Structural and Histochemical Characterization of Developing Rice Caryopsis

The development of pericarp, seed coat, starchy endosperm and aleurone of the rice caryopsis was investigated, histochemically and structurally, from the time of flowering to maturity. The results showed that during its growth, the maximum length of the caryopsis was attained first, followed by width and then thickness. Histochemical examination of the caryopsis showed that starch was mainly accumulated in the endosperm, but the endosperm showed no metabolic activity, while embryo and pericarp contained a few starch grains, and embryo and aleurone were strongly active. Aleuronic cells contained many aleurone grains and spherosomes, and aleurone in the dorsal region developed earlier and contained more layers of cells. Amyloplasts in endosperm contained many starch granules and were spherical at early stages but polyhedric at late stages. The protein bodies appeared later than amyloplasts, and the number of protein bodies in subaleurone was greater than those in the starchy endosperm. The white-belly portion of endosperm might be relative to the status of amyloplast development.     

小麦乙烯响应转录因子基因 wfzp突变体颖果发育的研究

小麦WFZP基因编码的乙烯响应因子是植物中特有的转录因子，参与果实成熟、花叶器官衰老、脱落和种子发育等过程。为探明 wfzp基因突变对小麦颖果发育的影响，以甲基磺酸乙酯诱变的两个 wfzp基因突变体（ wfzp1和 wfzp2）和野生型小麦品种济麦22为材料，运用树脂超薄切片和显微观察技术观察小麦WFZP基因突变体颖果的形态结构发育特征。结果显示， wfzp突变体颖果发育有如下特征：（1）在颖果发育早期，两个突变体的外果皮发育进程较快，中果皮降解时间提前； wfzp1内果皮管细胞消失较快，与 wfzp1突变体相比， wfzp2突变体的管细胞发育缓慢；（2）两个突变体胚乳细胞的分化和发育明显较快，细胞中淀粉和蛋白体的积累量明显增多，细胞的充实度高，尤其在 wfzp2突变体中表现较为明显；（3）两个突变体胚乳传递细胞与糊粉层传递细胞的生长分化进程加快。研究结果为进一步探究乙烯响应转录因子调控小麦颖果发育提供了形态学参考。     

干旱胁迫下小麦颖果内源激素的变化及其与胚乳发育的关系

为探明干旱胁迫下小麦颖果内源激素与胚乳发育的关系,以小麦品种扬麦16为材料,在幼苗返青至颖果成熟阶段进行干旱处理,采用树脂切片及显微摄影等技术观察小麦颖果和胚乳细胞发育的形态结构特征,并通过酶联免疫吸附法测定颖果生长素(IAA)、脱落酸(ABA)、赤霉素(GA_3、GA_4)、玉米素核苷(ZR)、二氢玉米素核苷(DHZR)的含量。结果显示,干旱胁迫缩短了小麦颖果发育进程,促进颖果早衰,导致颖果发育不良;显著降低了籽粒千粒重、可溶性糖含量和总淀粉含量,提高了蛋白质含量;促进颖果发育早期胚乳细胞分裂和淀粉积累,抑制了发育后期胚乳细胞的分裂、体积扩大和淀粉体充实,并提高了颖果整个发育过程中胚乳蛋白体的积累;提高了颖果发育前期IAA、GA_3、GA_4、ZR和DHZR的含量,ABA含量在整个发育时期均较高。说明干旱胁迫能通过影响颖果内源激素的积累来调控胚乳发育。     

The Biochemical Basis and Implications of Grain Strength in Sorghum and Maize

This review deals with the biochemical basis and implications of hardness and grain strength in sorghum and maize. Grain hardness affects various aspects of the growth and processing of cereal grain from resistance to fungal infection to cooking quality. It is clear that the prolamins play an important role with the γ-prolamins being the most important. It would appear that these prolamins help shape the protein bodies and form disulphide bonds within themselves or with other proteins. The γ-prolamins form the cement while the α-prolamins are the bricks. Both prolamins are present in greater proportions in hard grains and in the vitreous portion of hard grains. Genetic and environmental effects on the amounts of the different prolamins and on their distributions within the protein body and in different parts of the endosperm also determine grain hardness. Grains that will be hard appear to deposit prolamins and antifungal proteins earlier and in greater amount than do soft grains. The cell wall composition is also different between the two types of grains while there is a higher proportions of amylose in the starch of hard than in that of soft grains. Most of the differences that exist between hard and soft grains also exist between the outer and inner portion of the grain. It is postulated that there might be a master gene controlling the onset of strength in grains by simultaneously altering the levels of various apparently unrelated biochemical events. It is also suggested that solute availability may play an important role in the regulation of expression of genes for hardness-related proteins.     

两个玉米品种胚乳发育的比较

以农乐988和扬糯1号两个品种的玉米颖果为材料,利用树脂半薄切片、组织化学染色及生理测定等方法研究胚乳组织和细胞的发育过程。结果表明,两个玉米品种粒重及淀粉含量的变化呈S形生长曲线;可溶性糖变化呈单峰曲线,授粉后12 d含量最高;总蛋白含量在授粉后6 d较高。颖果粒重和总蛋白含量农乐988>扬糯1号;可溶性糖及淀粉含量扬糯1号>农乐988。授粉后0~2 d胚乳处在游离核期,授粉后3~5 d胚乳处在细胞化期,授粉6 d以后胚乳细胞开始分化,内胚乳细胞出现淀粉体和蛋白体。胚乳中淀粉的积累由颖果顶端向基部、由胚乳外层向中央推进。扬糯1号胚乳发育较提前,失活较晚;农乐988胚乳发育相对滞后,失活较早。玉米胚乳发育和颖果发育关系紧密,胚乳游离核期和细胞化期相当于颖果形成期,胚乳分化期相当于颖果乳熟期,胚乳成熟期相当于颖果蜡熟期与完熟期。     

Disulphide Bonds in Wheat Gluten Proteins

Disulphide bonds play a key role in determining the structure and properties of wheat gluten proteins. Comparison of the sequences of monomeric gliadins and polymeric glutenin subunits allows the identification of conserved and variant cysteine residues. Direct disulphide bond determination demonstrates that the conserved cysteine residues present in S-rich prolamins (α-type gliadins, γ-type gliadins and LMW subunits) form intra-chain disulphide bonds while additional cysteines residues present only in the LMW subunits form inter-chain bonds with cysteines in HMW subunits and other LMW subunits. Conserved and variant cysteine residues are also present in the HMW subunits but their patterns of disulphide bond formation are less well understood. Further information on the abilities of individual cysteine residues to form intra- and inter-chain disulphide bonds has also been obtained by heterologous expression of wild type and mutant proteins inE. coliand, in the case of the HMW subunits, by examination of the patterns of dimers recovered on partial reduction of glutenin or resulting from the expression of subunits in transgenic tobacco plants. Wheat gluten proteins are folded and assembled within the lumen of the endoplasmic reticulum of the developing endosperm cells, where disulphide bond formation and exchange may be catalysed by the enzyme protein disulphide isomerase. Similarly, disulphide bond reduction, for example to facilitate mobilisation during germination, may be catalysed by thioredoxinh. Understanding the mechanism and specificity of disulphide bond formation in gluten is crucial for the manipulation of its functional properties by genetic engineering or chemical modification.     

Antigenicity of Wheat Prolamins: Detailed Epitope Analysis using a Panel of Monoclonal Antibodies

Putative continuous epitopes, recognised by five panels of monoclonal antibodies (MAb) with differing specificities for gliadins and glutenin subunits, were identified using overlapping nonapeptides. These peptides corresponded to the entire sequence of an α/β-gliadin, a γ-gliadin, an ω-prolamin (homologous to ω-gliadin), a low molecular weight glutenin subunit (L M_rGS) and several high molecular weight glutenin subunits (HM_r GS). Antibodies that bound to γ- or ω-gliadins, L M_rGS or HM_r GS bound to the peptides at similar concentrations used normally in direct ELISA, but little binding to the peptides was seen for several antibodies that bound specifically to small groups of α/β-gliadins. Epitopes for these antibodies in α/β-gliadin may be discontinuous (i.e. derived from amino acid residues that are brought together by folding of the polypeptide chain or by juxtaposition of two polypeptide chains), since binding of these antibodies to gliadins was greatly decreased following the reduction of intra-molecular disulphide bonds. While some regions in particular subunits were immunodominant, such as the cysteine–cysteine containing peptide found in the central domain of many prolamins, a diversity of reaction patterns was found. Cross-reaction of antibody with peptides from other prolamin families was often due to binding to a peptide having significant sequence homology, but in some cases no homology was obvious. Some major trends were as follows. Antibodies which bound to most or all H M_rGS recognised the central repeat region, while those that were selective for one or two subunits bound to epitopes in the unique N- and/or C-terminal domains. A high proportion of the epitopes recognised by MAb to α-, β-, ω-gliadins and L M_rGS contained cysteine; these MAb may be useful in detecting covalent binding sites within or between subunits. Although a number of MAb bound a wide range of gliadins and GS, several of these recognised single (and differing) epitopes in the target proteins. However, comparatively few MAb recognised epitopes from either the N- or C-terminal regions of the target proteins. Several explanations are possible; either these regions are buried in the immunogen and not accessible for antibody production or alternatively the repeat sequences are immunodominant.     

Use of mechanics of cohesive granular media for analysis of hardness and vitreousness of wheat endosperm

The characterisation of the wheat endosperm by mechanical tests of compression highlighted a relation between the rupture energy and the elasticity modulus for different varieties of wheat; this relation allows us to distinguish mealy and vitreous endosperms. An approach based on the micromechanics of cohesive granular materials is used to analyse these experimental results. A geometrical model of the wheat endosperm made of grains linked by cohesive bonds is proposed. We introduced two parameters, the first one α represents the percentage of active bonds (bonds where the stiffness and strength are non-zero), and the second one β represents the threshold of the bond's rupture. The parameter β can be related to the cross-section of the bond. This model successfully describes the mechanical tests on the wheat endosperm. The comparison with the experimental tests makes it possible to clearly differentiate vitreous wheats and mealy wheats and then attribute this property to the parameter β. The model shows the same tendency as regards the evolution of the rupture energy and the elastic modulus with the parameter α. The modelling of endosperm by the mechanics of cohesive granular media provides a new theoretical framework to interpret the rheology of endosperm. This approach allows us to connect this rheology to the mechanical actions at the scale of the granules.     

施氮时期对扬稻6号颖果发育及稻米品质的影响

通过盆栽试验研究了相同施氮量下不同施氮时期（分蘖期和孕穗期）对扬稻6号颖果发育及稻米品质的影响。与对照相比,增施氮肥（尿素）特别是孕穗肥能显著提高稻米精米率、整精米率、蛋白质含量,降低垩白粒率和直链淀粉含量;在分蘖期和孕穗期施氮肥能明显影响颖果发育,提高粒重,而后者的效果更为明显;在颖果发育过程中不同时期增施氮肥能显著降低颖果的总淀粉和直链淀粉含量,但对支链淀粉含量影响较小;不同时期增施氮肥特别是孕穗肥对淀粉体和蛋白质体的发育及结构均有显著影响,能显著改变籽粒中不同部位特别是腹部淀粉体和蛋白质的分布、数目和形状。与对照相比,淀粉体和蛋白质体的排列更紧密、相互间空隙较少、数量增加、密度增大,淀粉体形状多数呈晶状体。     

水稻胚乳细胞发育的结构观察及其矿质元素分析

 为了探明水稻胚乳发育的过程和糊粉层形成的机理, 用光学显微镜和电子显微镜观察了水稻糊粉层细胞和内胚乳细胞在颖果发育过程中的结构变化,用能谱仪分析了胚乳细胞中元素的种类和相对含量。结果表明, 糊粉层细胞是由胚乳表层细胞转化而来的。糊粉层细胞中的P、K、Mg和Ca等矿质元素含量要明显高于内胚乳细胞。发育初期糊粉层细胞中富含线粒体、圆球体和小液泡;发育中后期小液泡积累蛋白质和矿质元素而形成糊粉粒。在发育中后期,内胚乳细胞随着细胞内淀粉体的充实,细胞核发生形变而衰亡;而糊粉层细胞的核在发育过程中不消亡。糊粉层的形成与表层细胞积聚矿质和脂类等“灌浆废物”(指非内胚乳细胞的贮藏物)有关。因而,转运灌浆物质多的胚乳背部,其糊粉层细胞的层数要比腹部和侧部多。谷物胚乳发育分为游离核期、细胞化期、分化期和成熟期四个时期。     

Dynamic viscoelastic, calorimetric and dielectric characteristics of wheat protein isolates

Dynamic oscillatory rheology of two wheat protein isolate (Prolite 100 and Prolite 200) doughs (≈48% moisture content, wet basis) were studied over a frequency range of 0.1–10 Hz during temperature sweep from 20 to 90 °C at a heating rate of 2 °C/min. Both doughs behaved similarly during heating; showed a threshold value and increased sharply, thereafter. Prolite 200 dough had a higher elastic modulus (G′) and lower phase angle (δ) whereas Prolite 100 showed a distinct gel point at 52.2 °C followed by significant increase up to 90 °C. Rheological data of doughs after isothermal heating at 90 °C for 15 min followed by cooling to 20 °C resulted in strong mechanical strength. However, Prolite 100 dough showed more viscoelastic characteristics with significant transformation from liquid-like to solid-like behavior after heating than Prolite 200. Thermal analysis of isolates indicated distinct endothermic peaks in wider temperature range (50–130 °C) at various moisture levels. Lower temperatures could be associated with denaturation of various fractions of proteins whereas higher temperature linked to glass transition temperature of isolates. SDS–PAGE did not show any clear distinction among protein subunits between two isolates. Dielectric measurements of isolates at frequencies from 500 to 3000 MHz and temperature range between 30 and 80 °C indicated Prolite 200 had higher dielectric constant (ε′) and loss factor (ε″) than Prolite 100. Isolates showed significant changes in dielectric properties above 50 °C indicating protein denaturation and supported rheological and calorimetric data.     

小麦胚乳传递细胞发育的结构观察

为从细胞学方面了解小麦产量和品质的形成机制,以扬麦5号为材料,利用光镜和透射电镜观察了小麦颖果发育过程中胚乳传递细胞的结构变化,并探讨了胚乳传递细胞的生理功能。结果表明：（1）胚乳传递细胞是胚乳发育过程中最早分化的细胞类型,它们发生在紧邻胚乳腔的胚乳表层,由位于外侧1~2层糊粉层传递细胞和位于内侧1~2层内胚乳传递细胞构成;（2）颖果发育成熟时,内胚乳传递细胞核衰亡,糊粉层传递细胞核依然完整;（3）胚乳传递细胞发育呈明显的极性,且具时空性;（4）糊粉层传递细胞胞质较浓,富含粗面内质网、线粒体、高尔基体和脂质体;质膜皱褶,在局部区域外翻,形成众多的原生质管;（5）内胚乳传递细胞胞质较稀,液泡化程度较高,富含淀粉体;（6）胚乳传递细胞未加厚以及未形成壁内突的壁区域分布有大量的胞间连丝;（7）胚乳传递细胞中线粒体呈极性分布,即质膜附近线粒体的密度较大。根据胚乳传递细胞的结构特点推测,经胚乳传递细胞的养分输送既可通过质外体途径又可通过共质体途径来完成。     

Effect of protease treatment on the baking quality of brown rice bread: From textural and rheological properties to biochemistry and microstructure

In this study, protease treatment of brown rice (BR) batters was investigated in order to evaluate its impact on the textural and baking properties of BR bread. The enzymatic treatment improved bread quality by significantly increasing specific volume (p < 0.05), while decreasing crumb hardness and chewiness (p < 0.05). Fundamental rheology and viscometry of batters revealed that protein hydrolysis induced lower complex modulus and initial viscosity, while phase angle was unaffected. Flour pasting properties were also affected, with a significant decrease in paste viscosity and breakdown (p < 0.05). Protein analysis of batters revealed that the enzymatic treatment induced the release of low molecular weight proteins from macromolecular protein complexes. In conclusion, a lower resistance to deformation of batters during proofing and in the early stages of baking as well as the preserved batter elasticity and the increased paste stability positively affected the breadmaking performance.     

Influence of allelic prolamin variation and localities on durum wheat quality

Thirty-seven varieties of a Mediterranean durum wheat collection grown in Tunisia and Spain were analysed for their allelic composition in prolamins, as well as their protein concentration, sodium dodecyl sulphate sedimentation (SDSS) test and mixograph parameters. Genotype was a greater source of variation in all measurements than locality. Uncommon high and low molecular glutenin subunits (HMW-GS and LMW-GS) were found (V and 2•• subunits at Glu-A1, 13 + 16 at Glu-B1, 5* subunit and ax allele at Glu-A3). The rare combinations 2 + 4+14 + 18 and 8 + 9+13 + 16+18 subunits at the Glu-B3 locus were found. Glu-A3ax had a positive influence on SDSS and mixograph parameters. Of all the prolamins, those that have the B-LMW-GS composition aaa (for Glu-A3, Glu-B3 and Glu-B2 loci, respectively), when associated with the Glu-A1c and Glu-B1d gave the best semolina quality. By contrast, semolina quality is poor when this same composition is associated with the Glu-A1c and Glu-B1e and even poorer when associated with the Glu-A1c and Glu-B1f. In addition, the cultivars with B-LMW-GS allelic composition aab (for Glu-A3, Glu-B3 and Glu-B2 loci, respectively), when associated with the Glu-A1c and Glu-B1d, gave high quality, whereas when associated with the Glu-A1c and Glu-B1e or with Glu-A1o and Glu-B1f, the quality was very poor.     

Investigation of the fracture mode for hard and soft wheat endosperm using the loading–unloading bending test

Investigation of the fracture mode for hard and soft wheat endosperm was aimed at gaining a better understanding of the fragmentation process. Fracture mechanical characterization was based on the three-point bending test which enables stable crack propagation to take place in small rectangular pieces of wheat endosperm. The crack length can be measured in situ by using an optical microscope with light illumination from the side of the specimen or from the back of the specimen. Two new techniques were developed and used to estimate the fracture toughness of wheat endosperm, a geometric approach and a compliance method. The geometric approach gave average fracture toughness values of 53.10 and 27.0 J m⁻² for hard and soft endosperm, respectively. Fracture toughness estimated using the compliance method gave values of 49.9 and 29.7 J m⁻² for hard and soft endosperm, respectively. Compressive properties of the endosperm in three mutually perpendicular axes revealed that the hard and soft endosperms are isotropic composites. Scanning electron microscopy (SEM) observation of the fracture surfaces and the energy–time curves of loading–unloading cycles revealed that there was a plastic flow during crack propagation for both the hard and soft endosperms, and confirmed that the fracture mode is significantly related to the adhesion level between starch granules and the protein matrix.     

Direct Evidence that the Number and Location of Cysteine Residues affect Glutenin Polymer Structure

In this study, the possible roles of three well-characterised model prolamins in the structure of the glutenin macropolymer were examined. Model prolamins were labelled with fluorescein isothiocyanate (FITC), and incorporated into the glutenin macropolymer of a base flour using a partial reduction-oxidation scheme. The effect of incorporation of the model prolamins on dough behaviour was determined by assessing differences in polymer size distribution, mixing properties, and distribution of the model prolamins in a dough after incorporation. Using this approach, the prolamins capable of forming inter-chain disulphide bonds were shown to be incorporated into the glutenin macroÍpolymer, while prolamins that were not capable of forming inter-chain disulphide bonds were retained as monomers. The distribution of fluorescently-labelled prolamins after their incorporation into the glutenin macropolymer of the dough was examined by confocal light scanning microscopy, in order to determine the possible roles of ω-gliadins and glutenin-like subunits with varied cysteine residue compositions in the structural organisation. The role of the model prolamins was a function of the disulphide-bonding capabilities of the polypeptides. Model ω-gliadins were retained as monomers and functioned as space fillers; model glutenin-like subunits containing a single cysteine residue incorporated into the glutenin macropolymer but functioned as chain terminators; and model glutenin-like subunits containing two cysteine residues incorporated into the glutenin macropolymer and acted as chain extenders.     

Near infrared spectra indicate specific mutant endosperm genes and reveal a new mechanism for substituting starch with (1→3,1→4)-β-glucan in barley

Near Infrared Reflectance spectroscopy was tested as a screening method to characterise high lysine mutants from a barley collection by classification through Principal Component Analysis (PCA). Mean spectra of the samples within each cluster identified gene-specific patterns in the 2270–2360 nm region. The characteristic spectral signatures representing the lys5 locus (Risø mutants 13 and 29) were found to be associated with large changes in percentage of starch and (1→3,1→4)-β-glucan. These alleles compensated for a low level of starch (down to 30%) by a high level of (1→3,1→4)-β-glucan (up to 15–20%), thus, maintaining a constant production of polysaccharides at 50–55%, within the range of normal barley.The spectral tool was tested by an independent data set with six mutants with unknown polysaccharide composition. Spectral data from four of these were classified within the high (1→3,1→4)-β-glucan BG lys5 cluster in a PCA. Their high (1→3,1→4)-β-glucan and low starch content was verified. It is concluded that genetic diversity such as from gene regulated polysaccharide and storage protein pathways in the endosperm tissue can be discovered directly from the phenotype by chemometric classification of a spectral library, representing the digitised phenome from a barley gene bank.     

Comparative Studies of High MrSubunits of Rye and Wheat. II. Partial Amino Acid Sequences

A panel of anti-peptide antibodies specific for each of the different N-terminal sequence types of B- and C-low molecular mass glutenin subunits (L M_rGS) were utilised in immunoblotting studies to identify the chromosomal location of genes encoding different sequences and to characterise the allelic variation of the encoding loci. The MET-type sequences were predominantly found among the B- subunits, while the α- and γ- sequences predominated in the C- subunits. The quantitatively major SHIPGLERPS sequence was found in both the B- and C- mobility regions. Using either biotypes in the cultivar, Aroona or genetic lines containing double rye chromosome 1 substitutions and thus expressing only single LM_r GS alleles, the sequences were determined for most of the major polypeptides expressed by each LM_r GS allele. The L M_rGS from different genomes encoded different numbers of each sequence type. Furthermore, different polypeptides within a particular «block» of subunits encoded by a given allele often had differing N-terminal sequences. However, subunits of similar electrophoretic mobilities encoded by different alleles at each locus usually had identical N-terminal sequences, suggesting that they may instead differ in the number of repeats. In Chinese Spring, genes encoding the SHIPGLERPS and METSHIPGL sequence types were predominantly present on chromosomes 1B and 1D, while the related METSRVPGL sequence was only encoded on 1D. In contrast, the METSCIPGL, α- and γ-sequences were encoded on each of chromosomes 1A, 1B and 1D. Several different electrophoretic and immunoblotting approaches using null lines suggested that some of the α-type L M_rGS may also be encoded by group 6 chromosomes, particularly 6D. The anti- SHIPGLERPS antibody also recognised chromosome 1B encoded β-, γ- and ω-gliadins, while the anti-METSRVPGL antibody recognised 1D encoded α- and β-gliadins. The absence of sequences within the major gliadin families that are highly homologous to the latter two N-terminal L M_rGS sequences may suggest that some monomeric L M_rGS could exist within the electrophoretically-resolved gliadins. These antibodies will provide valuable reagents for the study of the roles of particular L M_rGS families in the structure and function of the glutenin macropolymer, the role of different LM_r GS types in determining the influence of allelic variation of L M_rGS composition on dough properties, and potentially in the development of diagnostics for these flour components.