry (DSC) analyses because major peaks were absent. Abnormal crystallinity was also suggested by the lack of a polarized cross in SGP-1 null starch. The above results suggest that SGP-1 is responsible for amylopectin synthesis. Since the SGP-1 null wheat produced novel starch which has not been described before, it can be used to expand variation in wheat starch. Received: 30 April 1999 / Accepted: 9 November 1999