A New and Permanent Staining Method for Starch Granules Using Fluorescence Microscopy

A fluorescence technique has been developed for observing starch granules in plant tissues. Sections are stained with a mixture of dyes which we have named F.A.S.G.A. from the initials of the Spanish names of its components (fucsina, alcian blue, safranina, glicerina, agua), and viewed by epifluorescence microscopy. The starch granules fluoresce greenish yellow, allowing the degradative state to be observed. Cell structures which do not fluoresce are also differentiated. The stain permits identification of other structures when examined by visible light microscopy and is relatively resistant to fading over time.     

In situ Imaging of Pea Starch in Seeds

A method has been developed for the in situ imaging of starch in dry seeds by exploiting the tight packing of the starch and protein storage reserves within the cells of the embryo. The method can be adapted to prepare seed samples which are suitable for light microscopy (birefringence and iodine staining), scanning electron microscopy and atomic force microscopy. Its potential for imaging the internal structure of starch granules without any prior isolation process is demonstrated for round smooth peas. Using a standard ultramicrotome, thin sections were cut directly from selected regions of dry pea seeds and examined by light microscopy before and after hydration. The sectioning procedure left a planed surface with the internal structure of the starch granules exposed. This material was examined by scanning electron microscopy and atomic force microscopy directly or after controlled hydration. In the hydrated pea samples, the growth ring structure and blocklet sub-structure of individual starch granules within the seed were visualised directly by atomic force microscopy. Furthermore, the effects of hydration and staining were monitored and have been used to introduce contrast into the images. The observations have revealed new information on the blocklet distribution within pea starch granules and the physical origins of the growth ring structure of the granules: the blocklet distribution suggests that the granules contain alternating bands with different levels of crystallinity, rather than alternating amorphous and crystalline growth rings.     

The molecular deposition of transgenically modified starch in the starch granule as imaged by functional microscopy

The molecular deposition of starch extracted from normal plants and transgenically modified potato lines was investigated using a combination of light microscopy, environmental scanning electron microscopy (ESEM) and confocal laser scanning microscopy (CLSM). ESEM permitted the detailed (10 nm) topographical analysis of starch granules in their hydrated state. CLSM could reveal internal molar deposition patterns of starch molecules. This was achieved by equimolar labelling of each starch molecule using the aminofluorophore 8-amino-1,3,6-pyrenetrisulfonic acid (APTS). Starch extracted from tubers with low amylose contents (suppressed granule bound starch synthase, GBSS) showed very little APTS fluorescence and starch granules with low molecular weight amylopectin and/or high amylose contents showed high fluorescence. Growth ring structures were sharper in granules with normal or high amylose contents. High amylose granules showed a relatively even distribution in fluorescence while normal and low amylose granules had an intense fluorescence in the hilum indicating a high concentration of amylose in the centre of the granule. Antisense of the starch phosphorylating enzyme (GWD) resulted in low molecular weight amylopectin and small fissures in the granules. Starch granules with suppressed starch branching enzyme (SBE) had severe cracks and rough surfaces. Relationships between starch molecular structure, nano-scale crystalline arrangements and topographical-morphological features were estimated and discussed.     

New insights on the mechanism of acid degradation of pea starch

The degradation of pea starch granules by acid hydrolysis has been investigated using a range of chemical and structural methods, namely through measuring changes in amylose content by both the iodine binding and concanavalin A precipitation methods, along with small angle X-ray scattering (SAXS), wide angle X-ray diffraction (XRD) and field emission scanning electron microscopy (FE-SEM). The relative crystallinity, intensity of the lamellar peak and the low-q scattering increased during the initial stages of acid hydrolysis, indicating early degradation of the amorphous regions (growth rings and lamellae). In the first 2 days of hydrolysis, there was a rapid decline in amylose content, a concomitant loss of precipitability of amylopectin by concanavalin A, and damage to the surface and internal granular structures was evident. These observations are consistent with both amylose and amylopectin being located on the surface of the granules and attacked simultaneously in the early stages of acid hydrolysis. The results are also consistent with amylose being more concentrated at the core of the granules. More extensive hydrolysis resulted in the simultaneous disruption of amorphous and crystalline regions, which was indicated by a decrease in lamellar peak intensity, decrease in interhelix peak intensity and no further increase in crystallinity. These results provide new insights into the organization of starch granules.     

Optical microscopy in photosynthesis

Emerging as well as the most frequently used optical microscopy techniques are reviewed and image contrast generation methods in a microscope are presented, focusing on the nonlinear contrasts such as harmonic generation and multiphoton excitation fluorescence. Nonlinear microscopy presents numerous advantages over linear microscopy techniques including improved deep tissue imaging, optical sectioning, and imaging of live unstained samples. Nonetheless, with the exception of multiphoton excitation fluorescence, nonlinear microscopy is in its infancy, lacking protocols, users and applications; hence, this review focuses on the potential of nonlinear microscopy for studying photosynthetic organisms. Examples of nonlinear microscopic imaging are presented including isolated light-harvesting antenna complexes from higher plants, starch granules, chloroplasts, unicellular alga Chlamydomonas reinhardtii, and cyanobacteria Leptolyngbya sp. and Anabaena sp. While focusing on nonlinear microscopy techniques, second and third harmonic generation and multiphoton excitation fluorescence microscopy, other emerging nonlinear imaging modalities are described and several linear optical microscopy techniques are reviewed in order to clearly describe their capabilities and to highlight the advantages of nonlinear microscopy.     

魔芋球茎贮藏淀粉和甘露聚糖粒的形态观察

在光学显微镜和透射电镜下观察了魔芋（Ａｍｏｒｐｈｏｐｈａｌｕｓｃｏｎｊａｃ）球茎中甘露聚糖粒和淀粉粒的形态。两种贮藏多糖分别位于不同的细胞中。淀粉粒在造粉体内发育,以复粒存在,用魔芋球茎仔茎茎尖为材料观察显示,淀粉粒的形成早于甘露聚糖颗粒的形成。甘露聚糖粒形态多数近随圆形,一些甘露聚糖颗粒内包含了针晶体,但多数的甘露聚糖粒内部不包含针晶体,由纯净的甘露聚糖构成。     

Characterization of a Potato Starch-digesting Bacterium and Its Production of Amylase

A bacterium which can utilize potato starch granules as sole carbon source was isolated and identified as Bacillus circulans from its physiological and biochemical properties. Scanning electron microscopic observation of potato starch granules recovered from the culture broth revealed that granules were degraded gradually from their surface resulting in elongated granules with layered structures on their surface. This bacterium produced extracellular amylase which can digest potato starch granules in vitro. The amylase has a unique property in that it produces only maltohexaose from gelatinized starch in the early stage of the reaction. For the production of this amylase potato starch was found to be most effective while soluble sugars including gelatinized starch and maltose had little effect.     

Atomic force microscopy of pea starch: origins of image contrast

Atomic force microscopy (AFM) has been used to image the internal structure of pea starch granules. Starch granules were encased in a nonpenetrating matrix of rapid-set Araldite. Images were obtained of the internal structure of starch exposed by cutting the face of the block and of starch in sections collected on water. These images have been obtained without staining, or either chemical or enzymatic treatment of the granule. It has been demonstrated that contrast in the AFM images is due to localized absorption of water within specific regions of the exposed fragments of the starch granules. These regions swell, becoming "softer" and higher than surrounding regions. The images obtained confirm the "blocklet model" of starch granule architecture. By using topographic, error signal and force modulation imaging modes on samples of the wild-type pea starch and the high amylose r near-isogenic mutant, it has been possible to demonstrate differing structures within granules of different origin. These architectural changes provide a basis for explaining the changed appearance and functionality of the r mutant. The growth-ring structure of the granule is suggested to arise from localized "defects" in blocklet distribution within the granule. It is proposed that these defects are partially crystalline regions devoid of amylose.     

Effect of the alkaline treatment on the ultrastructure of C-type starch granules

The effect of alkaline treatment on the ultrastructure of C-type starch granules was investigated during the alkaline extraction of Araucaria angustifolia (pinhao) starch. The efficiency in protein removal was evaluated using intrinsic fluorescence and Kjeldahl's method. In parallel, morphological changes of starch granules were observed using scanning electron microscopy and atomic force microscopy. The starch crystallinity was monitored by wide-angle X-ray scattering and the lamellar structure was studied by small-angle X-ray scattering (SAXS). The paracrystalline model was employed to interpret the SAXS curves. It was found that the granular organization was significantly altered when alkaline solutions were used during the extraction. A partial degradation of B-type allomorph of starch and a significant compression of semicrystalline growth rings were observed.     

Scanning electron-microscopy of starch granules, with or without amylase attack

Scanning electron-microscopy (SEM) revealed that, for starch granules relatively susceptible to amylase, numerous pin holes could be observed on the surfaces of granules attacked by amylase. We also observed that the pores penetrated into the inner layers of granules during the enzyme action and some of the granules exhibited a terraced or step-shaped apperance in their inner portions. These internal characteristics are most probably indicative of layered internal structures of the granules. The other characteristic observations by SEM were striated structures on the surfaces of starch granules of banana, lily, and lotus attacked by pancreatin.     

Internal structure of the starch granule revealed by AFM

Atomic force microscopy images of sectioned native corn starch granules show evidence of the well-known radial organisation of the starch macromolecules, with the less-ordered hilum region near to the centre. Native granules show blocks 400-500 nm in size that span the growth rings. Lintnerised starch granules, where a mild acid hydrolysis has been used to remove the amorphous and less crystalline parts of the granule, clearly show smaller 'blocklets' within the rings approximately 10-30 nm in size. This level of organisation within the growth rings corresponds to the blocklet or superhelix structures that have been proposed in the literature for the association or clustering of amylopectin helices. Mechanical property imaging techniques have provided enhanced contrast to view this morphology, and shown the deformability of the starch structure under contact mode imaging conditions.     

Confocal laser scanning microscopy of dextran-rice starch mixtures

The microstructure of rice starch and dextran-rice starch mixtures was studied using confocal laser scanning microscopy (CSLM) and scanning electron microscopy (SEM). Surface pores and channels of rice starch were looked for. Channels could not be found in rice starch granules after reaction with 3-(4-carboxybenzoyl)-quinoline-2-carboxaldehyde (CBQCA). Fluorescein-labeled dextran was mixed with rice starch in order to locate hydrocolloid molecules in hydrocolloid-rice starch mixtures. The results showed that FITC-dextran (ave. Mw 4000; FD4) penetrated into raw rice starch granules, with the degree of penetration varying from granule to granule. FD4 could penetrate throughout cooked rice starch granules. FITC-dextran with an average Mw of more than 10,000 could not penetrate either raw or cooked rice starch granules.     

Genotype-specific spatial distribution of starch molecules in the starch granule: a combined CLSM and SEM approach

Starch granule types from a variety of botanical sources were selected to represent differences in crystalline polymorph, amylose and phosphate content, and amylopectin chain length distribution. Equimolar labeling of starch molecules with the fluorophore 8-amino-1,3,6-pyrenetrisulfonic acid (APTS) was used to construct a detailed map of the distribution of amylose and amylopectin within the granule by confocal laser scanning microscopy (CLSM) analysis. Medium- and high-resolution scanning electron microscopy (SEM) were used to provide detailed images of granule surface structures. By using a combined surface and internal imaging approach, interpretations of a number of previous structural observations is presented. In particular, internal images of high amylose maize and potato suggest that multiple initiations of new granules are responsible for the compound or elongated structures observed in these starches. CLSM optical sections of rice granules revealed an apparent altered distribution of amylose in relation to the proposed growth ring structure, hinting at a novel mechanism of starch molecule deposition. Well-described granule features, such as equatorial grooves, channels, cracks, and growth rings were documented and related to both the internal and external observations. A new method for probing the phosphate distribution in native granules was developed using a phosphate-binding fluorescent dye and CLSM.     

Architectural changes of heated mungbean, rice and cassava starch granules: Effects of hydrocolloids and protein-containing envelope

Architectural changes of starch granules induced by heat were demonstrated using light microscopy and confocal laser scanning microscopy. Heat treatment (80 °C, 30 min) on mungbean starch, cassava starch and rice flour suspensions resulted in the rearrangement of amylose and granule-associated proteins within the deformed granules. The presence of alginate and carrageenan influenced the RVA pasting characteristics of starch/flour-hydrocolloid mixed suspensions by maintaining the granular structure of amylose-rich swollen granules or inducing the aggregation of the swollen ones. Generally, the addition of hydrocolloid increased peak viscosity, lowered breakdown and reduced setback of the flour-hydrocolloid mixed paste. This study demonstrated that the heat treatment in excess water generated the protein-containing granule envelope encasing the mungbean and cassava starch content within the deformed granules.     

Structural features of non-granular spherulitic maize starch

Complementary analyses of the internal structure of spherulites crystallized from high-amylose maize starch were obtained using light, electron and atomic force microscopy. Radially oriented crystalline lamellae were observed in transmission and scanning electron microscopy, as well as AFM. Internal structures consistent with the central hilum region of starch granules were observed. Spherulites were composed largely of linear or lightly branched starch polymers. Degradation of amylopectin at gelatinization temperatures of 180 degrees C was evident, but iodine binding suggested a high molecular weight (>100 DP) for the spherulitic polymers.     

Synthesis and Development of Starch Granules in High Lysine Barley Grains

Electron microscopy studies of developing endosperm have shown differences in the synthesis and development of starch granules between high lysine mutant Notch-2 and parent NP 113. The starch granules in Notch-2 were modified and did not develop into characteristic oval granules. Based on iodine absorption and phospholipids analysis, it is suggested that the presence of phospholipids impairs the development of starch granule in Notch-2. This is further confirmed by higher lipid density across starch granule in mutant Notch-2, and its absence in NP 113. Amylopectin from mutant differs from that of NP 113. The results indicate that the lack of geometry and smaller size of starch granules in Notch-2 is ultimately due to specific interaction of lipids with developing starch granules, and this leads to decreased yield.     

Osservazioni Preliminari Sulla Struttura Submicroscopica dei Granuli di Amido dell'endosperma Maturo di Triticum Durum Desf

Abstract

Preliminary observations on the submicroscopic structure of the endospermic starch granules in TRITICUM DURUM desf. (The electronic transparency of the starch granules). — The starch granules of Triticum durum Desf. kernels have been examined by electron microscopy.

The high degree of transparency of the granules appears to depend upon the regular disposition of the structural units, while their opacity probably is in relation with their orderless arrangement. The opacity of amorphous portions of the layers, of the median fissure and of the black bands seems to be in relation with the amorphous condition of such formations.

The black bands and the bubbles which have been considered respectively as protein layers and as the result of enzymes action, are only artifacts. The bubbles are interpreted as pyrodestrines.     

Physical investigations of surface membrane-water relationship of intact and gelatinized wheat-starch systems

Water mobility in intact and dried gelatinized starch was investigated by gravimetric water sorption, scanning electron microscopy (SEM), and solid-state nuclear magnetic resonance (NMR). A multi-component exponential model quantitatively measured different spin-spin relaxation times of two water components, namely bound water (Tsi) at 3.16 ms and mobile or free water (Tsii) at 3.23 ms, as a function of water activity (aw). The starch samples were moistened to 30% moisture content. SEM confirmed the disrupted, absorbent microstructure in dried, gelatinized starch powder and revealed starch granules in an incomplete gelatinized state, as compared to the complete membrane surface of the intact starch granule. Starch granules sorbed significantly differently at low aw, but after aw=0.44, sorption leveled similarly with increasing aw. The presence and role of a surface membrane was concluded, in support of the hypothetical "water sink" properties of intact granules, and was considered to influence in part the sorption behavior of incompletely gelatinized 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.     

Degradation of Raw Starch by a Wild Amylolytic Strain of Lactobacillus plantarum

Lactobacillus plantarum A6, isolated from fermented cassava, can break down cassava raw starch that has not been subjected to preliminary physicochemical treatment. When the pH was kept at 6, the microorganism cultured in a bioreactor excreted a high α-amylase activity (60 U/ml). Synthesis of the enzyme occurred during the stationary phase and resulted in full hydrolysis of the cassava starch granules. This gave 41 g of lactic acid from 45 g of raw starch after 3 days of fermentation. Enzymatic attack was evident under scanning electron microscopy in the rougher appearance of the surface of starch granules and in the presence of large cavities in some of them. In contrast, when the pH was not regulated, only a small amount of α-amylase activity was produced (2 U/ml) and no decrease in the starch content of the medium was observed. However, under scanning electron microscopy, some granules displayed a rougher surface, which might have been the result of weak enzymatic attack.