Wheat endosperm cell walls: Spatial heterogeneity of polysaccharide structure and composition using micro-scale enzymatic fingerprinting and FT-IR microspectroscopy

Micro-scale enzymatic fingerprinting and FT-IR microspectroscopy were used to investigate changes of polysaccharide structure and composition in cell walls from wheat endosperm. These methods were applied to transverse and longitudinal sections of wheat grains harvested at maturity and 270°D. Principal component analysis treatment of the data revealed marked differences in the (1,3)-(1,4)-beta-glucans (BG)/arabinoxylans (AX) ratio and in the structure of AX depending on both cell position in the grain and stage of development. Cell walls close to the germ were enriched in BG in both developing and mature grain. AX in developing grain were characterized by a higher proportion of di-substitution by arabinose as compared to mature grain AX. In addition, AX in mature grains exhibited a different structure depending on prismatic or central origin of cell walls in the grain. These results indicated a high spatial and temporal control of the biosynthesis of AX and BG in wheat endosperm.     

Study of grain cell wall structures by microscopic analysis with four different staining techniques

Four different grain cell wall staining techniques were compared. Two techniques specifically detected arabinoxylan (AX). The first technique used a xylanase probe, while the other one was based on immunolabeling of AX using monoclonal antibodies. The two other staining techniques, one based on Calcofluor and the other on immunolabeling using monoclonal antibodies, stained mixed-linkage β-glucan. Cell walls of wheat, barley, oat and rye grains, differing both in content and location of AX and β-glucan, were examined. The staining methods were complementary to each other in revealing the location and distribution of the major cereal dietary fiber components AX and β-glucan in the different grains. AX was mostly concentrated in nucellar epidermis and aleurone cells, whereas β-glucan was concentrated more in subaleurone cells. Furthermore, in the case of barley and rye, the endosperm cell walls also contained high amounts of β-glucan. Interestingly, β-glucan in rye and barley endosperm cell walls was located adjacent to the cell contents, suggesting that it is not evenly distributed in the endosperm cell walls. The results give new insight into the structure of the cereal dietary fiber complex. Further development of microscopic techniques will help in elucidating the cereal cell wall structure even in more detail.     

Cell wall deposition and metabolism in wheat grain

There is an amazing diversity of cell wall composition and cell wall polysaccharide structure in wheat grain that is only partly explained by the complexity of plant tissues and cell type functions present in this organ. This review presents the state of the art on the structure and diversity of cell wall polymers in mature wheat grain and recent knowledge on cell wall metabolism with a focus on development-associated changes in wheat grain cell wall polysaccharides and genes controlling their biosynthesis. The diversity of polysaccharide structure observed in endosperm cell walls is tentatively explained on the basis of polysaccharide properties and cell type function.     

Wheat arabinoxylans: Exploiting variation in amount and composition to develop enhanced varieties

Arabinoxylans (AX) are the major polymers of wheat grain cell walls. The content and the structure of AX polymers show large differences between tissues and between wheat cultivars that affect the end-use properties and nutritional quality of the grain. The development of new wheat cultivars with enhanced quality, therefore, requires methods to exploit this variation and it is essential to understand and modulate the mechanisms controlling the key events of cell-wall polymer synthesis.This paper summarises recent knowledge on the structure and physicochemical properties of AX including variation between cultivars and tissues, methods for analysis and screening, biosynthetic mechanisms and approaches to identifying key genes. This knowledge is essential to understand AX properties and defined possible targets for plant breeding.     

Wheat arabinoxylans: Exploiting variation in amount and composition to develop enhanced varieties

Arabinoxylans (AX) are the major polymers of wheat grain cell walls. The content and the structure of AX polymers show large differences between tissues and between wheat cultivars that affect the end-use properties and nutritional quality of the grain. The development of new wheat cultivars with enhanced quality, therefore, requires methods to exploit this variation and it is essential to understand and modulate the mechanisms controlling the key events of cell-wall polymer synthesis.This paper summarises recent knowledge on the structure and physicochemical properties of AX including variation between cultivars and tissues, methods for analysis and screening, biosynthetic mechanisms and approaches to identifying key genes. This knowledge is essential to understand AX properties and defined possible targets for plant breeding.     

LC-MS analysis reveals the presence of graminan- and neo-type fructans in wheat grains

This is the first study describing the fine structure of the main, individual fructan oligosaccharides present in wheat grains. Wheat grain fructan structure was investigated in developing wheat grains and in different tissues of mature grains with liquid chromatography-mass spectrometry. Fructan oligosaccharides with a low degree of polymerization (<5) were mainly of the graminan- and inulin-type in developing wheat grains during the first week after anthesis. Starting from 14 days after anthesis, neo-type fructans, fructans with an internal glucose, were observed for the first time. Several neo-type fructan structures were identified and their portion in the total fructan pool gradually increased during grain development. In the mature kernel, almost no differences were noted between the fructan distributions of wheat flour and two wheat bran fractions enriched in either pericarp or aleurone tissue. Results are related to wheat fructan metabolizing enzymes and the nutritional implications are discussed.     

Investigation by Confocal Raman Microspectroscopy of the Molecular Factors Responsible for Grain Cohesion in theTriticum aestivum Bread Wheat. Role of the Cell Walls in the Starchy Endosperm

Confocal Raman microspectroscopy has previously been employed to investigate the protein content and composition of the starchy endosperm of the wheat grain. With the same objective, that is to determine the molecular basis of grain cohesion and more specifically of kernel hardness, the contribution of endosperm cell walls in the kernel structure and cohesion was explored. The technique showed that endosperm cell walls consist not only of arabinoxylan chains with ramifications of ferulic esters, but also of others components such as proteins and lipids that could play some role in the mechanical properties of the endosperm cell walls. A new model of interaction between ferulic ramifications and a phospholipid component was proposed. The investigation of cell wall composition at successive stages of grain development revealed a decrease in the protein to arabinoxylan ratio and simultaneously an increase of the ferulic acid to arabinoxylan ratio that could be associated with a strengthening of the cell wall structure. The study confirms the effectiveness of confocal Raman microspectroscopy to approach the structure of wheat grain at the micrometer scale and to identify specific molecular factors responsible for grain cohesion and involved in the fracture modes generated during the milling process.     

Isolation of wheat endosperm cell walls: Effects of non-endosperm flour components on structural analyses

We report the isolation of a pure form of cell walls from wheat endosperm ‘popped’ out from the whole, enzyme deactivated and soaked grain, and compare them with cell walls isolated from milled flours of extraction rates from 45% to 75%, at physiological (37 °C) and elevated (70 °C) temperatures. Cell walls isolated from flours all contained non-endosperm walls whereas walls from popped endosperm were apparently pure. The monosaccharide composition of ‘popped’ cell walls was different to that of cell walls isolated from flour, particularly glucose and mannose contents (34 and 7% for ‘popped’ cf 29 and 3% for flour respectively) and arabinose to xylose ratios (0.45 for ‘popped’ cf 0.58 for flour). Total phenolic contents of popped endosperm cell walls were three to four times lower than for cell walls from flour. Elevated isolation temperature also had a solubilising effect, altering the cell wall composition. This study provides a novel method of isolating pure wheat endosperm cell walls, and demonstrates how contaminating (thick cell walled) non-endospermic material in milled flours can have a major influence on cell wall compositional analyses.     

Chemical imaging: the distribution of ions and molecules in developing and mature wheat grain

The ability to combine topographical and in situ chemical analysis of individual cereal grains, without recourse to fractionation, offers an opportunity to determine the distribution of functionally- and nutritionally-important components. Three such technologies are reviewed, including immunolocation using monoclonal antibodies specific for different types of wheat prolamins, secondary ion mass spectroscopy (SIMS) to detect the presence of inorganic elements such as sodium and sulphur, and infrared (including Raman and Fourier-transform infrared [FT-IR]) microspectroscopy to determine the distribution of biopolymers across the grain. Immunolabelling has shown that the distribution of prolamin proteins changes across the endosperm, with the outer endosperm containing a much greater proportion of prolamins than the inner endosperm. SIMS has shown, for the first time, the presence of Na⁺ in the phytin granules and that sulphur is enriched at the boundary between the starch granules and the protein matrix. Raman microspectroscopy has been used to investigate the distribution of proteins and the phenolic compound, ferulic acid, across the grain, whilst FT-IR has been used to define the microheterogeneity of arabinoxylans in endosperm cell walls. These methods highlight how in situ analysis can yield new insights into grain composition and how this may be altered by environmental conditions during grain development.     

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。     

Study of the microstructure of durum wheat endosperm using X-ray micro-computed tomography

Durum wheat grains are used for producing food, such as pasta or couscous. The grain mechanical properties which are linked to its internal micro-structure (i.e. endosperm porosity) are known to determine its ability to produce semolina during milling. The proportion of grains having porous endosperm in a batch appears therefore as a critical quality factor for the durum wheat value chain. Our objective was to investigate the ability of X-ray micro-tomography (μCT) method to describe the porous or vitreous counterpart structures in the endosperm of durum wheat grains. We selected two different durum wheat samples displaying vitreous or partially porous endosperms. The grains were analyzed using μCT at two pixel sizes (1 μm or 7 μm). The μCT data collected at 7 μm pixel size were used for qualitative classification of grains according to apparent distribution curve of the porosity parameters. Analysis of μCT images at 1 μm pixel size allowed us to propose pore size classification in the vitreous and porous parts of the endosperm in three durum wheat grain. Results are used to better describe the durum-wheat endosperm microstructure, but requires long scanning periods.     

Endosperm and aleurone cell structure in barley and wheat as studied by optical and Raman microscopy

Grain ultrastructure is of utmost importance when designing grain processing procedures in the food industry. In this study, wheat and barley grain components were localised using optical and Raman microscopy. The optical microscopic analyses were performed using several selective stains to localise β-glucan, protein and starch or autofluorescence to image the ferulic acid and other fluorescing substances. Alternatively, Raman microscopy was applied to localise the grain components without any need for preceding staining or other sample pretreatment. Both methods provided consistent information on the grain structures, illustrating the distribution of polysaccharides, aromatics and protein in endosperm and aleurone layers. In aleurone layers of both barley and wheat, a distinct difference between the anticlinal and periclinal cell walls was observed. The anticlinal cell walls were enriched with aromatic substances which were present in remarkably lower concentrations in the inner periclinal cell walls but for barley, an even higher concentration in the outer periclinal cell wall was observed. In addition, Raman spectroscopy illustrated the detailed distribution of substances across the aleurone cell walls: β-glucan was adjacent to proteins and it was deficient in the middle lamella whereas arabinoxylan was enriched in the outer cell wall layers and middle lamella.     

Fracture Mechanics of Vitreous and Mealy Wheat Endosperm

The concepts of fracture mechanics have been applied to quantify the fracture behaviour of vitreous and mealy wheat endosperm in a single wheat cultivar. Two new techniques were developed and used to measure fracture toughness (energy per unit area of fracture) of individual grains: (1) load cycling of a notched grain; and (2) instrumented microtome cutting. The load cycling method gave average fracture toughness values for vitreous endosperm of 130 ± 42 J/m² and 50 ± 12 J/m² for mealy endosperm. Fracture toughness measured using the instrumented microtome gave values of 159 ± 7·4 J/m² for vitreous endosperm and 44 ± 4·6 J/m² for mealy endosperm. The results are consistent with the hypothesis that vitreous endosperm has stronger starch-protein matrix bonding than mealy endosperm. The effect of changing grain moisture on cutting fracture properties was investigated. As moisture decreased, values of fracture toughness increased for both mealy and vitreous endosperms at the same rate down to 11% moisture content, below which fracture toughness increased more rapidly for vitreous than for mealy endosperm. Intra-grain fracture toughness was also investigated by cutting successive sections across individual wheat grains. These showed a decrease in cutting force from the outside of the grain towards the centre, and then an increase near the crease. The critical particle size at which a transition from brittle to ductile failure occurs was calculated, giving predicted values of 1·2 mm for vitreous endosperm and 0·9 mm for mealy endosperm at 15% moisture content. This shows that vitreous endosperm undergoes more ductile deformation during deformation than does mealy endosperm, and that larger particle sizes are predicted for vitreous endosperm as a result of milling.     

Endogenous arabinoxylans variability in refined wheat flour and its relationship with quality traits

Arabinoxylans (AXs) are the major dietary fiber (DF) component in wheat and their consumption has been associated with several health benefits. Genetic improvement of the AX in refined wheat flour could be a good solution to improve the DF daily consumption while maintaining the flour desirable quality. In this study, 193 common wheat lines were analyzed for their AX content in refined flour and end-use quality. Wide variation in both the total arabinoxylan (TOT-AX) (10.8–16.5 mg/g) and water-extractable arabinoxylan (WE-AX) (3.2–7.6 mg/g) was identified and, in both cases, the genotype had the greatest impact on the observed phenotypes. Variation in the endogenous AX fractions appeared to have a moderate effect on wheat quality. The WE-AX, specifically, were positively correlated with gluten strength (r = 0.11 to 0.32) and bread loaf volume (r = 0.16), whereas the TOT-AX were negatively correlated with dough extensibility (r = −0.11) and bread making quality (r = −0.11). Overall, results of this study show that the genetic improvement of grain AX is feasible and that the AXs present in refined flour do not dramatically alter wheat quality indicating that it is possible to select varieties with high AX endosperm content end desired end-use quality.     

紫糯小麦籽粒石蜡切片制作技术优化及淀粉粒累积分析

为了探明糯性小麦授粉后籽粒淀粉粒的累积情况,以山农紫糯2号授粉后不同天数的籽粒为试验材料,通过对传统石蜡切片技术的脱水、透明和浸蜡等环节进行改进,制作切片后对小麦籽粒结构发育进行观察研究。结果显示,与传统石蜡切片方法相比较,技术改良70%乙醇和无水乙醇脱水时间与逐级透明和渐进式浸蜡环节后,小麦籽粒（授粉后0~14 d）不皱缩变形,石蜡切片完整,且染色分明,结构显示清晰。授粉后0~2 d,果皮中已经开始积累少量淀粉粒,随着授粉后天数推移而逐渐增多;授粉后第6天,胚乳内部逐渐由游离核期向细胞化过渡;授粉后第8天,胚乳细胞进一步增大,淀粉粒开始累积,但体积较小;授粉后第10~12天,胚乳细胞向无核化发展,不同部位胚乳细胞和淀粉粒的排列分布呈现明显差异;授粉后第14天,胚乳细胞中几乎完全充满淀粉粒。表明改进的石蜡切片制作方法适合于早期小麦籽粒的结构研究。     

施氮量对两个粒型小麦品种籽粒胚乳细胞增殖及糖含量的影响

为了解小麦胚乳细胞增殖、糖代谢在粒型间的差异及氮营养的调控效应,2009-2010年度以大粒型品种郑育麦9987和小粒型品种郑麦004为材料,研究了施氮量对小麦籽粒胚乳细胞增殖、细胞壁转化酶活性及糖含量的影响。结果表明,郑育麦9987胚乳细胞数(11.92×105)较郑麦004(10.73×105)多11.1%。施氮量影响籽粒胚乳细胞数,其中郑育麦9987低氮处理(150kg·hm-2)的胚乳细胞数目较高氮处理(300kg·hm-2)增加29.1%,而郑麦004则表现为高氮处理较低氮处理增加44.7%。两品种籽粒细胞壁转化酶活性均于花后5~8d达到峰值,花后20d则降到较低水平;籽粒可溶性糖和蔗糖含量均随灌浆进程整体呈先上升后下降趋势,在花后25d有一个小波峰。增施氮肥可增加籽粒细胞壁转化酶活性,提高后期籽粒可溶性糖和蔗糖含量。表明增施氮肥有利于小麦籽粒胚乳细胞增殖、光合产物的积累与运转,从而提高了粒重。     

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.     

小麦胚乳充实特性研究初报

通过剪叶去穗试验探讨了小麦籽粒从乳熟始到面团期不同小穗位、不同粒位籽粒胚乳的容积扩充和物质填充特性以及源库改变对不同小穗位、不同粒位籽粒胚乳充实的影响。结果表明:（1）不同部位相同粒数的小穗间,下部小穗籽粒胚乳的容积扩充量和物质填充量大于上部小穗同一粒位;不同类型小穗的相同粒位间,3粒小穗大于2粒小穗,2粒小穗又大于1粒小穗;同一小穗不同粒位间,3粒小穗的第2粒位最大,基部粒位次之,最上粒位最少;2粒小穗相应粒位的籽粒胚乳相差不大;胚乳充实量大的小穗位、粒位,更突出表现在胚乳的容积扩充量上,这导致胚乳充实比的下降,相反则导致上升。（2）剪叶去穗后,籽粒胚乳的各种参数都发生了较大变化。去穗处理籽粒胚乳容积扩充量和物质填充量均变大,但胚乳充实比变小;剪叶处理籽粒胚乳,与上面各种参数的变化相反。这是因为容积扩充量更为敏感,变化幅度更大。