FTIR imaging of wheat endosperm cell walls in situ reveals compositional and architectural heterogeneity related to grain hardness.

Endosperm cell walls of cultivars of wheat (Triticum aestivum L.) selected for their endosperm texture (two soft and two hard) were analysed in situ by Fourier transform infrared (FTIR) microspectroscopy. FTIR imaging coupled with statistical analysis was used to map the compositional and structural heterogeneity within transverse sections from which cell contents had been removed by sonication. In the majority of grains analysed, two distinct populations of endosperm cells could be identified by spectral features that were related to cell morphology and age, regardless of cultivar. The main cell-wall component responsible for these differences was the polysaccharide arabinoxylan. In a few samples, this heterogeneity was absent, for reasons that are not understood, but this was not correlated to endosperm texture or growth conditions. Within the same population of endosperm cells, cell walls of hard endosperm could be distinguished from those of soft endosperm by their spectral features. Compared to hard cultivars, the peripheral endosperm of soft cultivars was characterised by a higher amount of polymer, whose spectral feature was similar to water-extractable arabinoxylan. In contrast, no specific compound has been identified in the central endosperm: structural differences within the polysaccharides probably contribute to the distinction between hard and soft cultivars. In developing grain, a clear difference in the composition of the endosperm cell walls of hard and soft wheat cultivars was observed as early as 15 days after anthesis.     

Protein Body Inclusions in Developing Wheat Endosperm

Endosperm tissue of two wheat cultivars, Maris Freeman and Mardlerwas examined by light and electron microscopy from early stagesof development until maturity. Spherical electron-dense inclusionswere first seen embedded in the periphery of endosperm proteinbodies of both cultivars 11 days after anthesis. The inclusions,which gave a positive histochemical reaction for both proteinand lipid, persisted throughout endosperm development and werepresent in the protein matrix of mature grain. Two types ofmembranous inclusions were found. In Maris Freeman and Mardler,vesicles and myelin whorls were associated with the surfaceof the protein mass during development and maturation. In MarisFreeman, membrane lattices of branched tubules with a basiccubic repeat unit of 44.8 nm were found in close contact withthe protein mass, but these were not present in mature grain. Triticum aestivum L., wheat, endosperm, protein body inclusions, ultrastructure     

Endosperm cell number in cultivars of barley differing in grain weight

The number of endosperm cells in caryopses 30 ‘days’ after anthesis was determined and a positive correlation was found between endosperm cell number and 1000 grain weight, and between endosperm cell number and dry grain volume in a number of cultivars of field-grown barley. The genetic factors influencing grain weight are discussed in relation to these results and to observations made on transverse sections of immature caryopses.     

Method for hull‐less barley transformation and manipulation of grain mixed‐linkage beta‐glucan

Hull‐less barley is increasingly offering scope for breeding grains with improved characteristics for human nutrition; however, recalcitrance of hull‐less cultivars to transformation has limited the use of these varieties. To overcome this limitation, we sought to develop an effective transformation system for hull‐less barley using the cultivar Torrens. Torrens yielded a transformation efficiency of 1.8%, using a modified Agrobacterium transformation method. This method was used to over‐express genes encoding synthases for the important dietary fiber component, (1,3;1,4)‐β‐glucan (mixed‐linkage glucan), primarily present in starchy endosperm cell walls. Over‐expression of the HvCslF6 gene, driven by an endosperm‐specific promoter, produced lines where mixed‐linkage glucan content increased on average by 45%, peaking at 70% in some lines, with smaller increases in transgenic HvCslH1 grain. Transgenic HvCslF6 lines displayed alterations where grain had a darker color, were more easily crushed than wild type and were smaller. This was associated with an enlarged cavity in the central endosperm and changes in cell morphology, including aleurone and sub‐aleurone cells. This work provides proof‐of‐concept evidence that mixed‐linkage glucan content in hull‐less barley grain can be increased by over‐expression of the HvCslF6 gene, but also indicates that hull‐less cultivars may be more sensitive to attempts to modify cell wall composition.     

三种粒型小麦品种胚乳细胞增殖动态研究

以三种粒型小麦品种(系)为材料,观察了不同品种和同一品种不同粒位籽粒胚乳细胞增殖动态。结果表明,用Richards方程能较好地模拟胚乳细胞增殖动态。强势粒胚乳细胞分裂起始势高,达到最高增殖速率的时间短,活跃分裂期长,可分裂出更多的胚乳细胞。弱势粒胚乳细胞增殖起始势低,细胞分裂速率变化缓慢,其最终胚乳细胞数显著低于强势拉。不同品种间胚乳细胞数有一定的差异,表现为大粒饱满品种(鄂思1号)>不饱满品系(95A-10)>小粒饱满品种(华麦8号)。胚乳细胞增殖速率变化为单峰曲线,强势粒胚乳细胞增殖速率曲线偏左,弱势粒胚乳细胞增殖速率曲线偏右。     

Cell Production and DNA Accumulation in the Wheat Endosperm, and their Association with Grain Weight

Nuclear DNA content was measured in developing endosperm cellsof two wheat varieties, Chinese Spring and Spica. 3C, 6C, 12Cand 24C nuclei were detected, indicating that some form of endoreduplicationand/or endopolyploidization was occurring. The total amountof DNA in the endosperm continued to increase until 24 dayspost anthesis. This accumulation of DNA resulted both from productionof new nuclei and also from increases in the DNA content ofexisting nuclei. Estimates of endosperm cell numbers were made from the totalDNA content per endosperm and the mean DNA content per endospermnucleus for a range of genotypes differing in mature grain weight.Endosperm DNA content and cell number were both positively associatedwith mature grain weight among the genotypes examined. However,not all of the variation in grain weight could be attributedto variation in cell number because of differences in mean dryweight per endosperm cell. The large-grained variety, Spica, had a greater mean weightper endosperm cell than Chinese Spring and this difference aroseafter cell production in the endosperm had ceased. Triticum aestivum, grain weight, cell size, cell number, DNA content     

Remodelling of arabinoxylan in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis>) endosperm cell walls during grain filling

Previous studies using spectroscopic imaging have allowed the spatial distribution of structural components in wheat endosperm cell walls to be determined. FT-IR microspectroscopy showed differing changes in arabinoxylan (AX) structure, during grain development under cool/wet and hot/dry growing conditions, for differing cultivars (Toole et al. in Planta 225:1393–1403, 2007). These studies have been extended using Raman microspectroscopy, providing more details of the impact of environment on the polysaccharide and phenolic components of the cell walls. NMR studies provide complementary information on the types and levels of AX branching both early in development and at maturity. Raman microspectroscopy has allowed the arabinose:xylose (A/X) ratio in the cell wall AX to be determined, and the addition of ferulic acid and related phenolic acids to be followed. The changes in the A/X ratio during grain development were affected by the environmental conditions, with the A/X ratio generally being slightly lower for samples grown under cool/wet conditions than for those from hot/dry conditions. The degree of esterification of the endosperm cell walls with ferulic acid was also affected by the environment, being lower under hot/dry conditions. The results support earlier suggestions that AX is either delivered to the cell wall in a highly substituted form and is remodelled through the action of arabinoxylan arabinofuranohydrolases or arabinofuranosidases, or that low level substituted AX are incorporated into the wall late in cell wall development, reducing the average degree of substitution, and that the rate of this remodelling is influenced by the environment. ¹H NMR provided a unique insight into the chemical structure of intact wheat endosperm cell walls, providing qualitative information on the proportions of mono- and disubstituted AX and the levels of branching of adjacent units. The A/X ratio did not change greatly with either the development stage or the growth conditions, but the ratio of mono- to disubstituted Xylp residues increased markedly (by about fourfold) in the more mature samples, confirming the changes in branching levels determined using FT-IR. To the best of our knowledge, this is the first time that intact endosperm cell walls have been studied by ¹H NMR.     

不同穗型小麦光能利用和旗叶13C同化物分配特性及对补灌水平的响应

为明确不同穗型小麦冠层光能利用和¹³C同化物分配特性的差异及对补灌水平的响应,以中穗型品种‘青农2号’和‘济麦22’、大穗型品种‘山农23’和‘山农30’为材料,设置3个水分处理:小麦全生育期不灌水(W₀)、节水灌溉(W₁,拔节期和开花期0～40 cm土层土壤相对含水量分别补灌至65%和70%)和充分灌溉(W₂,拔节期和开花期0～40 cm土层土壤相对含水量分别补灌至85%和90%),研究了不同处理对两种穗型小麦冠层光能利用和旗叶¹³C同化物分配特性的影响.结果表明:W₁处理两种穗型小麦品种开花后2、11、20和31 d的叶面积指数、冠层PAR截获率和利用率均显著高于W₀处理,再增加灌水至W₂处理,上述各指标无显著变化.¹³C示踪表明,济麦22和山农23的W₁旗叶¹³C同化物在籽粒中的分配量比W₀分别高159.34和171.1 g·hm^-2,分配比例分别高6.5%和6.5%,与W₂无显著差异;两种穗型品种W₁的籽粒产量亦显著高于W₀,与W₂无显著差异.不同穗型品种比较,节水灌溉条件下中穗型品种在开花后2和11 d、大穗型品种在开花后20和31 d具有较高的冠层光合有效辐射(PAR)截获和利用能力;中穗型品种济麦22旗叶¹³C同化物在籽粒的分配量和分配比例比大穗型品种山农23分别低6.8%和2.7%.     

强筋与弱筋小麦籽粒蛋白质组分与加工品质对灌浆期弱光的响应

选用强筋小麦品种济麦20和弱筋小麦品种山农1391,在大田试验条件下,分别于籽粒灌浆前期(花后6—9 d)、中期(花后16—19 d)和后期(花后26—29 d)对小麦进行弱光照处理,研究了籽粒产量、蛋白质组分及加工品质的变化。灌浆期弱光显著降低小麦籽粒产量,灌浆中期对济麦20和灌浆后期对山农1391的产量降幅最大。弱光处理后,籽粒氮素积累量及氮素收获指数减少。但弱光使籽粒蛋白质含量显著升高,其中灌浆中期弱光升幅最大,原因可能是由于其粒重降低造成的。弱光对可溶性谷蛋白无显著影响,但增加不溶性谷蛋白含量,使谷蛋白聚合指数显著升高,面团形成时间和稳定时间亦升高,籽粒灌浆中、后期弱光对上述指标的影响较前期大。灌浆期短暂的弱光照对改善强筋小麦粉质仪参数有利,但使弱筋小麦变劣;并均伴随籽粒产量的显著降低这一不利影响。     

Effects of crop thinning and reduced grain numbers per ear on grain size in two winter wheat varieties given different amounts of nitrogen

To study the importance for final grain size in wheat (Triticum aestivum, L.) of assimilate supply and the storage capacity of the grain, two field experiments were done. In 1976 nitrogen was applied in the range from none to 180 kg ha^-1, part of the crop was thinned, and the top halves of some ears of the short variety Hobbit and of the tall variety Maris Huntsman were removed soon after anthesis. In 1977 ears of Maris Huntsman were halved 5 days after anthesis or at 30 days after anthesis when grain volume was maximum. Thinning the crop from 360 to 180 ear-bearing shoots m^-2 30 days before anthesis increased the number of grains per ear, except in the absence of nitrogen fertiliser, but did not increase grain size, grain dry weight per ear or total dry weight per culm. Removing the upper half of ears of Hobbit 5 days after anthesis increased dry weight per grain, but when this treatment was applied to Maris Huntsman either 5 days after anthesis in 1976 and 1977, or when grain volume was maximal in 1977, the grains failed to increase in dry weight. Non-grain dry weight of both varieties was increased by halving the ear. In both varieties the maximum volume of grains in halved ears was larger than in intact ears. Grain dry weight increased relatively less than volume after halving the ear of Hobbit, and the decrease in volume up to maturity was greater in halved than intact ears of both varieties. The larger grain volume in halved ears of Maris Huntsman in 1977 was associated with more endosperm cells.     

小麦开花后iPA和ABA含量的变化及与旗叶光合,根系活力和籽？…

以２个粒重不同的小麦品种为材料,研究了开花后旗叶、根系和籽粒异戊烯基腺苷（ｉＰＡ）和脱落酸（ＡＢＡ）含量的变化及其与旗叶光合速率、根系活力和籽粒样重的关系。结果表明,高粒重的鲁麦２２较低粒重的鲁麦１４,旗叶和根系ｉＰＡ含量高且速降始期推迟,ＡＢＡ含量低且速升始期推迟;与此相对应,旗叶光合速率和根系活力高且高值持续期长。鲁麦２２籽粒ｉＰＡ含量高于鲁麦１４,其ＡＢＡ含量在花后１６ｄ内高于鲁麦１４,１６     

Change in wall composition of transfer and aleurone cells during wheat grain development

In addition to the starchy endosperm, a specialized tissue accumulating storage material, the endosperm of wheat grain, comprises the aleurone layer and the transfer cells next to the crease. The transfer cells, located at the ventral region of the grain, are involved in nutrient transfer from the maternal tissues to the developing endosperm. Immunolabeling techniques, Raman spectroscopy, and synchrotron infrared micro-spectroscopy were used to study the chemistry of the transfer cell walls during wheat grain development. The kinetic depositions of the main cell wall polysaccharides of wheat grain endosperm, arabinoxylan, and (1–3)(1–4)-β-glucan in transfer cell walls were different from kinetics previously observed in the aleurone cell walls. While (1–3)(1–4)-β-glucan appeared first in the aleurone cell walls at 90°D, arabinoxylan predominated in the transfer cell walls from 90 to 445°D. Both aleurone and transfer cell walls were enriched in (1–3)(1–4)-β-glucan at the mature stage of wheat grain development. Arabinoxylan was more substituted in the transfer cell walls than in the aleurone walls. However, arabinoxylan was more feruloylated in the aleurone than in the transfer cell walls, whatever the stage of grain development. In the transfer cells, the ferulic acid was less abundant in the outer periclinal walls while para-coumarate was absent. Possible implications of such differences are discussed.     

Photosynthetic and translocation pattern in contrasting winter wheat varieties

The rates of photosynthesis and patterns of translocation of ¹⁴C from the flag leaves of tall and semi-dwarf wheat varieties, with and without awns, were compared in a field experiment. The rates of photosynthesis of the awnless varieties fell during the 14 days after anthesis but those of the awned varieties did not change. Wide differences in translocation pattern of ¹⁴C supplied at anthesis were also found between varieties, but the differences in distribution pattern of ¹⁴C supplied 14 days later were less. At anthesis, the high yielding semi-dwarf selection translocated most carbohydrate to the ears and awned selections least. It is suggested that varietal differences in translocation pattern are determined by differences in the capacity of the grain to store carbohydrate.     

Abscisic acid in developing grains of wheat and barley genotypes differing in grain weight

Two genetically related wheat lines growing in cabinets were given different temperatures during grain filling, and abscisic acid (ABA) was measured in whole grains by gas chromatography with an electron-capture detector. Three genetically related barley lines grown in the field were assayed for ABA content in endosperm and embryo fractions separately by radiommunoassay.Maximum grain growth rate and final weight per grain of the two wheat lines differed by 50–60% at low temperature and 30–40% at high temperature. During grain development two peaks in ABA level were observed at low temperature but only one at high temperature. At times when differences in grain growth rate between genotypes and between temperature treatments were large, the corresponding differences in ABA concentration were small. In barley, one line (Iabo 14) had 30% heavier grains than the other two (Onice and Opale). Endosperm ABA concentrations showed no clear differences between genotypes until grain filling was nearly complete. Embryo ABA levels were up to 10-times greater than those in the endosperm, with Opale having significantly less ABA in the embryo than the other two cultivars.Our experiments did not provide evidence for a causal relationship between ABA levels during grain filling and grain growth rate or final weight.Abbreviations ABA Abscisic acid - DAA days after anthesis - DW dry weight - FW fresh weight     

Arabinoxylan and (1→3),(1→4)-β-glucan deposition in cell walls during wheat endosperm development

Arabinoxylans (AX) and (1→3),(1→4)-β-glucans are major components of wheat endosperm cell walls. Their chemical heterogeneity has been described but little is known about the sequence of their deposition in cell walls during endosperm development. The time course and pattern of deposition of the (1→3) and (1→3),(1→4)-β-glucans and AX in the endosperm cell walls of wheat (Triticum aestivum L. cv. Recital) during grain development was studied using specific antibodies. At approximately 45°D (degree-days) after anthesis the developing walls contained (1→3)-β-glucans but not (1→3),(1→4)-β-glucans. In contrast, (1→3),(1→4)-β-glucans occurred widely in the walls of maternal tissues. At the end of the cellularization stage (72°D), (1→3)-β-glucan epitopes disappeared and (1→3),(1→4)-β-glucans were found equally distributed in all thin walls of wheat endosperm. The AX were detected at the beginning of differentiation (245°D) in wheat endosperm, but were missing in previous stages. However, epitopes related to AX were present in nucellar epidermis and cross cells surrounding endosperm at all stages but not detected in the maternal outer tissues. As soon as the differentiation was apparent, the cell walls exhibited a strong heterogeneity in the distribution of polysaccharides within the endosperm.     

Development of Embryo and Endosperm and Nutrient Accumulation in Vigna sesquipedalis (L.) Fruwirth

The structure of embryo sac, fertilization and development of embryo and endosperm in Vigina sesquipedalis (L.) Fruwirth were investigated. Pollization occures 7–10h before anthesis, and fertilization is completed 10 h after anthesis. After fertilization, wall ingrowths are formed at the micropylar and chalazal ends of the embryo sac. Embryo development conforms to the Onagrad type, and passes through 2 or more celled proembryo, long stick-shaped, globular, heart shaped, torpedo, young embryo, growing and enlarging embryo and mature embryo. Wall ingrowths are formed on the walls of basal cells and outer walls of the cells at basal region of suspenser. The suspensor remains as the seed reaches maturity. The starch grains accumulate in the cells of cotyledons by 9–16 days after anthesis, and proteins accumulate by 12–18 days after. The endosperm development follows the nuclear type. The endosperm ceils form at the micropylar end, and remain free nuclear phase at chalazal end. The outer cells are transfer cells. Those cells at the micropylar end form folded cells with wall ingrowths. At heartembryo stage, the endosperm begins to degenerate and disintegrates before the embryo matures.     

The pattern of amyloplast DNA accumulation during wheat endosperm development

The accumulation of amyloplast DNA during endosperm development was studied in two cultivars of spring wheat, Triticum aestivum L. Chinese Spring (CS) and Spica, small and relatively larger-grained cultivars, respectively. Endosperms were isolated between 9 and 45 days post anthesis (dpa) and the amyloplast DNA content of endosperm nucleic-acid extracts was measured by quantitative hybridisation with a homologous chloroplast-DNA probe. The endosperm cells of CS and Spica accumulated amyloplast DNA during development in a similar way. In both cultivars there was a large increase in the amount of plastid DNA (ptDNA) per endosperm between 9 and about 15 dpa, after which there was no further increase. Because nuclear DNA continued to accumulate until 24 dpa, the percentage contribution of amyloplast DNA to total DNA fluctuated in both cultivars during development, reaching maxima at 12 dpa of about 1.00% and 0.85%, and dropping to apparently constant levels of 0.60% and 0.52% in CS and Spica, respectively, by 24 dpa. In both cultivars, the average number of ptDNA copies per amyloplast was calculated to increase from about 10 copies at 9 dpa to about 50 copies in the mature amyloplasts at 31 dpa. However, the heavier endosperms of Spica contain more cells than those of CS and the varieties therefore differed in the amount of ptDNA that accumulated per endosperm: Spica endosperms accumulated 110 ng of ptDNA by 15 dpa, compared with only 85 ng in CS. The apparent accumulation of ptDNA copies in wheat amyloplasts during endosperm development contrasts with the decline in chloroplast-DNA copies in wheat chloroplasts during leaf development.Abbreviations CS Chinese Spring - ctDNA chloroplast DNA - dpa days post anthesis - kbp 10³ base pairs - nDNA nuclear DNA - ptDNA plastid DNA - mtDNA mitochondrial DNA     

Investigation of ferulate deposition in endosperm cell walls of mature and developing wheat grains by using a polyclonal antibody

A polyclonal antibody has been raised against ferulic acid ester linked to arabinoxylans (AX). 5-O-feruloyl-α-l-arabinofuranosyl(1→4)-β-d-xylopyranosyl was obtained by chemical synthesis, and was coupled to bovine serum albumin for the immunization of rabbit. The polyclonal antibody designated 5-O-Fer-Ara was highly specific for 5-O-(trans-feruloyl)-l-arabinose (5-O-Fer-Ara) structure that is a structural feature of cell wall AX of plants belonging to the family of Gramineae. The antibody has been used to study the location and deposition of feruloylated AX in walls of aleurone and starchy endosperm of wheat grain. 5-O-Fer-Ara began to accumulate early in aleurone cell wall development (beginning of grain filling, 13 days after anthesis, DAA) and continued to accumulate until the aleurone cells were firmly fixed between the starchy endosperm and the nucellus epidermis (19 DAA). From 26 DAA to maturity, the aleurone cell walls changed little in appearance. The concentration of 5-O-Fer-Ara is high in both peri- and anticlinal aleurone cell walls with the highest accumulation of 5-O-Fer-Ara at the cell junctions at the seed coat interface. The situation is quite different in the starchy endosperm: whatever the stage of development, a low amount of 5-O-Fer-Ara epitope was detected. Contrary to what was observed for aleurone cell walls, no peak of accumulation of feruloylated AX was noticed between 13 and 19 DAA. Visualization of labelled Golgi vesicles suggested that the feruloylation of AX is intracellular. The distribution of (5-O-Fer-Ara) epitope is further discussed in relation to the role of ferulic acid and its dehydrodimers in cell wall structure and tissue organization of wheat grain.     

Effects of the barley mutants Risø 1508 and 527 high lysine genes on the cellular development of the endosperm

The dry grain weight of the Risø barley ( Hordeum vulgare L. var. disticum ) high lysine mutants 1508 and 527 at maturity was 32 and 37% lower, respectively, than the grains of the cultivar Bomi. Dry grain weight of the double mutant 527/1508 was reduced by 57%. Total number of endosperm nuclei from cv. Bomi, mutants 1508, 527 and 527/1508 at 24 days after anthesis was 173 000,156 000,121 000 and 111 000, respectively. Transverse mid-grain sections from mutants 1508 and 527 contained fewer aleurone cells and approximately the same number of starchy endosperm cells as cv. Bomi. The cellular organization of the endosperm of the double mutant deviated substantially from the normal. Cell volume in the central starchy endosperm of cv. Bomi, mutants 1508 and 527 at 33 days after anthesis averaged 390 000, 270 000 and 180 000 um5, respectively. Cell volume in the double mutant was smaller than in 527, but could not be accurately estimated due to the irregular shape of the cells. The mean section area of individual large starch granules in the central endosperm of mutants 1508, 527 and 527/1508 at 33 days after anthesis was 30, 48 and 72% smaller, respectively, than those of cv. Bomi. The average aleurone cell volume in cv. Bomi at 33 days after anthesis was 6 200 μm³.