Characterization of free amino acid QTLs in maize opaque2 recombinant inbred lines

The opaque2 (o2) mutation in maize (Zea mays L.) increases the content of free amino acids (FAA) in the endosperm. We investigated the basis of this trait by using recombinant inbred lines from a cross of Oh545o2 (high FAA) and Oh51Ao2 (low FAA) to identify quantitative trait loci (QTL) for FAA content and to determine their effect on FAA composition and protein accumulation. Mapping identified six QTLs that accounted for 71% of the phenotypic variation. Two QTLs in bins 4.01 and 7.02 are close to α-zein genes; high FAA individuals with these QTLs had reduced accumulation of α-zein 19 kDa isoforms and increased FAA abundant in α-zeins. A QTL in bin 3.03 is close to a gene encoding triose phosphate isomerase (tpi4) and a higher expression of this enzyme was found in high FAA individuals. Other differentially expressed proteins included vicilin-like globulins and the enzymes glyceraldehyde-3-phosphate dehydrogenase, enolase-2, sorbitol dehydrogenase and granule-bound starch synthase. The results suggest that the increased levels of FAA in o2 endosperm are mainly due to the reduction of storage proteins and the failure to incorporate their amino acids into other proteins, as well as the alteration of carbohydrate metabolism that may favor amino acid biosynthesis.     

Transition from vitreous to floury endosperm in maize (Zea mays L.) kernels is related to protein and starch gradients

Relationships between kernel vitreousness and proteins and starch partitioning to the floury and vitreous regions of the endosperm were monitored in a set of 13 maize inbred lines. Decrease of protein contents from the vitreous to the floury endosperms were mainly assigned to α-zeins. Using Raman microspectroscopy, we observed a protein gradient from the periphery to the center of endosperms that well fitted with the inverse relationships between vitreousness and protein content of the vitreous and floury regions. In addition, Raman microspectroscopy highlighted an increase of starch crystallinity from the periphery to the center of the maize endosperms. This agrees with the higher amylose and associated lipid contents within starches of vitreous than in those of floury endosperms. Finally, starch granules from vitreous regions displayed more channels than the floury ones. These channels contain proteins that might favor adhesion of proteins to starch granules or granule–granule contacts to form the close packing of the vitreous endosperm. Therefore transition from vitreous to floury endosperm is at least the result of both protein and starch gradients. These gradients are probably associated with metabolic gradients that have been observed during endosperm development.     

Effect of Grain Structure and Cooking on Sorghum and Maize in vitro Protein Digestibility

Uncooked and cooked sorghum showed improvement in in vitro protein digestibility as the structural complexity of the sample reduced from whole grain flour through endosperm flour to protein body-enriched samples. This was not the case for maize. Cooking reduced protein digestibility of sorghum but not maize. Treating cooked sorghum and maize whole grain and endosperm flours with alpha -amylase to reduce sample complexity before in vitro pepsin digestion slightly improved protein digestibility. The reduction in sorghum protein digestibility on cooking was not related to the total polyphenol content of samples. Pericarp components, germ, endosperm cell walls, and gelatinised starch were identified as possible factors limiting sorghum protein digestibility. Electrophoresis of uncooked and cooked protein-body-enriched samples of sorghum and maize, and prolamin fractions of sorghum under non-reducing conditions showed oligomeric proteins with molecular weights (M_r) 45, 66 and >66 kDa and monomeric kafirins and zeins. Protein-body-enriched samples of sorghum had more 45–50 kDa oligomers than those of maize. In cooked sorghum, some of these were resistant to reduction. Pepsin-indigestible residues from protein-body-enriched samples consisted mainly of α-zein (uncooked and cooked maize) or α-kafirin (uncooked sorghum), whilst cooked sorghum had in addition, β- and γ-kafirin and reduction-resistant 45–50 kDa oligomers. Cooking appears to lead to formation of disulphide-bonded oligomeric proteins that occurs to a greater extent in sorghum than in maize. This may explain the poorer protein digestibility of cooked sorghum.     

Effect of pH and ethanol content of solvent on rheology of zein solutions

Zein, a corn protein, is a mixture of the polypeptides α-, γ-, β-, and δ-zein. α-Zein and γ-zein comprise 70–85% and 10–20% of total zein mass, respectively. Both peptides have similar amino acid composition, except γ-zein is rich in cysteine. The presence of cysteine has been associated with gelation of zein solutions. A common solvent for zein is aqueous ethanol. Preliminary results suggested that pH and ethanol content affect the rheology of zein solutions. Our objective was to investigate the effect of ethanol content (65–90%) and pH of the solvent (2, 6, and 12) on rheological properties of zein solutions (20% w/w) containing γ-zein. Steady shear tests and oscillatory time sweeps were performed to determine flow behavior and gelation time of zein solutions. Results indicated that α-zein solutions were nearly Newtonian while those containing γ-zein showed shear thinning behavior. At high pH, γ-zein increased the consistency index (K) and shortened gelation time. Results were attributed to the cysteine in γ-zein. High pH promoted formation of disulfide bonds leading to higher K values and shorter gelation times. Results of this work are expected to be useful in the design of zein extraction processes and the development of new zein applications.     

Effects of endosperm texture and cooking conditions on the in vitro starch digestibility of sorghum and maize flours

The effects of endosperm vitreousness, cooking time and temperature on sorghum and maize starch digestion in vitro were studied using floury and vitreous endosperm flours. Starch digestion was significantly higher in floury sorghum endosperm than vitreous endosperm, but similar floury and vitreous endosperm of maize. Cooking with 2-mercaptoethanol increased starch digestion in both sorghum and maize, but more with sorghum, and more with vitreous endosperm flours. Increasing cooking time progressively reduced starch digestion in vitreous sorghum endosperm but improved digestibility in the other flours. Pressure-cooking increased starch digestion in all flours, but markedly more in vitreous sorghum flour; probably through physical disruption of the protein matrix enveloping the starch. Irrespective of vitreousness or cooking condition, the alpha-amylase kinetic constant (k) for both sorghum and maize flours remained similar, indicating that differences in their starch digestion were due to factors extrinsic to the starches. SDS-PAGE indicated that the higher proportion of disulphide bond-cross-linked prolamin proteins and more extensive polymerisation of the prolamins on cooking, resulting in polymers of M_r>100k, were responsible for the lower starch digestibility of the vitreous sorghum endosperm flour.     

Quantification and distribution of kafirins in the kernels of sorghum cultivars varying in endosperm hardness

Kafirins extracted from the endosperm of seven sorghum [Sorghum bicolor (L.) Moench] cultivars were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and enzyme-linked immunosorbent assay (ELISA). Kafirins extracted from the vitreous and floury endosperm portions within the kernel were also analysed by these techniques. The ELISA results indicated that the level of all the three kafirins was high in the hard endosperm kernels. The level of γ-kafirin was particularly higher in the vitreous endosperm portions of these kernels. The ratio of γ-kafirin to the α-kafirin was, however, higher for the floury portions of soft kernels. Tissue print immunoblotting revealed that the β- and γ-kafirins were concentrated in the central floury endosperm portions of soft kernels, whilst α-kafirin was distributed more uniformly throughout the endosperm. In contrast, all three kafirins were distributed uniformly throughout the endosperms of hard kernels. The data indicate that the content, as well as the distribution, of kafirins within the kernel is different in grains varying in endosperm hardness.     

基于元分析的抗玉米灰斑病QTL比较定位

以玉米遗传连锁图谱IBM2 2008 Neighbors为参考图谱,整合65个抗玉米灰斑病QTL,构建QTL综合图谱。采用元分析方法优化65个QTL,获得11个"一致性"QTL区间,分别位于染色体bin区的1.05、1.06、2.03、2.07、3.02、4.05、5.03、5.05、7.02、8.07、9.03位置,其在遗传连锁图谱上对应的位置分别为442.21、528.27、228.10、478.00、74.65、311.59、169.62、302.35、252.19、422.70、257.93 cM。对两个具有较多报道和较高表型贡献的"一致性"QTL区间bin1.05和bin1.06,从MaizeGDB网站搜索得到324个基因。因抗病基因在结构上具有高度保守性,将324个基因分别与水稻和拟南芥基因组进行同源比对,在bin1.05和bin1.06内分别确定了7个和3个基因作为玉米抗灰斑病候选基因。     

Mapping Quantitative Trait Loci for Starch Granule Traits in Barley

Starch accumulates in barley (Hordeum vulgareL.) endosperm in large (type A) and small (type B) granules. The sizes, shapes and relative proportions of A and B granules may affect the quality of barley malt for brewing. The objective of this study was to use genetic markers to map quantitative trait loci (QTL) affecting starch granule traits in a cross between a malting barley cultivar, Morex, and a feed barley cultivar, Steptoe. Data on starch granule dimensions were obtained using digital image analysis. With simple interval mapping, a region of chromosome 2 (2H) was found to contain QTL affecting the overall mean granule volume, the proportion of A granules, the mean volume of A granules, the mean maximum diameter of A granules and the mean F-shape of B granules. This region also affected days to heading and plant height, but contained no QTL for grain or malt quality traits. With composite interval mapping, QTL affecting starch granule traits were detected in two additional regions, one on chromosome 4 (4H) affecting the mean F-shape of B granules and one on chromosome 7 (5H) affecting the mean maximum diameter of A granules.     

Biochemical Basis of Kernel Milling Characteristics and Endosperm Vitreousness of Maize

Chemical characteristics of 18 normal maize grain samples, having a wide range of physical properties (endosperm vitreousness, kernel friability and milling characteristics), were analysed and related to physical properties. Measurement of damaged starch showed that starch behaved as a passive filler in endosperm. Endosperm protein content and class, as determined from extractability properties and reversed-phase high-performance liquid chromatography, were correlated with kernel physical properties. Endosperm vitreousness and kernel mechanical properties (kernel friability and milling characteristics) were related to different components, however. Vitreousness seems to be linked to the proportion (%) of the two γ-zein fractions, whereas friability increased when α-zein content decreased and when salt extractable protein content increased.     

The composition of floury and vitreous endosperm affects starch digestibility kinetics of the whole maize kernel

Maize grain starch is the major energy source in animal nutrition, and its high digestion and utilization largely depend on endosperm traits and the structure of the starch-lipoprotein matrix. The aim of this work was to determine floury and vitreous endosperm traits and its relation to starch digestibility rate. In total, kernels of 30 hybrids were manually dissected, and amylose, total zein and starch and non-starch lipids were determined in both vitreous and floury endosperm. Starch digestibility of the whole kernel was determined based on glucose released during a two-step in vitro pig model of enzymatic digestion, and starch digestibility rate was calculated according to the first-order kinetics. The vitreous endosperm of tested hybrids had higher contents of amylose (204.6 vs 190.4 g/kg), zein (63.2 vs 40.4 k/kg) and starch lipids (5.6 vs 4.9 g/kg), and lower content of non-starch lipids (7.3 vs 9.6 g/kg) than floury endosperm. Digestibility coefficients varied among hybrids, and starch digestibility rate varied from 0.73 to 1.63 1/h. Lipids in both vitreous and floury endosperm negatively correlated with the most of digestion coefficients, whereas zein correlated in vitreous and amylose in the floury endosperm (P < 0.05). Starch digestibility rate negatively correlated with all traits, except amylose content in vitreous endosperm. As a result, a linear regression model with four variables including contents of zein and starch lipids in vitreous and zein and amylose in floury endosperm can predict more than 65% variability of starch digestibility rate of tested hybrids.     

Protein quality and endosperm modification of quality protein maize (Zea mays L.) under two contrasting soil nitrogen environments

Quality protein maize (QPM) breeding involves the combined use of the opaque-2 (o2) gene and the genetic modifiers of the o2 locus to develop cultivars with modified kernel endosperm, and increased concentrations of lysine and tryptophan. This study was designed to assess grain yield performance, endosperm modification, and protein quality and quantity under two contrasting soil nitrogen environments. A 15-parent diallel cross was evaluated under one low nitrogen stress and one optimal nitrogen environment each at Harare (Zimbabwe) and Bako (Ethiopia). Most QPM hybrids showed higher protein quality levels than the best non-QPM check under both conditions. Protein concentration tended to vary across nitrogen levels, but not endosperm type. Significant differences were found for the test of main effect (nitrogen-level) for endosperm modification and tryptophan concentration. This indicated that QPM maintains quality even under low soil nitrogen, a widespread condition in Africa. General combining ability (GCA) mean squares were highly significant for most protein quality traits for each environment and across environments whereas specific combining ability (SCA) mean squares were not significant in most cases. This indicated that additive gene effects were primarily responsible for variation of most traits evaluated and hence progeny performance can adequately be predicted on the basis of parental performance. Inbred lines P2, P4 and P12 had desirable GCA effects for endosperm modification while P1 and P3 had the best GCA for tryptophan concentration in grain. The current study suggests that hybrids with desirable endosperm modification, protein quality and stable performance under low nitrogen stress and optimal conditions can be produced with careful selection.     

西藏冬青稞胚乳淀粉粒发育特征研究

为了解青稞胚乳淀粉及淀粉粒形成与生长特征,以西藏育成青稞品种冬青8号为材料,对籽粒发育过程中胚乳灌浆速度、淀粉积累量及淀粉粒的产生、二维面积、粒径变化进行了分析。结果表明,胚乳发育过程中,干物质和淀粉积累量呈S型曲线,两者呈正相关。花后5d,青稞胚乳中可零星见到粒径和二维面积较小的A型小淀粉粒;花后10~15d,A型小淀粉粒数量显著增大,但粒径和二维面积增加较慢;花后20d,B型小淀粉粒可能已经形成,与A型小淀粉粒组成一个复合群体;花后25d,形成了粒径10μm的A型淀粉粒、粒径5~10μm的B型淀粉粒和粒径5μm的C型淀粉粒;花后30~55d,不同粒径的淀粉粒数量和二维面积逐渐增加,其中,C型淀粉粒数量不断增加直至籽粒成熟。青稞成熟籽粒胚乳中,淀粉粒的粒径为1.45~44.76μm,二维面积为3.56~1 750.8μm2;A型淀粉粒形状为近圆形,表面光滑,个别表面附着有C型淀粉粒,B型淀粉粒形状有近圆形、椭圆形多种类型,C型淀粉粒形状比B型淀粉粒的变异更大。     

Starch granule morphology in oat endosperm

Mature and developing oat (Avena sativa) grains were sectioned and image analysis methods used to estimate the starch granule-size distribution and morphology in endosperm cells. This showed that oat endosperm cells contain two types of starch granule: compound starch granules such as those seen in rice endosperm and in most other grasses; and simple granules similar to the B-type starch granules seen in the endosperm of Triticeae species such as wheat (Triticum aestivum). The simple granules in oats are similar in size and relative abundance to B-type granules in Triticeae suggesting that they may share a common evolutionary origin. However, there is a fundamental difference between oats and Triticeae in the timing of granule initiation during grain development. In Triticeae, the B-type granules initiate several days after the A-type granules whereas in oats, both the simple and compound granule types initiate at the same time, in early grain development.     

Two fraction extraction of α-zein from DDGS and its characterization

Zein was recovered from corn distiller's dried grains with solubles (DDGS) by a modified method using 70% (w/w) aqueous 2-propanol (70-IPA) or 70% (v/v) aqueous ethanol (70-EtOH) solvents, and a commercial method using 88% (w/w) aqueous 2-propanol (88-IPA). Yield, purity, and film properties of the isolated zein were determined. The modified procedure extracted two fractions of zeins: a mostly α-zein fraction, and a mostly γ-zein fraction. The modified method increased α-zein yield from 4% to 14%. Enzyme cellulase pretreatment did not improve zein yield, but grinding did. The α-zein fraction showed electrophoretic bands at 40, 22, 19, and 10 kDa, corresponding to α-zein dimer, α₁-zein, α₂-zein, and δ-zein, respectively. The α-zein of DDGS retained its film forming capability. The α-zein film of unmodified DDGS was cloudy and rough, unlike the clear and smooth films of α-zeins isolated from corn gluten meal and enzyme-treated DDGS.     

Editing of Rice Endosperm Plastidial Phosphorylase Gene OsPho1 Advances Its Function in Starch Synthesis

正Os Pho1 in Zhonghua 11(ZH11) was edited using the clustered regularly interspaced short palindromic repeatsassociated endonuclease 9(CRISPR/Cas9) system. Two homozygous T1 mutants(cr-pho1-34 and cr-pho1-37) displayed a chalky endosperm with a white core, which significantly decreased 1000-grain weight.     

The effects of puroindoline b on the ultrastructure of endosperm cells and physicochemical properties of transgenic rice plant

Endosperm texture is an important factor governing the end-product quality of cereals. The texture of wheat (Triticum aestivum L.) endosperm is controlled by puroindoline a and b genes which are both absent in rice (Oryza sativa L.). It has been reported that the endosperm texture of rice can be modified by puroindoline genes. The mechanism, however, by which puroindolines affect the ultrastructure of rice endosperm cells remains to be investigated. In this study, we observed the ultrastructure of endosperm cells and the morphology of isolated starch granules of the transgenic rice expressing the puroindoline b gene. SEM and TEM observations indicated that compound starch granules were embedded within the matrix material in non-transgenic rice, Nipponbare, whereas they were surrounded by spaces in the transgenic rice. The morphology and size of each starch granule were not different between non-transgenic and the transgenic rice. However, the transgenic rice flour showed smaller particle size, higher starch damage, and lower viscosity during gelatinization than that of non-transgenic rice. These results confirm that puroindoline b reduces the grain hardness in rice. Moreover, the results also suggest that puroindoline b functions at the surface of compound starch granules, and not on polygonal starch granules in rice endosperm.     

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.     

Effect of High Temperature at Ripening Stage on the Reserve Accumulation in Seed in Some Rice Cultivars

Abstract

The resistance to high-temperature stress and the structural appearance of the imperfect grains caused by a high temperature at the ripening stage were studied using 13 selected cultivars of rice. High temperature treatment (daily maximum temperature range, 32-40°C) given from the 4th day after heading caused the decrease in panicle weight in all of the cultivars examined. The number of empty grains in the upper and lower parts of a panicle was increased by the high temperature in 10 cultivars. Cultivars KRN, Citanduy, Belle patna and BPB were tolerant to the high-temperature treatment at the ripening stage, and cvs. Koshihikari, Sanlicun, Tainung 67, Yamada-nishiki and Lady Wright were sensitive. Light microscopic observation showed that, the whole endosperm was covered with a nucellar epidermis (NE) under both high and natural temperature (26-31°C) conditions at the first week after heading (WAH). Under high-temperature conditions the NE degenerated earlier than under natural temperature conditions. Scanning electron microscopic observation showed that, the endosperm cells of the seeds with a specific gravity (s.g.) of higher than 1.06 had large amyloplasts filled with starch granules. However, the endosperm cells of seeds with a s.g. of 1.00-1.06 had many small amyloplasts containing small single starch granules and had numerous spaces among the amyloplasts. In the endosperm cells at the dorsal side of imperfect grain, layered structures showing progressive decomposition of starch granules were observed.     

不同类型专用小麦品种胚乳发育的比较研究

为揭示不同类型专用小麦品种胚乳发育的差异,以强筋小麦皖麦38和弱筋小麦扬麦9号为材料,比较了两者胚乳细胞游离核分裂状况、细胞数目的增殖变化、细胞体积的变化、糊粉层发育、淀粉体和蛋白质体发育等内容,主要结果如下：（1）皖麦38游离核有丝分裂所占比例较大,扬麦9号无丝分裂所占比例较大,且受温度影响较大;（2）胚乳细胞增殖均呈"S"型曲线变化,皖麦38胚乳细胞增殖较快,胚乳体积较大,最终粒重较高;（3）皖麦38胚乳细胞中大淀粉体数目多于扬麦9号,而小淀粉体数目表现却相反;（4）皖麦38与扬麦9号相比较,糊粉层出现时间较晚,而且糊粉层细胞壁较厚,被甲苯胺蓝染色更浓;（5）成熟籽粒中淀粉粒形状有饼形、椭圆形和近圆球形三种。皖麦38胚乳中饼形和椭圆形淀粉粒较多,且相互结合较紧密;扬麦9号饼形和近球形淀粉粒较多,胚乳结构疏松。