Electron paramagnetic resonance study of water distribution in starch granules

An electron paramagnetic resonance (EPR) study was performed for potato and wheat starch containing Cu2+ ions as a paramagnetic probe. Distribution of water in the starch granules as well as the interactions between the copper and starch matrix of different crystalline structures were determined. EPR spectra of the native starches consisted of two different centers of Cu2+. One of them, giving at 293 and 77 K an EPR signal of axial symmetry with a well-resolved hyperfine structure (HFS), was assigned to the Cu2+ -starch complex in which Cu2+ ions strongly interacted with oxygen atoms of the starch matrix. Another Cu2+ species, exhibiting an isotropic signal at 293 K and an axial signal with resolved HFS at 77 K, was attributed to a [Cu(H2O)6]2+ complex freely rotating at room temperature and immobilized at low temperatures. Interaction of Cu2+ with the starch matrix and the relative number of the particular copper species depended on the crystallographic type of starch. Dehydration at 393 K resulted in elimination of the rotating complex signal and decrease of the total intensity of the EPR spectrum caused by clustering of the Cu2+ ions. Freezing at 77 K and thawing led to restoring of the spectrum intensity and reappearing of the signal of the [Cu(H2O)6]2+ complex. This effect, related to liberation of water molecules from the granule semicrystalline growth rings on freezing/thawing, was especially visible for wheat starch, indicating differences in the water retention ability of starch granules of different crystallographic structure.     

Tritiated starch granules

1. Surface-labelling of starch granules by tritiation seems feasible, and a technique is described that could be useful in structure determination. The impurities that are produced must be taken into account but the fact that a high polymer can be successfully tritiated seems very promising. 2. The surfaces of corn-starch granules must contain both amylose and amylopectin.     

Soluble isoforms of starch synthase and starch-branching enzyme also occur within starch granules in developing pea embryos

Developing wild-type pea embryos contain two major isoforms of starch synthase and two isoforms of starch-branching enzyme. One of the starch synthases and both starch-branching enzymes occur both in the soluble fraction and tightly bound to starch granules. The other starch synthase, which is very similar to the waxy proteins of other species, is exclusively granule-bound. It is inactive when solubilized in a native form from starch granules, but activity is recovered when the SDS-denatured protein is reconstituted from polyacrylamide gels.
Evidence is presented which indicates that all of these proteins become incorporated within the structure of the granule as it grows. It is proposed that the granule-bound waxy protein is active in vivo at the granule surface, whereas the remaining proteins are active in the soluble fraction of the amyloplast. The proteins become trapped within the granule matrix as the polymers they synthesize crystallize around them, and they probably play no further part in polymer synthesis.     

Tracking sulfur and phosphorus within single starch granules using synchrotron X-ray microfluorescence mapping

Background

Native starch accumulates as granules containing two glucose polymers: amylose and amylopectin. Phosphate (0.2–0.5%) and proteins (0.1–0.7%) are also present in some starches. Phosphate groups play a major role in starch metabolism while granule-bound starch synthase 1 (GBSS1) which represents up to 95% of the proteins bound to the granule is responsible for amylose biosynthesis.

Methods

Synchrotron micro-X-ray fluorescence (μXRF) was used for the first time for high-resolution mapping of GBSS1 and phosphate groups based on the XRF signal of sulfur (S) and phosphorus (P), respectively. Wild-type starches were studied as well as their related mutants lacking GBSS1 or starch-phosphorylating enzyme.

Results

Wild-type potato and maize starch exhibited high level of phosphorylation and high content of sulfur respectively when compared to mutant potato starch lacking glucan water dikinase (GWD) and mutant maize starch lacking GBSS1. Phosphate groups are mostly present at the periphery of wild-type potato starch granules, and spread all over the granule in the amylose-free mutant. P and S XRF were also measured within single small starch granules from Arabidopsis or Chlamydomonas not exceeding 3–5 μm in diameter.

Conclusions

Imaging GBSS1 (by S mapping) in potato starch sections showed that the antisense technique suppresses the expression of GBSS1 during biosynthesis. P mapping confirmed that amylose is mostly present in the center of the granule, which had been suggested before.

General significance

μXRF is a potentially powerful technique to analyze the minor constituents of starch and understand starch structure/properties or biosynthesis by the use of selected genetic backgrounds.     

Relationship between the distribution of the chain length of amylopectin and the crystalline structure of starch granules

The distributions of chain lengths in the amylopectins of starches from 20 species (11 A-, 6 B-, and 3 C-type) were characterised by h.p.l.c. in terms of the relationship between the molecular structure of the amylopectin and the crystalline structure of the starch granule. The weight-average chain-lengths of the amylopectins of the A-, B-, and C-type starches were in the ranges 23–29, 30–44, and 26–29, respectively. Gel-permeation chromatograms of the amylopectins debranched with isoamylase showed bimodal distributions of fractions containing long and short chains for 17 specimens (including corn, rice, potato, etc.) and trimodal distributions, of which the fraction containing short chains had twin peaks, for wheat, tapioca, and tulip amylopectins. The correlation coefficients between the average chain-lengths of amylopectins and the fractions of long and short chains and the ratio of the fractions of short and long chains by weight were 0.90, 0.69, and ?0.95, respectively. In general, amylopectin molecules of A-type starches have shorter chains in both the long- and short-chain fractions and larger amounts of the short-chain fractions than those of the B-type starches. The chain lengths of amylopectins of the C-type starches were intermediate and it is inferred that these starches possibly yield any type of crystalline structure depending on the environmental temperature and other factors, whereas the A- and B-type starches are insensitive to temperature.     

The distribution of water in native starch granules—a multinuclear NMR study

The microscopic distribution and dynamic state of water in native potato, maize and pea starch granules are investigated with NMR relaxometry and diffusometry. Besides extra-granular water, three water populations can be identified inside native potato starch granules. These are assigned to water in the amorphous growth rings; water in the semi-crystalline lamellae and “channel water”, which is located in the hexagonal channels within the B-type amylopectin crystals. The first two water populations are orientationally disordered and exchange with each other on a millisecond timescale at 290 K. NMR diffusometry shows that the water in packed granule beds is undergoing translational diffusion in a 2-dimensional space, either in thin layers between granules and/or in amorphous growth rings within the granules. The “channel water” is uniquely characterised by a 1 kHz deuterium doublet splitting and is in slow exchange with water in the other compartments on the NMR timescale. In the smaller maize granules all intra-granular water populations are in fast exchange and there is no evidence for “channel water” in the A-type crystal lattice. The NMR water proton and deuterium data for pea starch are consistent with a composite A and B-type crystal structure.     

Specific adsorption of starch oligosaccharides in the gel phase of starch granules

The gel phase of native starch-granules is penetrable by such low-molecular-weight solutes as oligosaccharides, amino acids, and salts [Lathe and Ruthven, Biochem. J., 62 (1956) 665]. Molecules larger than about 1000 daltons are effectively excluded. Starch oligosaccharides (maltotriose through maltoheptaose and perhaps higher) exhibit anomalous behavior in that they are taken up by the gel phase far in excess of the amount expected on the basis of their molecular size. Adsorption was measured by using radioactive starch oligosaccharides and counting weighed amounts of solution before and after equilibration with starch granules. The measurements were corrected for water sorption by the starch granules and for exclusion effects as ascertained by controls with nonstarch types of oligosaccharides. Maximum adsorption was observed with maltotetraose. The results indicate a specific binding between the starch oligosaccharides and molecular chains in the starch, presumably those chains in the gel phase. We suggest that these chains constitute interbranch regions of branched molecules, or segments of linear molecules in the gel or amorphous phase, the segments being of sufficient length to form a double helix or other association with the linear oligosaccharides.     

Glucan, water dikinase activity stimulates breakdown of starch granules by plastidial beta-amylases

Glucan phosphorylating enzymes are required for normal mobilization of starch in leaves of Arabidopsis (Arabidopsis thaliana) and potato (Solanum tuberosum), but mechanisms underlying this dependency are unknown. Using two different activity assays, we aimed to identify starch degrading enzymes from Arabidopsis, whose activity is affected by glucan phosphorylation. Breakdown of granular starch by a protein fraction purified from leaf extracts increased approximately 2-fold if the granules were simultaneously phosphorylated by recombinant potato glucan, water dikinase (GWD). Using matrix-assisted laser-desorption ionization mass spectrometry several putative starch-related enzymes were identified in this fraction, among them beta-AMYLASE1 (BAM1; At3g23920) and ISOAMYLASE3 (ISA3; At4g09020). Experiments using purified recombinant enzymes showed that BAM1 activity with granules similarly increased under conditions of simultaneous starch phosphorylation. Purified recombinant potato ISA3 (StISA3) did not attack the granular starch significantly with or without glucan phosphorylation. However, starch breakdown by a mixture of BAM1 and StISA3 was 2 times higher than that by BAM1 alone and was further enhanced in the presence of GWD and ATP. Similar to BAM1, maltose release from granular starch by purified recombinant BAM3 (At4g17090), another plastid-localized beta-amylase isoform, increased 2- to 3-fold if the granules were simultaneously phosphorylated by GWD. BAM activity in turn strongly stimulated the GWD-catalyzed phosphorylation. The interdependence between the activities of GWD and BAMs offers an explanation for the severe starch excess phenotype of GWD-deficient mutants.     

Three-dimensional ultrastructural analysis of RNA distribution within germinal granules of Xenopus.

The germ plasm is a specialized region of oocyte cytoplasm that contains determinants of germ cell fate. In Xenopus oocytes, the germ plasm is a part of the METRO region of mitochondrial cloud. It contains the germinal granules and a variety of coding and noncoding RNAs that include Xcat2, Xlsirts, Xdazl, DEADSouth, Xpat, Xwnt11, fatVg, B7/Fingers, C10/XFACS, and mitochondrial large and small rRNA. We analyzed the distribution of these 11 different RNAs within the various compartments of germ plasm during Xenopus oogenesis and development by using whole-mount electron microscopy in situ hybridization. Serial EM sections were used to reconstruct a three-dimensional image of germinal granule distribution within the METRO region of the cloud and the distribution of RNAs on the granules in oocytes and embryos. We found that, in the oocytes, the majority of RNAs were associated either with the precursor of germinal granules or with the germ plasm matrix. Only Xcat2, Xpat, and DEADSouth RNAs were associated with the mature germinal granules in oocytes, while only Xcat2 and Xpat were associated with germinal granules in embryos. However, Xcat2 was the only RNA that was consistently sequestered inside the germinal granules, while the others were located on the periphery. Xdazl, which functions in germ cell migration/formation, was detected on the matrix between granules. Later in development, Xcat2 mRNA was released from the germinal granules. This coincides with the timing of its translational derepression. These results demonstrate that there is a dynamic three-dimensional architecture to the germinal granules that changes during oogenesis and development. They also indicate that association of specific RNAs with the germinal granules is not a prerequisite for their serving a germ cell function; however, it may be related to their state of translational repression.     

Enzyme modification of starch granules: formation and retention of cyclomaltodextrins inside starch granules by reaction of cyclomaltodextrin glucanosyltransferase with solid granules

Cyclomaltodextrin glucanosyltransferase (CGTase) was adsorbed into starch granules and allowed to react at 37 degrees C. The reaction was conducted with the granules removed from an aqueous environment, but containing 50% w/w water inside the granule. Reaction for 20 h gave a maximum of 1.4%, w/w of cyclodextrins (CDs) inside the granule. Waxy maize and maize starches gave the highest amounts of CDs (1.3 and 1.4%, respectively), with tapioca and amylomaize-7 starches giving about 50% less (0.9 and 0.6%, respectively). Reaction of a combination of CGTase and isoamylase with solid starch granules gave a 2.6-fold increase in the formation of CDs, with a maximum yield of 3.4 and 100% retention inside waxy maize starch granules.     

Ecology of Legionella pneumophila within water distribution systems

The reservoir for hospital-acquired Legionnaires disease has been shown to be the potable water distribution system. We investigated the influence of the natural microbial population and sediment (scale and organic particulates) found in water systems as growth-promoting factors for Legionella pneumophila. Our in vitro experiments showed that: (i) water from hot-water storage tank readily supported the survival of L. pneumophila, (ii) the concentration of sediment was directly related to the survival of L. pneumophila, (iii) the presence of environmental bacteria improved the survival of L. pneumophila via nutritional symbiosis, (iv) the combination of sediment and environmental bacteria acted synergistically to improve the survival of L. pneumophila, and (v) the role of sediment in this synergistic effect was determined to be nutritional. Sediment was found to stimulate the growth of environmental microflora, which in turn stimulated the growth of L. pneumophila. These findings confirm the empiric observations of the predilection of L. pneumophila for growth in hot-water tanks and its localization to sediment. L. pneumophila occupies an ecological niche within the potable water system, with interrelationships between microflora, sediment, and temperature.     

Solubilization of starch synthetase bound to Oryza sativa starch granules

Starch synthetase was solubilized from purified starch granules of ripening grains of rice at the midmilky stage. The procedure consisted of making the granules amorphous and dispersing the amorphous starch by sonication in 75% dimethysulfoxide. A starch synthetase-amylose complex was isolated by discontinuous sucrose density gradient centrifugation, which does not require added primer and can utilize both ADP glucose and UDP glucose. A starch-free protein fraction was obtained by treatment with sodium dodecyl sulfate and β-mercaptoethanol.     

Ecology of Legionella pneumophila within water distribution systems.

The reservoir for hospital-acquired Legionnaires disease has been shown to be the potable water distribution system. We investigated the influence of the natural microbial population and sediment (scale and organic particulates) found in water systems as growth-promoting factors for Legionella pneumophila. Our in vitro experiments showed that: (i) water from hot-water storage tank readily supported the survival of L. pneumophila, (ii) the concentration of sediment was directly related to the survival of L. pneumophila, (iii) the presence of environmental bacteria improved the survival of L. pneumophila via nutritional symbiosis, (iv) the combination of sediment and environmental bacteria acted synergistically to improve the survival of L. pneumophila, and (v) the role of sediment in this synergistic effect was determined to be nutritional. Sediment was found to stimulate the growth of environmental microflora, which in turn stimulated the growth of L. pneumophila. These findings confirm the empiric observations of the predilection of L. pneumophila for growth in hot-water tanks and its localization to sediment. L. pneumophila occupies an ecological niche within the potable water system, with interrelationships between microflora, sediment, and temperature.     

Growth ring formation in the starch granules of potato tubers

Starch granules from higher plants contain alternating zones of semicrystalline and amorphous material known as growth rings. The regulation of growth ring formation is not understood. We provide several independent lines of evidence that growth ring formation in the starch granules of potato (Solanum tuberosum) tubers is not under diurnal control. Ring formation is not abolished by growth in constant conditions, and ring periodicity and appearance are relatively unaffected by a change from a 24-h to a 40-h photoperiod, and by alterations in substrate supply to the tuber that are known to affect the diurnal pattern of tuber starch synthesis. Some, but not all, of the features of ring formation are consistent with the involvement of a circadian rhythm. Such a rhythm might operate by changing the relative activities of starch-synthesizing enzymes: Growth ring formation is disrupted in tubers with reduced activity of a major isoform of starch synthase. We suggest that physical as well as biological mechanisms may contribute to the control of ring formation, and that a complex interplay of several factors may by involved.     

Enzymatic removal of zeins from the surface of maize starch granules

Alcohol-extractable, hydrophobic zein proteins contaminate starch granule surfaces and can be removed by enzymatic digestion with thermolysin. The goal of this research was to find practical alternatives to thermolysin that might be used during the corn wet-milling process. All of the commercial thermostable alkaline proteases studied (SP 709, Neutrase, and Spezyme FAN) removed the zein proteins from various types of cornstarch, as demonstrated by the lack of protein bands below 30 kDa under the reducing conditions of SDS-PAGE gel. Each enzyme removed the zein proteins as effectively as thermolysin removed them. However, the removal of the zein protein did not reduce the quantity of free fatty acids associated with the starch. Journal of Industrial Microbiology & Biotechnology (2000) 24, 71–74. Received 27 May 1999/ Accepted in revised form 01 October 1999