A simple and rapid method for preparing the whole section of starchy seed to investigate the morphology and distribution of starch in different regions of seed

BackgroundStorage starch in starchy seed influences the seed weight and texture, and determines its applications in food and nonfood industries. Starch granules from different plant sources have significantly different shapes and sizes, and even more the difference exists in the different regions of the same tissue. Therefore, it is very important to in situ investigate the morphology and distribution of starch in the whole seed. However, a simple and rapid method is deficient to prepare the whole section of starchy seed for investigating the morphology and distribution of starch in the whole seeds for a large number of samples.ResultsA simple and rapid method was established to prepare the whole section of starchy seed, especially for floury seed, in this study. The whole seeds of translucent and chalky rice, vitreous and floury maize, and normal barley and wheat were sectioned successfully using the newly established method. The iodine-stained section clearly exhibited the shapes and size of starch granules in different regions of seed. The starch granules with different morphologies and iodine-staining colors existed regionally in the seeds of high-amylose rice and maize. The sections of lotus and kidney bean seeds also showed the feasibility of this method for starchy non-cereal seeds.ConclusionThe simple and rapid method was proven effective for preparing the whole sections of starchy seeds. The whole section of seed could be used to investigate the morphology and distribution of starch granules in different regions of the whole seed. The method was especially suitable for large sample numbers to investigate the starch morphology in short time.

Related papers:

title: Starch synthesis in the cereal endosperm.
abstract: The pathway of starch synthesis in the cereal endosperm is unique, and requires enzyme isoforms that are not present in other cereal tissues or non-cereal plants. Recent information on the functions of individual enzyme isoforms has provided insight into how the linear chains and branch linkages in cereal starch are synthesized and distributed. Genetic analyses have led to the formulation of models for the roles of de-branching enzymes in cereal starch production, and reveal pleiotropic effects that suggest that certain enzymes may be physically associated. For the first time, tools for global analyses of starch biosynthesis are available for cereal crops, and are heralded by the draft sequence of the rice genome.

title: Physico‐chemical properties of starches from Indian kidney bean (Phaseolus vulgaris) cultivars
abstract: Starches isolated from four Kidney bean cultivars (French Yellow, Contender, Master Bean, Local Red) grown in temperate climate were studied for their physico-chemical, morphological, thermal, pasting, textural and retrogradation properties. Physico-chemical properties such as composition, amylose content, water absorption capacity, swelling power, syneresis, freeze–thaw stability and light transmittance showed significant differences among starches. Amylose content (36.4–41.7%) showed strong correlations with peak, trough, breakdown, final and setback viscosity, gel hardness, gumminess and chewiness. The starch granule morphology of these starches showed considerable variation when studied by scanning electron microscopy. Starch granules were observed to be round, irregular or elliptical with smooth surfaces. Master Bean starch granules were larger than those of other kidney bean starches. Pasting and textural properties of French Yellow starches were found to be higher than other kidney bean starches. Local Red starches showed the highest gelatinisation transition temperatures, whereas Master Bean starches showed the lowest transition temperatures.

title: Structural and physicochemical properties of lotus seed starch treated with ultra-high pressure.
abstract: Aqueous lotus seed starch suspensions (15%, w/w) were subjected to ultra-high pressure treatment (UHP, 100-600 MPa) for 30 min. The effects of UHP treatment on the structural and physicochemical properties of starch were investigated. The SEM and laser diffraction particle size analysis revealed that UHP treatment affected the shape and size distribution of starch granules. The morphological structure of starch was completely destroyed at 600 MPa, indicating complete gelatinization. Analysis of HPSEC-MALLS-RI suggested that the dispersity index of UHP-treated starch were decreased from 1.28 to 1.11. According to XRD analyses, UHP treatment converted native starch (C-type) into a B-type pattern. The swelling power and solubility presented a significant decrease at 85 and 95 °C, but opposite trends were found at 55-75 °C. The DSC results indicated a reduction in gelatinization temperatures and enthalpy with increasing pressure treatment. The RVA viscograms revealed that UHP-treated starch showed a decreased breakdown and setback viscosity, reflecting lower retrogradation tendency compared to native starch.

title: A rapid, direct observation method to isolate mutants with defects in starch grain morphology in rice.
abstract: Starch forms transparent grains, referred to as starch grains (SGs), in amyloplasts. Despite the simple glucose polymer composition of starch, SGs exhibit different morphologies depending on plant species, especially in the endosperm of the Poaceae family. This study reports a novel method for preparing thin sections of endosperm without chemical fixation or resin embedding that allowed us to visualize subcellular SGs clearly. Using this method, we observed the SG morphologies of >5,000 mutagenized rice seeds and were able to isolate mutants in which SGs were morphologically altered. In five mutants, named ssg (substandard starch grain), increased numbers of small SGs (ssg1-ssg3), enlarged SGs (ssg4) and abnormal interior structures of SGs (ssg5) were observed. Amylopectin chain length distribution analysis and identification of the mutated gene suggested a possible allelic relationship between ssg1, ssg2, ssg3 and the previously isolated amylose-extender (ae) mutants, while ssg4 and ssg5 seemed to be novel mutants. Compared with conventional observation methods, the methods developed here are more effective for obtaining fine images of subcellular SGs and are suitable for the observation of a large number of samples.

title: Maize endosperm-specific transcription factors O2 and PBF network the regulation of protein and starch synthesis
abstract: Nutritional quality and yield are equally important considerations in crop breeding, although they sometimes appear at odds. In this work we made the discovery that these traits are linked through regulation by two transcription factors. Mutations that affect the expression of these transcription factors can improve the nutritional quality of the seed but also can reduce kernel yield and hardness. Therefore future corn-breeding programs should silence zein genes directly, not by blocking transcription factors. The maize endosperm-specific transcription factors opaque2 (O2) and prolamine-box binding factor (PBF) regulate storage protein zein genes. We show that they also control starch synthesis. The starch content in the PbfRNAi and o2 mutants was reduced by ∼5% and 11%, respectively, compared with normal genotypes. In the double-mutant PbfRNAi;o2, starch was decreased by 25%. Transcriptome analysis reveals that >1,000 genes were affected in each of the two mutants and in the double mutant; these genes were mainly enriched in sugar and protein metabolism. Pyruvate orthophosphate dikinase 1 and 2 (PPDKs) and starch synthase III (SSIII) are critical components in the starch biosynthetic enzyme complex. The expression of PPDK1, PPDK2, and SSIII and their protein levels are further reduced in the double mutants as compared with the single mutants. When the promoters of these genes were analyzed, we found a prolamine box and an O2 box that can be additively transactivated by PBF and O2. Starch synthase IIa (SSIIa, encoding another starch synthase for amylopectin) and starch branching enzyme 1 (SBEI, encoding one of the two main starch branching enzymes) are not directly regulated by PBF and O2, but their protein levels are significantly decreased in the o2 mutant and are further decreased in the double mutant, indicating that o2 and PbfRNAi may affect the levels of some other transcription factor(s) or mRNA regulatory factor(s) that in turn would affect the transcript and protein levels of SSIIa and SBEI. These findings show that three important traits—nutritional quality, calories, and yield—are linked through the same transcription factors.

title: Anthology of Starch Granule Morphology by Scanning Electron Microscopy
abstract: Scanning electron micrographs of the granules of 54 starches obtained from a wide variety of plant sources, consisting of roots and tubers, grains, maize, peas and beans, fruits and nuts, and small granule starches, are presented as a comparative study of their sizes and morphologies. All micrographs are presented at a magnification of 1500X with the addition of micrographs of 600X for the very large granules and micrographs of 10,000X for the very small granules.

title: Characteristics of the different corn types and their grain fractions: physicochemical, thermal, morphological, and rheological properties of starches
abstract: Pop corn, dent corn and baby corn (dent type) grains were fractionated into different fractions on the basis of their size. The starches were separated from these fractions and evaluated for physicochemical, morphological, thermal and rheological properties. Significant difference was observed in various properties among different fractions of dent corn and pop corn. Mean granule diameter of the starches separated from different fractions ranged between 6.33 and 13.64 μm. The shape of starch granules varied from oval to polyhedral. Baby corn starch showed the presence of oval shape granules whereas polyhedral shape granules were observed in starches from other corn types. Amylose content of starches from different corn types ranged between 15.3% and 25.1%. Baby corn starch showed lowest swelling power, solubility, amylose content and mean granule diameter. The transition temperatures ( T o , T p and T c ) and enthalpy of gelatinization (Δ H gel ) of starches were determined using differential scanning calorimetry. T o , T p , T c and Δ H gel varied from 66.3 to 69.3, 71.5 to 73.1, 76.5 to 78 °C and 8.9 to 10.9 J/g, respectively. Baby corn starch showed lowest T o , T p , Δ H gel and PHI. The values of these parameters were highest for both the fractions of dent corn and large grain fraction of pop corn starch. The rheological properties of the starches from different fractions of pop corn and dent corn measured using a dynamic rheometer, showed significant variation in the peak G ′ , G ″ and peak tan δ values. Pop corn large grain fraction showed highest values for peak G ′ , G ″ and breakdown in G ′ , whereas pop corn small grain fraction showed lower values of these parameters. The turbidity of the gelatinized aqueous starch pastes from all the corn types increased with increase in storage period. Baby corn starch showed the lowest and pop corn large grain fraction showed highest retrogradation values during storage.

title: Comparison of physicochemical properties of starches from seed and rhizome of lotus
abstract: Lotus seed and rhizome starches have been used as functional foods in east Asia for thousands of years. In this paper, starches were isolated from lotus seed and rhizome, and their physicochemical properties were compared. Seed starches were small oval granules, rhizome starches had small oval granules and large elongated granules. Seed starches showed significantly higher amylose content and gelatinization temperature and lower swelling power than rhizome starches. Seed starches exhibited an A-type X-ray diffraction pattern with higher crystalline degree, while rhizome starches showed a C-type pattern which changed from C- to A-type with gradually increasing crystalline degree during acid hydrolysis. The degree of order in starch external region was higher in rhizome than in seed. The external region structure of rhizome starches became more ordered during acid hydrolysis. Rhizome starches had higher rate of acid hydrolysis and lower rate of porcine pancreatic α-amylase hydrolysis than seed starches.

title: Comparison of starch granule development and physicochemical properties of starches in wheat pericarp and endosperm.
abstract: The objectives of this study were: (i) to characterize structural development of starch granule in pericarp and endosperm during wheat caryopsis growth; (ii) to compare physicochemical properties of starches in pericarp and endosperm; (iii) to further discover the relationships between pericarp starches and endosperm starches. Wheat pericarp and endosperm at different development stages were observed by light microscopy and scanning electron microscopy, respectively. Structural properties of starches were determined using X-ray power diffraction and (13) C solid nuclear magnetic resonance.



Pericarp starch granules (PSG) accumulated in amyloplasts and chloroplasts, and showed a typical accumulation peak at 5 days after fertilization (DAF), and then gradually decomposed during 5-22 DAF. PSG in the abdominal region showed a higher rate of decomposition compared to the dorsal region of pericarp. Endosperm starch granules (ESG) accumulated in amyloplasts, and occurred in endosperm cells at 5 DAF, then rapidly enriched the endosperm cells until 22 DAF. Compared with ESG, PSG were compound granules of irregular shape and small size distribution. The results also suggested lower amylose content and V-type single-helix content and higher proportions of double helices for PSG compared to ESG.



Based on the structural development of PSG and ESG, we speculated that the saccharides resulting from decomposition of PSG, on one hand, enabled the pericarp to survive before maturity of wheat caryopsis and, on the other hand, provided extra nutrition for the growth of ESG.

title: Development of Small Starch Granule in Barley Endosperm
abstract: The structural characteristics and physicochemical properties of barley starch differ with granule size, which are important in final product applications of barley flours. A barley (Hordeum vulgare L.) cultivar, Yangsimai 3, was used to investigate the development of small starch granule in endosperm under electron microscope to provide references to barley breeding. Large starch granules were formed and developed at the early stage of endosperm development. Large amyloplasts with large starch granules were divided and increased in number through binary fission. One large amyloplast had only 1 large starch granule. Small starch granules could be formed and develop at the middle and late stage of endosperm development. Endosperm cells had large amyloplasts that exhibited protrusion, and some of the protrusions contained small starch granules. Small amyloplasts with small starch granules formed new small amyloplasts to produce small starch granules by the way of protruding their envelopes. Many small starch granules were formed and developed in 1 small amyloplast. The amyloplast envelope began to degrade and release starch granules into cell matrix when amyloplast was full of starch granules. These results showed that small amyloplasts came from the protrusion of amyloplast envelopes. Many small starch granules, which were compound starch granules, were formed and developed in 1 small amyloplast.

title: Physicochemical properties and development of wheat large and small starch granules during endosperm development
abstract: Wheat mature seeds have large, lenticular A-type starch granules, and small, spherical B-type and irregular C-type starch granules. During endosperm development, large amyloplasts came from proplastid, divided and increased in number through binary fission from 4 to 12 days after flowering (DAF). Large starch granules formed and developed in the large amyloplast. One large amyloplast had only one large starch granule. Small amyloplasts came from the protrusion of large amyloplast envelope, divided and increased in number through envelope protrusion after 12 DAF. B-type starch granules formed and developed in small amyloplast from 12 to 18 DAF, C-type starch granules formed and developed in small amyloplast after 18 DAF. Many B- and C-type starch granules might form and develop in one small amyloplast. The amyloplast envelopes were asynchronously degraded and starch granules released into cell matrix when amyloplasts were full of starch granules. Apparent amylose contents of large starch granules were higher than that of small starch granules, and increased with endosperm development. The swelling powers and crystallinity of large starch granule were lower than that of small starch granules, and decreased with endosperm development. Small starch granules displayed broader gelatinization temperature ranges than did large starch granules.

title: Structural variability between starch granules in wild type and in ae high-amylose mutant maize kernels.
abstract: Starch granule structure within wild-type and ae high-amylose mutant maize kernels has been mapped in situ using light, electron and atomic force microscopy, and both Raman and infra-red spectroscopy. The population of wild-type starch granules is found to be homogenous. The ae mutant granule population is heterogeneous. Heterogeneity in chemical and physical structure is observed within individual granules, between granules within cells, and spatially within the kernel. The highest level of heterogeneity is observed in the region where starch is first deposited during kernel development. Light microscopy demonstrates structural diversity through use of potassium iodide/iodine staining and polarised microscopy. Electron and atomic force microscopy, and infra-red and Raman spectroscopy defined the nature of the structural changes within granules. The methodology provides novel information on the changes in starch structure resulting from kernel development.

title: Heterogeneous structure and spatial distribution in endosperm of high-amylose rice starch granules with different morphologies.
abstract: Starch granules from high-amylose cereal mutants or transgenic lines usually have different morphologies. It is not clear whether the structure and spatial distribution of starch granules with different morphologies in endosperm is homogeneous or heterogeneous. In the present study, the structure and spatial distribution in endosperm of morphologically different starch granules from high-amylose transgenic rice line (TRS) were investigated. The TRS endosperm had individual, aggregate, elongated, and interior hollow starch granules. The individual and interior hollow granules had the lowest and the highest amylose content and gelatinization resistance, respectively, among the four types of granules. The individual granules were mainly distributed in the middle of the endosperm; the aggregate granules in the starchy endosperm cells between the subaleurone layer and the middle of the endosperm; the elongated granules in the peripheral starchy endosperm cells adjacent to the subaleurone layer; and the interior hollow granules in the subaleurone layer cells.

title: Formation of elongated starch granules in high-amylose maize
abstract: GEMS-0067 maize starch contains up to 32% elongated starch granules, much higher than amyloseextender (ae) single-mutant maize starch (7%) and normal (non-mutant) maize starch (0%). These elongated granules are highly resistant to enzymatic hydrolysis at 95–100 C, which function as resistant starch. The structure and formation of these elongated starch granules, however, were not known. In this study, light, confocal laser-scanning, scanning electron, and transmission electron microscopy were used to reveal the structure and formation of these elongated starch granules. The transmission electron micrographs showed fusion through amylose interaction between adjacent small granules in the amyloplast at the early stage of granule development. A mechanistic model for the formation of elongated starch granules is proposed.

title: Different structures of heterogeneous starch granules from high-amylose rice.
abstract: High-amylose cereal starches usually have heterogeneous starch granules in morphological structure. In the present study, the polygonal, aggregate, elongated, and hollow starch granules were separated from different regions of the kernels of high-amylose rice, and their structures were investigated. The results showed that the polygonal starch granules had low amylose content and high short branch-chain and branching degree of amylopectin, and exhibited A-type crystallinity. The aggregate starch granules had high long branch-chain of amylopectin, relative crystallinity, and double helix content, and exhibited C-type crystallinity. The elongated starch granules had high amylose content and low branching degree of amylopectin and relative crystallinity, and exhibited C-type crystallinity. The hollow starch granules had very high amylose content, proportion of amorphous conformation, and amylose-lipid complex, and very low branch-chain of amylopectin, branching degree of amylopectin, and double helix content, and exhibited no crystallinity. The different structures of heterogeneous starch granules from high-amylose rice resulted in significantly different thermal properties.

title: Application of whole sections of mature cereal seeds to visualize the morphology of endosperm cell and starch and the distribution of storage protein.
abstract: The morphology of endosperm cell and starch and the distribution of storage protein affect the kernel weight and endosperm quality of cereal crops, and are of interest for crop breeders. They are significantly different in different regions of endosperm. Therefore, it is very important for in situ investigation of the morphology of endosperm cell and starch and the distribution of storage protein in mature cereal seed. In the present study, we reported a method for preparing the whole section of mature rice, maize, and wheat seeds. The section could be easily stained with fluorescent brightener 28, iodine solution, and coomassie brilliant blue R250 to exhibit the cell wall, starch, and storage protein. The observation of whole section of seed showed that the morphology of endosperm cell and starch and the distribution of storage protein were significantly different in different regions of endosperm of rice, maize, and wheat seed. The method could in situ investigate the morphology of endosperm cell and starch and the distribution of storage protein in not only mature cereal seed but also developing, germinated, and gelatinized cereal seed. This study was very useful for investigation of cereal seed development and utilization.

title: Chemical Composition and Microstructure of Two Naked Waxy Barleys
abstract: Two naked waxy barley cultivars, Bz 489-30 and Prowashonupana were analysed in order to study the chemical composition and the microstructure. Prowashonupana contained more protein, fat and ash, and less starch than Bz 489-30. The content of mixed-linked β-glucan was almost three times higher in Prowashonupana (14·9%) than in Bz 489-30 (5·6%), but extractability at 40 °C was much lower. Microscopy studies showed that kernels and endosperm cells of Prowashonupana were irregular, and that cell walls were thicker with a higher content of mixed-linked β-glucan than in Bz 489-30. In both barleys the sub-aleurone layer contained black starch granules, indicating a higher amylose content in this structure compared to the rest of the kernel. Starch was isolated from both barleys using two different procedures and analysed for amylose content. The amylose content was higher, while volume % of A-granules was lower and mean diameter of A-granules was smaller in starch isolated from Prowashonupana than from Bz 489-30. Both composition and yield of starch were however influenced by the isolation procedure used. The procedure which gave higher starch yield resulted in a higher amylose content, probably because of greater recovery of starch from outer parts of the kernel with a higher amylose content.

title: Endosperm and aleurone cell structure in barley and wheat as studied by optical and Raman microscopy
abstract: Grain ultrastructure is of utmost importance when designing grain processing procedures in the food industry. In this study, wheat and barley grain components were localised using optical and Raman microscopy. The optical microscopic analyses were performed using several selective stains to localise β-glucan, protein and starch or autofluorescence to image the ferulic acid and other fluorescing substances. Alternatively, Raman microscopy was applied to localise the grain components without any need for preceding staining or other sample pretreatment. Both methods provided consistent information on the grain structures, illustrating the distribution of polysaccharides, aromatics and protein in endosperm and aleurone layers. In aleurone layers of both barley and wheat, a distinct difference between the anticlinal and periclinal cell walls was observed. The anticlinal cell walls were enriched with aromatic substances which were present in remarkably lower concentrations in the inner periclinal cell walls but for barley, an even higher concentration in the outer periclinal cell wall was observed. In addition, Raman spectroscopy illustrated the detailed distribution of substances across the aleurone cell walls: β-glucan was adjacent to proteins and it was deficient in the middle lamella whereas arabinoxylan was enriched in the outer cell wall layers and middle lamella.

title: In situ Raman microscopy of starch granule structures in wild type and ae mutant maize kernels
abstract: A method developed for in situ imaging of starch granule structure in dry seeds has been applied to compare the starch granule structures found in wild type and ae mutant maize kernels. In the isogenic ae mutant the activity of the starch branching enzyme IIb is inhibited, which gives rise to a high amylose starch. The granule structures in the wild type samples have been found to be homogeneous, whereas those in the ae mutant are grossly heterogeneous within individual granules, between granules within individual cells, and between cells across the endosperm. The level of heterogeneity observed in situ appears to be more marked than that previously reported for studies on isolated ae mutant starches. Iodine/potassium iodide staining and polarised light microscopy have been used together with Raman microscopy, which has allowed high-resolution mapping of the composition and physical state of the structures within the granules, to probe the origins of the heterogeneity of the starch structures. Although the mutation inhibits the activity of the branching enzyme within the granules, and both the composition and level of crystallinity within and between granules is variable, the major origin of the heterogeneity of the granule architecture appears to result from significant changes in the assembly and packaging of the crystalline structures within the granule. It is suggested that this arises due to the mutation of the starch branching enzyme introducing defects into the self-assembly of the crystalline structure, resulting in an accumulation of defects and increased randomisation of the granule structure.

title: Chalk5 encodes a vacuolar H+-translocating pyrophosphatase influencing grain chalkiness in rice
abstract: Grain chalkiness is a highly undesirable quality trait in the marketing and consumption of rice grain. However, the molecular basis of this trait is poorly understood. Here we show that a major quantitative trait locus (QTL), Chalk5, influences grain chalkiness, which also affects head rice yield and many other quality traits. Chalk5 encodes a vacuolar H+-translocating pyrophosphatase (V-PPase) with inorganic pyrophosphate (PPi) hydrolysis and H+-translocation activity. Elevated expression of Chalk5 increases the chalkiness of the endosperm, putatively by disturbing the pH homeostasis of the endomembrane trafficking system in developing seeds, which affects the biogenesis of protein bodies and is coupled with a great increase in small vesicle-like structures, thus forming air spaces among endosperm storage substances and resulting in chalky grain. Our results indicate that two consensus nucleotide polymorphisms in the Chalk5 promoter in rice varieties might partly account for the differences in Chalk5 mRNA levels that contribute to natural variation in grain chalkiness.

title: Chalky part differs in chemical composition from translucent part of japonica rice grains as revealed by a notched‐belly mutant with white‐belly
abstract: Chalkiness has a deleterious influence on rice appearance and milling quality. We identified a notched‐belly mutant with a high percentage of white‐belly, and thereby developed a novel comparison system that can minimize the influence of genetic background and growing conditions. Using this mutant, we examined the differences in chemical composition between chalky and translucent endosperm, with the aim of exploring relations between occurrence of chalkiness and accumulation of starch, protein and minerals.  Comparisons showed a significant effect of chalkiness on chemical components in the endosperm. In general, occurrence of chalkiness resulted in higher total starch concentration and lower concentrations of the majority of the amino acids measured. Chalkiness also had a positive effect on the concentrations of As, Ba, Cd, Cr, Mn, Na, Sr and V, but was negatively correlated with those of B, Ca, Cu, Fe and Ni. By contrast, no significant chalkiness effect on P, phytic acid‐P, K, Mg or Zn was observed. In addition, substantial influence of the embryo on endosperm composition was detected, with the embryo showing a negative effect on total protein, amino acids such as Arg, His, Leu, Lys, Phe and Tyr, and all the 17 minerals measured, excluding Ca, Cu, P and Sr.  An inverse relation between starch and protein as well as amino acids was found with respect to chalkiness occurrence. Phytic acid and its colocalized elements K and Mg were not affected by chalkiness. The embryo exerted a marked influence on chemical components of the endosperm, in particular minerals, suggesting the necessity of examining the role of the embryo in chalkiness formation.

title: Formation of semi-compound C-type starch granule in high-amylose rice developed by antisense RNA inhibition of starch-branching enzyme.
abstract: Cereal starch granules with high-amylose and resistant starch (RS) always show irregular morphology and special crystalline structure, but their formation during grain development is not yet clear. In our previous studies, we had generated a transgenic rice line (TRS) enriched with amylose and RS, which contained semi-compound starch showing a C-type crystalline structure. In this study, the formation of semi-compound C-type starch granule during TRS endosperm development was carefully investigated with light, scanning electron, and transmission electron microscopes and X-ray powder diffraction. The results showed that the TRS starch subgranules, each with a central hilum, were individually initiated in amyloplast and showed an A-type crystal at the early stage of starch granule development, which was similar to that in its wild type. However, with the endosperm development, the amylose content in TRS endosperm starch increased and the B-type starch crystal was deposited in the periphery of subgranules; then, the adjacent subgranules fused together and finally formed a continuous outer layer band surrounding the entire circumference of the starch granule. Accordingly, a mechanistic model for the formation of semi-compound C-type starch granules is proposed.

title: The effect of high temperature on kernel dimensions and the type and occurrence of kernel damage in rice
abstract: Rice (Oryza sativa L. cv. Calrose) growing at 27/22"C was either transferred to day/night temperatures ranging from 24/19 to 39/34"C 7days after heading and held at these temperatures until maturity, or transferred to a temperature of 36/31°C for 8 day periods at regular intervals commencing from heading. Kernel dimensions were measured directly and the types of kernel damage at maturity were characterized by direct viewing under the light microscope of intact and sectioned kernels, or by scanning electron microscopy of the exposed surface of kernels cut transversely with a razor blade. Kernel thickness was reduced most by high temperature treatments commencing 12 days after heading, but other kernel dimensions (length and width) were most sensitive to high temperature earlier in development. Sterility and pathenocarpy were most evident when temperature treatments commenced at heading (approximately 2 days before anthesis) and were greatest at the highest temperature (39/34"C). Abortive and opaque kernels were most evident when the high temperature commenced 4 days after heading and were also most numerous at the highest temperature tested. From observations on the morphology of the kernels it appears that temperatures above 27/22"C can interfere with the early stages of cell division and development in the endosaerm. Chalky endosperm tissue occurred in several forms depending on both the temperature level and the timing of the temperature treatment. White-core kernels were only evident at a temperature of 27/2Z°C. White-back kernels were most numerous at 36/31°C and when the high temperature treatment commenced 16 days after heading. Milky-white kernels were found in all but the lowest temperature treatment (24/1g0C), with a peak at 36/31°C and this type of damage was most evident when the high temperature treatment commenced 12 days after heading. Differences in endosperm cellular morphology were observed between the different types of damage, and in accord with other published data the chalky appearance was associated with the development of numerous air spaces between loosely packed starch granules and a change in light refraction.

title: Endosperm Structure of White-Belly and White-Core Rice Grains Shown by Scanning Electron Microscopy
abstract: White-belly and white-core are the major two types of grain chalkiness in japonica rice. This study aims to compare the morphological features of white-belly and white-core using a scanning electron microcope (SEM). A japonica rice cultivar Wuyujing3 and its mutants were used as materials. Nearly 1000 SEM images were observed, and 12 representative photos were selected. SEM images showed contrasting differences between white-belly and white-core in endosperm microstructure including the shape of endosperm cell, the size distribution of starch granules, and the amount of protein bodies. White-belly and white-core also varied markedly in morphological features of the cracked compound starch granules. Our findings should help to advance our understanding of the multi-faceted nature of grain chalkiness from the perspective of starch and protein accumulation, and should be of value for future work on rice grain chalkiness.

title: Comprehensive Expression Profiling of Rice Grain Filling-Related Genes under High Temperature Using DNA Microarray[OA]
abstract: To elucidate the effect of high temperature on grain-filling metabolism, developing rice (Oryza sativa) ‘Nipponbare’ caryopses were exposed to high temperature (33°C/28°C) or control temperature (25°C/20°C) during the milky stage. Comprehensive gene screening by a 22-K DNA microarray and differential hybridization, followed by expression analysis by semiquantitative reverse transcription-PCR, revealed that several starch synthesis-related genes, such as granule-bound starch synthase I (GBSSI) and branching enzymes, especially BEIIb, and a cytosolic pyruvate orthophosphate dikinase gene were down-regulated by high temperature, whereas those for starch-consuming α-amylases and heat shock proteins were up-regulated. Biochemical analyses of starch showed that the high temperature-ripened grains contained decreased levels of amylose and long chain-enriched amylopectin, which might be attributed to the repressed expression of GBSSI and BEIIb, respectively. SDS-PAGE and immunoblot analysis of storage proteins revealed decreased accumulation of 13-kD prolamin, which is consistent with the diminished expression of prolamin genes under elevated temperature. Ripening under high temperature resulted in the occurrence of grains with various degrees of chalky appearance and decreased weight. Among them, severely chalky grains contained amylopectin enriched particularly with long chains compared to slightly chalky grains, suggesting that such alterations of amylopectin structure might be involved in grain chalkiness. However, among high temperature-tolerant and sensitive cultivars, alterations of neither amylopectin chain-length distribution nor amylose content were correlated to the degree of grain chalkiness, but rather seemed to be correlated to grain weight decrease, implying different underlying mechanisms for the varietal difference in grain chalkiness. The possible metabolic pathways affected by high temperature and their relevance to grain chalkiness are discussed.

title: Endosperm and starch granule morphology in wild cereal relatives
abstract: Australia's native grass species contain a diverse array of wild cereal relatives which are adapted to a broader range of environmental conditions than current commercial cereals and may contain novel alleles which have utility in commercial production systems. Characterizing the available variation in endosperm morphology is one of the first steps towards in planta manipulation of endosperm by either the introgression of novel alleles or bioengineering cereal starch and protein. The endosperm of 19 crop wild relatives (CWR) was examined using scanning electron microscopy (SEM). Mature caryopses were fixed, dehydrated, critical-point dried and then snap fractured transversely through the grain. Wild relatives exhibited similar types of starch granules to that of their respective cultivated species, though in general the wild species retained a greater proportion of the endosperm cell wall at maturity. The two species examined with no closely related cultivated species exhibited a rice-like endosperm. Wild sorghum relatives exhibited an abundance of endosperm variations described as variations in starch granule size, shape and surface morphology, and the distribution of protein bodies. This is particularly important because the grain of Sorghum bicolor has inherently low starch and protein digestibility. These variations within the wild relatives of commercial cereals may provide novel sources of genetic diversity for future grain improvement programmes.

title: Physicochemical properties and starch digestibility of whole grain sorghums, millet, quinoa and amaranth flours, as affected by starch and non-starch constituents.
abstract: Minor grains such as sorghum, millet, quinoa and amaranth can be alternatives to wheat and corn as ingredients for whole grain and gluten-free products. In this study, influences of starch structures and other grain constituents on physicochemical properties and starch digestibility of whole flours made from these grains were investigated. Starches were classified into two groups according to their amylopectin branch chain-length: (i) quinoa, amaranth, wheat (shorter chains); and (ii) sorghum, millet, corn (longer chains). Such amylopectin features and amylose content contributed to the differences in thermal and pasting properties as well as starch digestibility of the flours. Non-starch constituents had additional impacts; proteins delayed starch gelatinization and pasting, especially in sorghum flours, and high levels of soluble fibre retarded starch retrogradation in wheat, quinoa and amaranth flours. Enzymatic hydrolysis of starch was restricted by the presence of associated protein matrix and enzyme inhibitors, but accelerated by endogenous amylolytic enzymes.

title: Amylolysis of large and small granules of native triticale, wheat and corn starches using a mixture of α-amylase and glucoamylase
abstract: Starch granules from different sources differ in their morphology and structure, resulting in diverse amylolysis, which is closely related to bioconversion efficiency of starch to fermentable sugars during ethanol production. In this study, large and small granules were fractionated from native triticale, wheat and corn starches using a centrifugal sedimentation protocol and were hydrolyzed by a granular starch hydrolyzing enzyme at sub-gelatinization temperatures. Native triticale starches had bimodal granule size distribution with highest sizes of 14.9–15.9, 19.1–20.2, and 7.2 μm in diameter for overall, large and small granules, respectively, when compared to those of wheat and corn starches. The large granules in triticale starches contributed higher proportion of the total granules either by number (36–45%) or by weight (93–95%) than did those of wheat and corn starches. Surface pores and internal channels viewed by SEM and CLSM were more visible in large granules than in small granules of triticale, wheat, and corn starches, but corn starch generally displayed more pores and channels than triticale and wheat starches. Large granules contained significantly higher apparent amylose content and higher relative crystallinity than did small granules (23.0–28.5% vs. 12.4–21.0% and 24.9–26.8% vs. 20.8–24.4%, respectively) in all starches. Small granules were hydrolyzed significantly faster than did large granules initially (DH 9.3–18.0%, 24.7–40.3%, 17.7% higher in small granules than in large counterparts of triticale, wheat, and corn starches, respectively, at 55 °C for 1 h), however slowed down in later stage of hydrolysis. The final DH of large and small granules were similar in triticale (82.3–84.5%) and corn (80.8–83.4%) starches except wheat starches (90.8–95.1% in large and 79.6–86.2% in small granules) at 72 h hydrolysis. SEM and CLSM further revealed that the hydrolysis pattern differed between large and small starch granules and among starches, indicating diverse structure of large and small granules existed at macro, micro, and nano levels within and between species and cultivars. The study may be beneficial for precise control of liquefaction and saccharification in simultaneous hydrolysis and fermentation process of ethanol production.

title: The susceptibility of large and small granules of waxy, normal and high-amylose genotypes of barley and corn starches toward amylolysis at sub-gelatinization temperatures
abstract: Starches from waxy (CDC Candle), normal (CDC McGwire) and high-amylose (SH 99250) genotypes of hull-less barley were isolated and fractionated into large and small granules. The unfractionated and fractionated barley starches were then characterized toward their composition, morphology and structure, and compared with commercial corn starches of similar genotypes. The effect of starch properties on degree of hydrolysis (DH) at 55 °C for 1 h followed by at 30 °C for 72 h using granular starch hydrolyzing enzymes (mixture of α-amylase and glucoamylase) was also evaluated. Morphological changes of starches before and after hydrolysis were visualized by scanning electron microscopy (SEM) and confocal laser scanning microscopy (CLSM). The proportion (% weight) of small granules in high-amylose (HA) genotypes of barley (46.5%) and corn (32.4%) starches were higher than in the waxy (WX) and normal (NM) genotypes. Apparent amylose (AM) content of corn genotypes ranged from 1 to 1.1% (WX), 21 to 26% (NM), and 66 to 70% (HA). Whereas, the corresponding ranges for barley genotypes were 4–6% (WX), 17–24% (NM), and 35–40% (HA). HA corn starch exhibited a mixed ‘A + V’-type X-ray pattern, whereas the other genotypes of corn and barley starches exhibited an ‘A’-type X-ray pattern. The relative crystallinity (RC) of the genotypes of corn and barley starches followed the order: WX > NM > HA. The AM content and RC of small granules in corn and barley starches were lower than in large granules. Regardless of genotypes and starch sources, smaller granules had a higher gelatinization temperature range (15.5–42.8 °C) and a lower enthalpy of gelatinization (9.7–21.2 J/g) than those of larger granules (11.1–41.9 °C and 13.4–23.9 J/g, respectively). Compared to large granules, the small granules were initially hydrolyzed to a greater extent (18% higher). However after 72 h, hydrolysis was 10% higher in the large granules. SEM and CLSM images revealed that the distribution of surface pores and internal channels varied between genotypes of corn and barley starches. Although small variations exist between the NM corn and NM barley starches with respect to their proximate composition and amylose content, the observed large variations in the extent of amylolysis at the initial stages of hydrolysis is indicative that the molecular architecture and granule porosity influence amylolysis.

title: Comparative structure of starches from high-amylose maize inbred lines and their hybrids
abstract: High-amylose maize starch is of interest because of its health benefits and industrial uses. Hybrid maize is widely grown for the practical and economical value of heterosis. However, starch structure of high-amylose hybrid maize has seldom been reported compared with that of high-amylose inbred maize. In this study, structure of starches from high-amylose maize inbred lines and their hybrids was investigated and compared. Starch granule became smaller in size and more spherical, oval or elongated in shape with increasing amylose content. Hybrid maize starch had lower amylose content and long branch-chain of amylopectin and higher short branch-chain and branching degree of amylopectin than inbred maize starch. Inbred maize starch had B-type crystalline structure with low relative crystallinity, but hybrid maize starch had CB-type crystalline structure and high relative crystallinity. Hybrid maize starch had lower degree of order at a short-range scale on the edge of starch granule than inbred maize starch. Hybrid maize starch had higher double helix content and lower amorphous starch than inbred maize starch. Hybrid maize starch had higher peak intensity and longer Bragg spacing of lamellar structure than inbred maize starch. Hybrid maize starch had lower gelatinization temperature and higher gelatinization enthalpy and swelling power than inbred maize starch. The different structural properties of starches had significant correlation.

title: Cotyledon cells of Vigna mungo seedlings use at least two distinct autophagic machineries for degradation of starch granules and cellular components
abstract: α-Amylase is expressed in cotyledons of germinated Vigna mungo seeds and is responsible for the degradation of starch that is stored in the starch granule (SG). Immunocytochemical analysis of the cotyledon cells with anti–α-amylase antibody showed that α-amylase is transported to protein storage vacuole (PSV) and lytic vacuole (LV), which is converted from PSV by hydrolysis of storage proteins. To observe the insertion/degradation processes of SG into/in the inside of vacuoles, ultrastructural analyses of the cotyledon cells were conducted. The results revealed that SG is inserted into LV through autophagic function of LV and subsequently degraded by vacuolar α-amylase. The autophagy for SG was structurally similar to micropexophagy detected in yeast cells. In addition to the autophagic process for SG, autophagosome-mediated autophagy for cytoplasm and mitochondria was detected in the cotyledon cells. When the embryo axes were removed from seeds and the detached cotyledons were incubated, the autophagosome-mediated autophagy was observed, but the autophagic process for the degradation of SG was not detected, suggesting that these two autophagic processes were mediated by different cellular mechanisms. The two distinct autophagic processes were thought to be involved in the breakdown of SG and cell components in the cells of germinated cotyledon.

title: Effect of cooking methods on selected physicochemical and nutritional properties of barlotto bean, chickpea, faba bean, and white kidney bean
abstract: The effects of atmospheric pressure cooking (APC) and high-pressure cooking (HPC) on the physicochemical and nutritional properties of barlotto bean, chickpea, faba bean, and white kidney bean were investigated. The hardness of the legumes cooked by APC or HPC were not statistically different (P > 0.05). APC resulted in higher percentage of seed coat splits than HPC. Both cooking methods decreased Hunter “L” value significantly (P < 0.05). The “a” and “b” values of dark-colored seeds decreased after cooking, while these values tended to increase for the light-colored seeds. The total amounts of solid lost from legume seeds were higher after HPC compared with APC. Rapidly digestible starch (RDS) percentages increased considerably after both cooking methods. High pressure cooked legumes resulted in higher levels of resistant starch (RS) but lower levels of slowly digestible starch (SDS) than the atmospheric pressure cooked legumes.

title: Physicochemical properties of high-amylose rice starches during kernel development
abstract: In this paper, endosperm starches were isolated from a high-amylose transgenic rice line (TRS) and its wild type rice Teqing (TQ) kernels at different developmental stages. TQ and TRS starches showed similar amylose contents and shapes at early developmental stage, then the amylose content increased with kernel development. The rate of increase in amylose content was much faster in TRS starches than that in TQ starches. TRS starches showed heterogenous granules at the middle and late developmental stages. TQ starch crystallinity remained A-type, but TRS starch crystallinity changed from A- to C- via CA-type. TRS starches showed higher gelatinization temperatures, lower gelatinization enthalpies, lower swelling powers, and lower hydrolysis rates at middle and late developmental stages compared with TQ starches. The amylose content had a significantly negative correlation with crystallinity, gelatinization enthalpy, swelling power, enzyme digestibility, and acid hydrolysis.