Activities of enzymes involved in starch synthesis in wheat grains differing in starch content

This work was carried out to characterize starch accumulation and activities of key enzymes during grain filling in two wheat cultivars differing in starch content. The results showed that the starch accumulation rate (SAR) and activities of sucrose synthase, ADP-glucose pyrophosphorylase, soluble starch synthase, granule-bound starch synthase, and starch branching enzyme in the cultivar with a high starch content were significantly higher than those in the cultivar with a low starch content. The simulation with Richards’ equation showed that it was average starch accumulation rate but not active starch accumulation duration that determined starch accumulation. As compared with the cultivar with a low starch content, plants of the cultivar with a high starch content maintained the higher SAR and greater activities of related enzymes during mid and late grain filling stages. Consequently, the cultivar with a high starch content had advantages over that with a low starch content in terms of the amount of starch accumulation at mid and late grain filling stages.     

Composition and Structure of Starch from Taproots of Contrasting Genotypes of Medicago sativa L

The objective of this study was to examine the composition and branch chain lengths of alfalfa (Medicago sativa L.) taproot starch during starch utilization and reaccumulation in response to defoliation. Genotypes were propagated vegetatively and well-established plants were sampled at defoliation and at weekly intervals thereafter. Starch granules from root tissues were dispersed in dimethyl sulfoxide and starch components separated using gel permeation chromatography. Root starches also were debranched enzymically, and branch chain lengths were examined. Results indicate that, irrespective of starch concentration, starch from taproots of the high starch genotype was composed of approximately 80% high molecular weight starch with I₂-Kl absorbance characteristics similar to amylopectin. The remaining 20% of the starch was low molecular weight with I₂-Kl absorbance characteristics similar to amylose. Starches of the low starch genotype contained approximately 85% high molecular weight polysaccharide at high root starch concentrations (>50 grams per kilogram). At low root starch concentrations (<10 grams per kilogram), starch from the low starch genotype had nearly equal proportions of low and high molecular weight polysaccharide. The I₂-Kl absorbance properties of the low molecular weight starches from roots of the low starch genotype indicated that some branching may be present. The distribution of chain lengths from amylopectin did not change during starch degradation and reaccumulation for the high starch genotype. In the low starch genotype, the proportion of low molecular weight branches having a degree of polymerization between 1 and 30 was decreased at the very low starch concentrations observed on the 14th day of regrowth. Higher concentrations and/or quantities of starch in roots of the high starch genotype were not associated with greater rate of herbage regrowth, when compared to the low starch genotype.     

Estimation of the amount of dietary starch available to different populations

The daily per capita availability of dietary starch was estimated for 38 selected countries using food disappearance data from the FAO provisional food balance sheets (1972-1974 average); starch availability was also estimated for Canada from 1960 to 1987 using Agriculture Canada disappearance data. Total starch availability varies fourfold among the difference countries; the availability of different sources of starch vary up to 80-fold. Wheat and roots-tubers were the major sources of starch in countries with low total starch availability. Either corn and pulses, rice and pulses, or wheat and potatoes were the major sources of starch in those countries with high total starch availability. Starch availability in Canada, both total and by source, has been relatively constant over the 27 years examined. Slight increases in total starch and starch from cereals and pulses in 1987 will need to be followed to determine whether these represent the beginning of a real change in per capita starch availability.     

Physicochemical properties and development of wheat large and small starch granules during endosperm development

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.     

Functional properties of yam bean (Pachyrhizus erosus) starch

The study was carried out in order to determine and establish the functional characters of starch extracted from yam bean (Pachyrhizus erosus (L) Urban) compared with cassava starch. Yam bean is a tropical tuber legume easily grown and holds a great potential as a new source of starch. Yam bean starch shows functional properties which are peculiar to those of most starch root crops. Gelatinization temperature (53-63 degrees C) and the pasting temperature (64.5 degrees C) are less than those of cereal starch, however, the swelling power is high (54.4 g gel/g dried starch). Yam bean starch paste presents a high viscosity profile, high retrogradation tendency and low stability on cooking. The functional properties of yam bean starch, similar to those of cassava starch, allows yam bean to be used as a potential new source of starch.     

Enhancing the expression of starch synthase class IV results in increased levels of both transitory and long-term storage starch

Starch is an important renewable raw material with an increasing number of applications. Several attempts have been made to obtain plants that produce modified versions of starch or higher starch yield. Most of the approaches designed to increase the levels of starch have focused on the increment of the amount of ADP-glucose or ATP available for starch biosynthesis. In this work, we show that the overexpression of starch synthase class IV (SSIV) increases the levels of starch accumulated in the leaves of Arabidopsis by 30%-40%. In addition, SSIV-overexpressing lines display a higher rate of growth. The increase in starch content as a consequence of enhanced SSIV expression is also observed in long-term storage starch organs such as potato tubers. Overexpression of SSIV in potato leads to increased tuber starch content on a dry weight basis and to increased yield of starch production in terms of tons of starch/hectare. These results identify SSIV as one of the regulatory steps involved in the control of the amount of starch accumulated in plastids.     

Relationship Between Isozymes of Starch Synthases and Starch Composition in Germination Pinyon Seedlings

The relation between starch synthases and starch composition in the germinating pinyon ( Pinus edulis Engelm) seedlings was studied. Using the method of 14C-glucose transferred from 14C-ADPG in the assay of starch synthases activity. Starch was extracted with 32% HC1O4, separated on glass fiber with DMSO, and assayed with the sulfuric acid-phenol method. After the emergence of radicle, starch content increased rapidly accompanied with the increase of starch grains in number and size, the increase of both soluble and granulebound starch synthase activity and the change of the pattern of Western-blot. Amylopectin was the major composition in pinyon starch, accounted for 84% of the total starch. The activity of soluble starch synthase was 1.3 times higher than that of the granule-bound starch synthase, corresponding to the ratio of amylopectin to amylose. This result supports the conventional theory that soluble starch synthase is the major enzyme responsive for the synthesis of amylopectin, and also supports that granule-bound starch synthase is functional in the synthesis of amylopectin.     

Starch Grain Distribution in Taproots of Defoliated Medicago sativa L

Defoliation of alfalfa (Medicago sativa L.) results in a cyclic pattern of starch degradation followed by reaccumulation in taproots. Characterization of changes in anatomical distribution of starch grains in taproots will aid our understanding of biochemical and physiological mechanisms involved in starch metabolism in taproots of this species. Our objectives were to determine the influence of defoliation on starch grain distribution and size variation in taproots of two alfalfa lines selected for contrasting concentrations of taproot starch. In addition, we used electron microscopy to examine the cellular environment of starch grains, and computer-based image optical analysis to determine how cross-sectional area of tissues influenced starch accumulation. Taproots of field-grown plants were sampled at defoliation and weekly thereafter over a 28-day period. Taproot segments were fixed in glutaraldehyde and prepared for either light or electron microscopy. Transverse sections were examined for number and size of starch grains and tissue areas were measured. Starch grains were located throughout bark tissues, but were confined primarily to ray parenchyma cells in wood tissues. During the first week of foliar regrowth after defoliation, starch grains in ray cells near the cambium disappeared first, while degradation of those near the center of the taproot was delayed. During the third and fourth weeks of regrowth, there was a uniform increase in number of starch grains per cell profile across the rays, but by 28 days after defoliation there were more starch grains in ray cells near the cambium than in cells near the center of the taproot (low starch line only). Bark tissues from both lines showed synchronous degradation and synthesis of starch grains that was not influenced greatly by cell location. Diameter of starch grains varied with cell location in medullary rays during rapid starch degradation, but was not influenced by cell position in bark tissues. Therefore, during foliar regrowth there is a spatial separation in starch degradation and synthesis in alfalfa taproots. Amyloplasts from alfalfa taproots contained numerous starch grains, prolamellar-, and electron-dense bodies. The high starch line had 23% more cross-sectional area as ray cells in wood tissues when compared to the low starch line, which may explain part of the difference in starch accumulation between these alfalfa lines.     

秸秆还田对旱作马铃薯块茎淀粉合成关键酶活性及基因表达的影响

为探讨秸秆还田下旱作马铃薯块茎形成过程中淀粉合成关键酶活性及基因表达特性,以马铃薯栽培品种"青薯9号"为材料,以露地栽培为对照,设置秸秆还田处理,研究了马铃薯块茎形成过程中淀粉合成关键酶活性、基因表达、淀粉糊化及累积指标。结果表明:秸秆还田显著提高了旱作马铃薯SSS酶活性,降低了AGPP、GBSS酶活性,而对SBE酶活性没有显著影响;显著提高了SSⅡ、SSⅢ基因表达量,降低了AGPase、GBSSⅠ、SBEⅠ、SBEⅡ基因表达量;显著增加了淀粉崩解值,减少了淀粉各阶段粘度、回生值,而对淀粉糊化温度没有显著影响;显著增加了直链淀粉含量及直/支链淀粉比,减少了总淀粉含量;GBSS酶活性与AGPase、SBEⅠ基因表达量呈显著正相关,与直链淀粉含量、直/支链淀粉比呈显著负相关;SBE酶活性与SSⅡ基因表达量、峰值粘度、低谷粘度、最终粘度、总淀粉含量呈显著正相关,与崩解值、糊化温度呈显著负相关;AGPase基因表达量与直链淀粉含量呈显著负相关;GBSSⅠ基因表达量与最终粘度、回生值呈显著正相关,与糊化温度呈显著负相关;淀粉糊化与累积无显著相关性。     

Starch Phosphorylation in Potato Tubers Proceeds Concurrently with de Novo Biosynthesis of Starch

The in vivo phosphorylation of starch was studied in Solanum tuberosum cv Dianella and Posmo. Small starch granules contain 25% more ester-bound phosphate per glucose residue than large starch granules. The degree of phosphorylation was found to be almost constant during tuber development. Isolated tuber discs synthesize starch from externally supplied glucose at a significant rate. Tuber discs supplied with glucose and [32P]orthophosphate incorporate radiolabeled phosphorus into the starch. The level of 32P incorporation is proportional to the amount of starch synthesized. The incorporation of 32P from orthophosphate is correlated to de novo synthesis of starch, since the incorporation of 32P is diminished upon inhibition of starch synthesis by fluoride. Based on the amount of [14C]glucose phosphate isolated after hydrolysis of purified starch from tuber discs incubated in the presence of [U-14C]glucose, approximately 0.5% of the glucose residues of the de novo-synthesized starch are phosphorylated. This value is in general agreement with the observed levels of phosphorus in starch accumulated during tuber development. Thus, the enzyme system responsible for starch phosphorylation is fully active in the isolated tuber discs, and the starch phosphorylation proceeds as an integrated part of de novo starch synthesis.     

Effect of waxy rice flour and cassava starch on freeze-thaw stability of rice starch gels

Repeatedly frozen and thawed rice starch gel affects quality. This study investigated how incorporating waxy rice flour (WF) and cassava starch (CS) in rice starch gel affects factors used to measure quality. When rice starch gels containing 0-2% WF and CS were subjected to 5 freeze-thaw cycles, both WF and CS reduced the syneresis in first few cycles. However CS was more effective in reducing syneresis than WF. The different composite arrangement of rice starch with WF or CS caused different mechanisms associated with the rice starch gel retardation of retrogradation, reduced the spongy structure and lowered syneresis. Both swollen granules of rice starch and CS caused an increase in the hardness of the unfrozen and freeze-thawed starch gel while highly swollen WF granules caused softer gels. These results suggested that WF and CS were effective in preserving quality in frozen rice starch based products.     

The binding of α-amylase to starch plays a decisive role in the initiation of storage starch degradation in turions of Spirodela polyrhiza

Degradation of reserve starch in turions, perennation organs of the duckweed Spirodela polyrhiza , is induced by continuous red light (cR). Irradiation of the turions with this light results in the autophosphorylation of starch-associated glucan water dikinase (GWD). The ensuing phosphorylation of the starch by this enzyme was proposed to result in the enhanced association of starch-degrading enzymes to the starch granules and in the initiation of starch breakdown. The present results confirm that the irradiation of dark-adapted turions with cR results in phosphorylation of the starch, accompanying changes in the capacity of the granule starch to bind turion endogenous α-amylase, as well as changes in the starch degradation level. All three effects show very similar dependence on the time of irradiation, suggesting that they may be linked. The α-amylase is a plausible candidate for effecting starch breakdown initiation. However, the increased binding capacity of the starch granules for this enzyme is insufficient to account for the initiation of the starch breakdown as this capacity is already high prior to the irradiation. The decisive effect of cR irradiation on starch degradation may lie in enabling α-amylase to gain access to otherwise sequestered starch granules or in activating α-amylase bound to the granules.     

Production of a freeze-thaw-stable potato starch by antisense inhibition of three starch synthase genes

The use of unmodified starches in frozen foods is severely limited by the undesirable textural changes that occur after freezing and thawing. Retrogradation of glucan chains leads to syneresis, a separation of the starch gel and water phases. Stabilization of the starch structure is normally achieved by chemical modification to prevent these changes from occurring. We have now created a freeze-thaw-stable potato starch by alteration of starch composition and structure by genetic modification. An amylose-free starch with short-chain amylopectin was produced by simultaneous antisense downregulation of three starch synthase genes. This starch is extremely freeze-thaw-stable and shows no syneresis even after five freeze-thaw cycles. The use of this starch has potential for environmental and consumer benefits because its production requires no chemical modification.     

Measurement of starch absorption in humans

Although starch provides a large fraction of human caloric intake, there is limited information concerning the efficiency of intestinal absorption of this nutrient. Owing to the fermentation of starch by colonic bacteria, there is no quantitative test for starch absorption comparable to the fecal fat determination. The most accurate estimation of starch absorption has been obtained by intubating the terminal ileum and aspirating ileal contents following ingestion of a meal containing starch plus a nonabsorbable marker. Starch absorption is calculated from the ratio of starch:marker in the ileal aspirate relative to the ratio in the meal. Disadvantages of the technique are the requirement for ileal intubation and the possible adverse effect of intubation on the absorptive process. A more widely used technique to assess starch absorption involves measurement of breath hydrogen (H2) excretion after ingestion of starch. Malabsorbed starch is fermented by colonic bacteria with liberation of H2 that is absorbed and excreted in expired air. This test is simple and noninvasive and can provide quantitative measurements of starch malabsorption. Application of this technique has demonstrated that 5-10% of starch in wheat, potatoes, and corn is not absorbed by healthy subjects, while rice starch is nearly completely absorbed.     

淀粉合酶的酶学与分子生物学研究进展

淀粉合酶作为淀粉合成的关键酶之一,一直是淀粉研究的重要内容,这些研究多集中在对其同工型的研究,淀粉合酶的两类主要同工型分别为淀粉粒结合的淀粉合酶和可溶性淀粉合酶,这两类同工型的作用极为复杂,本文介绍了淀粉合酶同工型的酶学和分子生物学近年来的研究进展,同时也讨论了这些同工型的分类,相互关系及其在淀粉合成过程中的生理功能等内容。     

淀粉合酶的酶学与分子生物学研究进展

淀粉合酶作为淀粉合成的关键酶之一,一直是淀粉研究的重要内容。这些研究多集中在对其同工型的研究,淀粉合酶的两类主要同工型分别为淀粉粒结合的淀粉合酶和可溶性淀粉合酶,这两类同工型的作用极为复杂。本文介绍了淀粉合酶同工型的酶学和分子生物学近年来的研究进展,同时也讨论了这些同工型的分类、相互关系及其在淀粉合成过程中的生理功能等内容。     

Association of alpha-amylase and the R1 protein with starch granules precedes the initiation of net starch degradation in turions of Spirodela polyrhiza

In turions of Spirodela polyrhiza (L.) Schleiden, net degradation of storage starch is controlled by a special low fluence response of phytochrome requiring illumination for several days. This light effect has been used to study protein-starch interactions that occur prior to and during net degradation of starch. Following various pretreatments on S. polyrhiza turions, native starch granules were isolated and two fractions of starch-related proteins were distinguished: proteins enclosed within the starch particles (starch-internalized proteins) and those attached to the surface (starch-associated proteins). The pattern of starch-associated proteins as resolved by SDS-PAGE was more complex than that of starch-internalized proteins and varied depending upon the pretreatment of the turions. Two starch associated proteins were identified immunochemically as alpha-amylase (EC 3.2.1.1) and the R1 protein (Lorberth et al. (1998) Nature Biotechnology 16: 473-477). Dark-pretreatment of non-dormant turions does not induce starch net degradation. Under these conditions, alpha-amylase and R1 were bound to the surface of the starch granules. Continuous illumination with red light induces a rapid degradation of starch. Within the first 24 h of illumination the level of starch-associated alpha-amylase transiently increased and subsequently decreased rapidly. Similarly, the amount of the starch-associated R1 also decreased during illumination. The dissociation of both alpha-amylase and R1 from the starch granules preceded the decrease in starch content. However, binding of the two proteins to starch granules remained unchanged when the turions did not perform net starch degradation (as observed during continuous darkness, orthophosphate deficiency, or dormancy of the turions). Thus, during net starch degradation, so far unidentified changes are postulated to occur at the surface of the starch particles that are relevant for protein binding. This conclusion was supported by in vitro studies in which the binding of purified beta-amylase (EC 3.2.1.2) to starch granules isolated from turions following various pretreatments was monitored. The enzyme did bind to starch granules prepared from dark-stored turions (in which starch degradation had not been initiated), but not to those isolated from illuminated (starch degrading) turions.     

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.     

萌发中食松幼苗淀粉合酶同工酶与淀粉成分的关系

利用14C－ADPG标定法测定可溶性及与淀粉粒结合的淀粉合酶活性,采用过氯酸抽提、DMSO玻璃纤维纸层析、硫酸水解法定量测定各类淀粉成分,探讨了食松（PinusedulisEngelm）幼苗生长过程中淀粉合酶与淀粉成分间的关系。结果表明,在胚根出现以后,淀粉含量迅速增加,伴随着淀粉颗粒数目和质量的增加,两类淀粉合酶活性的增加以及淀粉合酶免疫印迹图谱的变化。支链淀粉是食松淀粉的主要成分,占总淀粉的84％。可溶性淀粉合酶峰值比淀粉粒结合的淀粉合酶活性峰值高1．3倍,与支链淀粉和直链淀粉的比例相对应。结果支持食松可溶性淀粉合酶是负责支链淀粉合成的主要酶的假说,同时表明淀粉粒结合的淀粉合酶在支链淀粉的合成中也有作用。     

The influence of various small plasticisers and malto-oligosaccharides on the retrogradation of (partly) gelatinised starch

Ageing of gelatinised and partly gelatinised potato starch and wheat starch were investigated in the presence of plasticisers with increasing size and number of OH groups (ethylene glycol, glycerol, threitol, xylitol, glucose, and for potato starch also maltose). The influences of these plasticisers and of granular remnants (ghosts) on recrystallisation were determined by using X-ray diffraction. Recrystallisation of potato starch samples in the presence of plasticisers resulted in crystallinity indices of 0.5. The largest reduction in potato starch recrystallisation is found for threitol (4 OH) and xylitol (5 OH). In the plasticiser range examined, the crystallisation inducing effect of granular potato starch remnants is reduced better when the plasticiser contains more OH groups. Wheat starch recrystallises to a lesser extent than potato starch, resulting in crystallinity indices of 0.4. The results for wheat starch do not show clear trends for the influences of plasticiser size and of ghosts. The difference in behaviour of the two starches is probably caused by wheat starch having shorter amylopectin chains. Resulting from these shorter amylopectin chains, the remaining structure in wheat starch ghosts may resemble A-type crystallinity, making it more difficult to form B-type crystals. Alternatively, the trends as found for potato starch may occur, but are less manifest for wheat starch, due to the lower total extent of recrystallisation. Solid state CP/MAS NMR spectra of the wheat starch samples containing ethylene glycol were obtained, in order to compare completely and partly gelatinised systems. The spectra were identical, confirming that the ghost structures do not influence wheat starch recrystallisation. Apparently, wheat starch ghosts do not act as nuclei for crystallisation.

Similarly, the influence of various malto-oligosaccharides in combination with granular remnants (ghosts) was investigated on wheat starch ageing. Gelatinised and partly gelatinised wheat starch were plasticised with maltose, maltotriose, maltotetraose, maltopentaose or maltohexaose. This resulted in crystallinity indices of 0.2, with the largest reduction in recrystallisation for maltotriose and maltotetraose. No trend was found for the influence of ghosts. The presence of ghosts did not influence the ¹³C solid state HP/DEC NMR spectra. Less recrystallisation took place than with the previously mentioned smaller plasticisers that resulted in crystallinity indices of 0.4. The finding that maltose was able to reduce retrogradation better than glucose could be of practical importance.