Characterisation of the endospermal trypsin inhibitor of barley

A trypsin inhibitor was isolated from grains of two row barley (cv. Proctor). The purified protein was identical with the corresponding inhibitor of a six row barley (cv. Pirkka); both proteins showed, a Pi of 7.4. The N-terminal amino acid was phenylalanine and an arginine residue was involved in the active site. Effects of substrate concentration showed that the inhibition was noncompetitive with a K_i of about 0.9 × 10^?7M. An enzyme-inhibitor complex was demonstrated by disc electrophoresis.     

Influence of amylopectin structure and degree of phosphorylation on the molecular composition of potato starch lintners

Morphology, molecular structure, and thermal properties of potato starch granules with low to high phosphate content were studied as an effect of mild acid hydrolysis (lintnerization) to 80% solubilization at two temperatures (25 and 45°C). Light microscopy showed that the lintners contained apparently intact granules, which disintegrated into fragments upon dehydration. Transmission electron microscopy of rehydrated lintners revealed lacy networks of smaller subunits. The molecular composition of the lintners suggested that they largely consisted of remnants of crystalline lamellae. When lintnerization was performed at 45°C, the lintners contained more of branched dextrins compared to 25°C in both low and intermediate phosphate‐containing samples. High‐phosphate‐containing starch was, however, unaffected by temperature and this was probably due to an altered amylopectin structure rather than the phosphate content. After lintnerization, the melting endotherms were broad with decreased onset and increased peak melting temperatures. The relative crystallinity was lower in lintners prepared at 45°C. A hypothesis that combines the kinetics of lintnerization with the molecular and thermal characteristics of the lintners is presented. © 2013 Wiley Periodicals, Inc. Biopolymers 101: 257–271, 2014.     

Manipulation of starch granule size distribution in potato tubers by modulation of plastid division

Starch granule size is an important parameter for starch applications in industry. Starch granules are formed in amyloplasts, which are, like chloroplasts, derived from proplastids. Division processes and associated machinery are likely to be similar for all plastids. Essential roles for FtsZ proteins in plastid division in land plants have been revealed. FtsZ forms the so-called Z ring which, together with inner and outer plastid division rings, brings about constriction of the plastid. It has been shown that modulation of the expression level of FtsZ may result in altered chloroplast size and number. To test whether FtsZ is also involved in amyloplast division and whether this, in turn, may affect the starch granule size in crop plants, FtsZ protein levels were either reduced or increased in potato. As shown previously in other plant species, decreased StFtsZ1 protein levels in leaves resulted in a decrease in the number of chloroplasts in guard cells. More interestingly, plants with increased StFtsZ1 protein levels in tubers resulted in less, but larger, starch granules. This suggests that the stoichiometry between StFtsZ1 and other components of the plastid division machinery is important for its function. Starch from these tubers also had altered pasting properties and phosphate content. The importance of our results for the starch industry is discussed.     

农艺与气候因素对青藏高原栽培大麦淀粉含量空间分布的影响

为了揭示不同环境因子对青藏高原栽培大麦籽粒淀粉含量(GSC)积累的影响程度,完善大麦GSC空间分异与环境因子的关系,明确青藏高原不同地区大麦品种GSC的环境效应,利用83个样点的地理、气候、土壤、农艺因子数据,研究了青藏高原栽培大麦GSC的分布特征,结果表明:(1)在地理水平方向上,青藏高原栽培大麦GSC的水平分布总体呈现出斑块状交错分布和南高北低的格局,并形成了以西藏拉孜、隆子、堆龙德庆、曲水、尼木、定日、萨迦、达孜、扎囊、日喀则、墨竹工卡、贡嘎、琼杰为中心的青藏高原中南部和以青海共和、贵德、海晏、同德为中心的青藏高原东北部等2个栽培大麦GSC高值区;(2)在地理垂直方向上,栽培大麦GSC的变化呈现出"S"型分布格局,即在海拔3300.0—3600.0 m以上,随着海拔的升高,栽培大麦GSC逐渐增加,在海拔4200.0 m与4500.0 m之间达到最高值,此后随着海拔的升高略有下降;(3)影响栽培大麦GSC的因子从大到小的顺序是穗密度﹥6月平均气温日较差芒长﹥9月平均气温1月平均气温年日照时数﹥≥0℃积温5月平均气温﹥8月平均气温日较差8月平均气温6月平均气温≥10℃积温6月平均月降水量5月平均月降水量7月平均相对湿度8月平均相对湿度7月平均气温。这一研究结果显示,对栽培大麦GSC影响最大的是基因型,其次是气候因素,土壤因素对GSC的影响不明显。影响栽培大麦GSC的农艺因子主要是穗密度和芒长,气候因子主要是拔节抽穗期的气温日较差和籽粒灌浆成熟期的平均气温,日照和降水的影响相对较小。     

小麦花后弱光引起籽粒淀粉的粒度分布及组分含量的变化

在籽粒灌浆阶段(花后1～30 d)对小麦进行光强为自然光照45%的弱光处理,研究了小麦籽粒淀粉粒度分布和组分含量的变化.结果表明,小麦籽粒淀粉粒体积分布呈双峰曲线,峰值分别在5.1～6.1 μm和20.7～24.9 μm,两峰值间的低谷出现在9.9 μm左右.表面积分布和数目分布分别表现为双峰和单峰曲线.小麦花后弱光显著降低2.8～9.9 μm淀粉粒体积百分比,增加22.8～42.8 μm淀粉粒体积百分比.同时花后弱光显著降低<0.8 μm和2.8～9.8 μm淀粉粒表面积百分比,增加0.8～2.8 μm和>9.9 μm淀粉粒表面积百分比.可见灌浆期弱光显著降低籽粒B型(＜9.9 μm)淀粉粒体积和表面积百分比,而A型(＞9.9 μm)淀粉粒比例相对增加.与A型淀粉粒相比,B型淀粉粒对弱光的反映更敏感.小麦弱光处理籽粒淀粉及其组分含量显著低于对照,但其直/支比较对照高.相关分析表明,籽粒直/支比与2.8～9.9 μm淀粉粒体积百分比呈显著负相关,而与22.8～42.8 μm淀粉粒体积百分比呈显著正相关.花后不同阶段弱光显著增加A型淀粉粒体积百分比、降低B型淀粉粒体积百分比,其中灌浆中、后期弱光影响程度较前期大.表明,弱光条件下小麦籽粒淀粉合成底物优先供应淀粉粒的生长,而非形成更多的淀粉粒.     

不同供水条件对小麦强、弱势籽粒中淀粉粒度分布的影响

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

青藏高原栽培青稞淀粉粒蛋白多态性及其与淀粉含量的关系

淀粉粒蛋白(Starch granule proteins, SGPs)是一类与淀粉粒结合的微量蛋白, 其变异可能与淀粉特性相关。实验率先研究了我国青藏高原栽培青稞的SGP组成, 初步探索了所分离的SGP蛋白与淀粉合成的关系。青藏高原青稞的SGP组成存在多态性, 66份青稞材料中分离了10种主要的SGPs, 16种组合带谱。西藏青稞和四川青稞的SGP组成有很大差异, SGP组成具有地域差异性。不同组合带谱材料间淀粉含量差异显著性检验显示, 实验所分离的淀粉粒蛋白可能与淀粉的合成相关。     

Antisense inhibition of isoamylase alters the structure of amylopectin and the physicochemical properties of starch in rice endosperm

This is the first report on regulation of the isoamylase1 gene to modify the structure of amylopectin and properties of starch by using antisense technology in plants. The reduction of isoamylase1 protein by about 94% in rice endosperm changed amylopectin into a water-insoluble modified amylopectin and a water-soluble polyglucan (WSP). As compared with wild-type amylopectin, the modified amylopectin had more short chains with a degree of polymerization of 5-12, while their molecular sizes were similar. The WSP, which structurally resembled the phytoglycogen in isoamylase-deficient sugary-1 mutants, accounted for about 16% of the total alpha-polyglucans in antisense endosperm, and it was distributed throughout the whole endosperm unlike in sugary-1 mutant. The reduction of isoamylase activity markedly lowered the gelatinization temperature from 54 to 43 degrees C and the viscosity, and modified X-ray diffraction pattern and the granule morphology of the starch. The activity of pullulanase, the other type of starch debranching enzyme, in the antisense endosperm was similar to that in wild-type, whereas it is deficient in sugary-1 mutants. These results indicate that the isoamylase1 is essential for amylopectin biosynthesis in rice endosperm, and that alteration of the isoamylase activity is an effective means to modify the physicochemical properties and granular structure of starch.     

Effect of granule size on the interference colors of starch in polarized light

Abstract

A Nikitin-Berek compensator tilted at 5.5° in a polarizing microscope was used to create a background second-order blue interference color against which starch granules were examined. A grating monochromator showed the first interference minimum of the background was at 590 nm. Starch granules have a radial molecular structure. Thus, some radii were in line with the axis of the compensator while others were across the compensator axis. Where radial birefringence counteracted the background birefringence, starch granules had two quadrants with a bright yellow first-order interference color. Where radial birefringence added to the background birefringence, there were two quadrants of second-order blue (higher than the background). In yellow quadrants where birefringence was reduced, the wavelength of the first interference minimum was reduced. In blue quadrants where birefringence was increased, the wavelength of the first interference minimum was increased. The extent to which the interference minimum of the background birefringence was shifted by starch granules was strongly dependent on the size of the starch granules. For yellow quadrants, the shifts were: r = ?0.87, P < 0.001, n = 22 for corn starch; r = ? 0.94, P <0.001, n = 22 for tapioca starch; and r = ?0.94, P <0.001, n = 12 for potato starch. For blue quadrants, the shifts were: r = 0.80, P < 0.001, n = 22 for corn; r = 0.81, P < 0.001, n = 22 for tapioca; and r = 0.93, P < 0.001, n = 16 for potato. When interference colors are used to evaluate starch granules, the granules should be similar in size or a correction must be made for granule size, and the Michel-Lévy chart of interference colors may be used to collect data subjectively.     

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.     

Effects of the activities of key enzymes involved in starch biosynthesis on the fine structure of amylopectin in developing rice (Oryza sativa L.) endosperms

The dynamic changes of the activities of enzymes involving in starch biosynthesis, including ADP-glucose pyrophosphorylase (AGPase), soluble starch synthases (SSS), starch branching enzyme (SBE) and starch debranching enzymes (DBE) were studied, and changes of fine structure of amy- lopectin were characterized by isoamylase treatment during rice grain development, using trans anti-waxy gene rice plants. The relationships between the activities of those key enzymes were also analyzed. The amylose synthesis was significantly inhibited in transgenic Wanjing 9522, but the total starch content and final grain weight were less affected as compared with those of non-transgenic Wanjing 9522 rice cultivar. Analyses on the changes of activities of enzymes involving in starch bio- synthesis showed that different enzyme activities were expressed differently during rice endosperm development. Soluble starch synthase is relatively highly expressed in earlier stage of endosperm de- velopment, whilst maximal expression of granule-bound starch synthase (GBSS) occurred in mid-stage of endosperm development. No obvious differences in changes of the activities of AGPase and SBE between two rice cultivars investigated, except the DBEs. Distribution patterns of branches of amy- lopectin changed continually during the development of rice grains and varied between two rice culti- vars. It was suggested that amylopectin synthesis be prior to the synthesis of amylose and different enzymes have different roles in controlling syntheses of branches of amylopectin.     

灌溉与旱作条件下源库关系对小麦籽粒淀粉粒度分布的调节效应

在灌溉和旱作两种栽培条件下,研究了源库关系对小麦籽粒淀粉粒度分布特征的影响.结果表明,山农8355(大穗型)各处理A型淀粉粒体积分布、表面积分布百分比成熟期较灌浆中期明显提高,灌溉栽培条件下增幅分别在17.65%～22.88%、35.8%～39.05%,旱作栽培条件下增幅分别在1.46%～2.82%、7.05%～8.12%;山农8355各处理B型淀粉粒体积分布、表面积分布百分比成熟期较灌浆中期明显降低,灌溉栽培条件下降幅分别在34.78%～40.47%、11.73%～13.77%,旱作栽培条件下降幅分别在5.08%～7.67%、2.52%～3.43%.济南17(多穗型)各处理下成熟期与灌浆中期的A、B淀粉粒体积分布、表面积分布百分比,其变化趋势与山农8355相同,其中A型淀粉粒灌溉栽培条件下增幅分别在1.56%～5.98%、2.96%～9.92%,旱作栽培条件下增幅分别在1.76%～4.52%、1.28%～8.63%;B型淀粉粒灌溉栽培条件下降幅分别在3.46%～12.27%、1 02%～4.18%,旱作栽培条件下增幅分别在5.31%～9.87%、0.58%～3.13%.在灌溉和旱作栽培条件下源库调节对两品种A、B型淀粉粒粒度分布的影响趋势表现为,减源处理A型淀粉粒较同期同品种对照处理的体积分布、表面积分布百分比显著提高,减库处理较同期同品种对照处理显著降低,B型淀粉粒粒度分布变化趋势则与之相反.     

Changes in structure of starch and enzyme activities affected by sugary mutations in developing rice endosperm. Possible role of starch debranching enzyme (R-enzyme) in amylopectin biosynthesis

In maturing endosperms of a variety of sugary mutants of rice, phytoglycogen-like polysaccharides with highly branched a -glucans were accumulated instead of amylopectin. while the amylose content greatly decreased. Measurement of activities per endosperm of the 10 major enzymes involved in starch and sucrose metabolism revealed that the activity of starch debranching enzyme (R-enzyme) was specifically reduced in the sugary mutants. The activity of starch branching enzyme I (Q-enzyme I) was also significantly decreased, but less so than the R-enzyme, in the mutants, suggesting some coordination of the expression of the genes coding for R-enzyme and Q-enzyme I. Western blot analysis showed that the sugary mutations of rice resulted in a decrease in the amount of R-enzyme protein, but not in major modification of the enzyme. These findings strongly suggest that R-enzyme plays a critical role in determining the amylopectin fine structure, since at the extremely low level of R-enzyme activity as compared with Q-enzyme activity, as found in sugary mutants, the rice endosperm produced phytoglycogen. We hypothesize that balance of activities or interaction between Q-enzyme and R-enzyme may be responsible for the fine structure of a -polyglucans in plant tissues.     

Influence of sucrose on the rheology and granule size of cross-linked waxy maize starch dispersions heated at two temperatures

Cross-linked waxy maize (CWM) starch dispersions (STDs) of concentration 50 g kg⁻¹ were heated in sucrose solutions containing 0–600 g kg⁻¹ (g sucrose/kg dispersion) at 85 °C at low shear and in intermittently agitated cans at 110 °C. The STDs heated in 0–300 g kg⁻¹ sucrose exhibited antithixotropic behavior, while those heated in 400–600 g kg⁻¹ sucrose exhibited thixotropic behavior. The mean starch granule diameter of the starch dispersions did not show strong dependence on sucrose concentration. The dispersions, especially those with high sucrose concentrations and heated at 110 °C, exhibited G′ versus frequency (ω) profiles of gels. The STDs exhibited first normal stress differences that increased in magnitude with the concentration of sucrose. Values of the first normal stress coefficient of canned dispersions calculated from dynamic rheological data plotted against ω and experimental values plotted against shear rate of some of the STDs overlapped.     

A sucrose non‐fermenting‐1‐related protein kinase‐1 gene,IbSnRK1, improves starch content,composition, granule size,degree of crystallinity and gelatinization in transgenic sweet potato

Sucrose non‐fermenting‐1‐related protein kinase‐1 (SnRK1) is an essential energy‐sensing regulator and plays a key role in the global control of carbohydrate metabolism. The SnRK1 gene has been found to increase starch accumulation in several plant species. However, its roles in improving starch quality have not been reported to date. In this study, we found that the IbSnRK1 gene was highly expressed in the storage roots of sweet potato and strongly induced by exogenous sucrose. Its expression followed the circandian rhythm. Its overexpression not only increased starch content, but also decreased proportion of amylose, enlarged granule size and improved degree of crystallinity and gelatinization in transgenic sweet potato, which revealed, for the first time, the important roles of SnRK1 in improving starch quality of plants. The genes involved in starch biosynthesis pathway were systematically up‐regulated, and the content of ADP‐glucose as an important precursor for starch biosynthesis and the activities of key enzymes were significantly increased in transgenic sweet potato. These findings indicate that IbSnRK1 improves starch content and quality through systematical up‐regulation of the genes and the increase in key enzyme activities involved in starch biosynthesis pathway in transgenic sweet potato. This gene has the potential to improve starch content and quality in sweet potato and other plants.     

Influence of cross-linked potato starch treated with POCl3 on DSC, rheological properties and granule size

Differential scanning calorimetry (DSC), rheological measurements and granule size analyses were performed to characterize the influence of phosphorylation substitution levels on the properties of cross-linked potato starch. Phosphorus oxychloride (POCl₃) was used to produce the cross-linked potato starch. The levels of the reagent used for the reaction ranged between 40 and 5000 ppm (dwb). Storage (G′) and loss (G″) moduli were measured for a 5% (w/w) gelatinized starch dispersion stored at 20 °C for 24 h after heating at 85 °C for 30 min. The samples from 80 to 500 ppm were recognized as ‘strong gel'systems, whereas native potato starch showed ‘weak gel'behavior. Steady shear and dynamic viscoelastic properties of gelatinized starch dispersion were compared. Furthermore, granule mean diameter was measured by laser scattering for a 1% (w/w) dispersion heated at 85 °C for 30 min. The granules in the 100 ppm sample swelled to a maximum of about 2.6 times the native starch granule mean diameter.     

Studies on the distribution, re-translocation and homeostasis of inorganic phosphate in barley leaves

Changes in inorganic phosphate (P_i) concentrations in barley leaves during growth of plants with sufficient or deficient supplies of P_i were studied. Measurements of the P_i distribution from subcellular levels to the leaf tissue level under the same experimental conditions allowed us to analyse the relationship between the P_i homeostasis of various compartments and P_i re-translocation in the whole plant. Under P_i deficiency, the finding of growth-dependent changes in the P_i concentrations of whole leaves established that P_i was re-translocated from the older leaves to the young leaves. Translocation of ³²P_i was also confirmed with an ‘imaging plate’ system, which made it possible to follow P_i movement in the same plantlet. To analyse the mechanism of P_i re-translocation, the P_i distribution amongst various compartments of the leaves was measured. Under P_i deficiency, the cytoplasmic P_i concentration of the first leaf remained constant until 16d after sowing, while vacuolar P_i was completely exhausted after 8 to 10d. Exhaustion of vacuolar P_i in the first leaf coincided with the appearance of the second leaf. The P_i concentration in the apoplast changed similarly to that of the whole leaf. However, the apoplastic P_i concentration was affected to some extent by the vacuolar P_i concentration and the growth of the younger leaf, because the main change in apoplastic P_i concentration coincided with the time of the disappearance of the vacuolar P_i and the appearance of the younger leaf. The P_i concentration in the apoplast was about 0.1 to I molm^?3, even in the absence of P_i, which was much higher than that in the usual soil environment (a few mmolin^?3). This suggests that the P_i absorbed by root cells is concentrated in the transport process from the root to the leaf apoplast. The content of P_i in the xylem exudate was constant irrespective of growth culture conditions. The root may be functioning as the constant P_i supplier to the above tissues.