Spatial distribution of proteins and metabolites in developing wheat grain and their differential regulatory response during the grain filling process

Grain filling and grain development are essential biological processes in the plant’s life cycle, eventually contributing to the final seed yield and quality in all cereal crops. Studies of how the different wheat (Triticum aestivum L.) grain components contribute to the overall development of the seed are very scarce. We performed a proteomics and metabolomics analysis in four different developing components of the wheat grain (seed coat, embryo, endosperm, and cavity fluid) to characterize molecular processes during early and late grain development. In-gel shotgun proteomics analysis at 12, 15, 20, and 26 days after anthesis (DAA) revealed 15 484 identified and quantified proteins, out of which 410 differentially expressed proteins were identified in the seed coat, 815 in the embryo, 372 in the endosperm, and 492 in the cavity fluid. The abundance of selected protein candidates revealed spatially and temporally resolved protein functions associated with development and grain filling. Multiple wheat protein isoforms involved in starch synthesis such as sucrose synthases, starch phosphorylase, granule-bound and soluble starch synthase, pyruvate phosphate dikinase, 14-3-3 proteins as well as sugar precursors undergo a major tissue-dependent change in abundance during wheat grain development suggesting an intimate interplay of starch biosynthesis control. Different isoforms of the protein disulfide isomerase family as well as glutamine levels, both involved in the glutenin macropolymer pattern, showed distinct spatial and temporal abundance, revealing their specific role as indicators of wheat gluten quality. Proteins binned into the functional category of cell growth/division and protein synthesis/degradation were more abundant in the early stages (12 and 15 DAA). At the metabolome level all tissues and especially the cavity fluid showed highly distinct metabolite profiles. The tissue-specific data are integrated with biochemical networks to generate a comprehensive map of molecular processes during grain filling and developmental processes.     

Endogenous abscisic acid and wheat germ agglutinin content in wheat grains during development

Abscisic acid (ABA) and wheat germ agglutinin content of immature wheat grains and embryos was determined by immunoassay throughout the development of a field-grown wheat crop ( Triticum aestivum cv. Timmo). Wheat germ agglutinin accumulation in the embryo was not preceded by an increase in endogenous abscisic acid amount or concentration in either embryos or grains. At a later stage in development the endogenous concentration of abscisic acid in both embryos and grains was found to be two orders of magnitude lower than the endogenous levels required to inhibit precocious germination and promote wheat germ agglutinin accumulation in excised embryos cultured in vitro. These findings are discussed in the context of the control of embryo development in vivo by both ABA and the water status of the grain and embryo.     

Changes in water relations and endogenous abscisic acid content of wheat and barley grains and embryos during development

Abstract. Immature cereal embryo development can be controlled by in vitro culture on media containing ABA, or by media of low osmotic potential. To assess the possible in vivo roles of these factors, endogenous ABA levels and water relations of embryos and grains of wheat ( Triticum aestivum L.) and barley ( Hordeum vulgare L.) were determined during development. ABA concentrations remained consistent with those required to inhibit precocious germination in vitro of early stage embryos but not of more mature embryos. With increasing maturity, a difference in water potential developed between grain and embryo, suggestive of an in vivo role for water status in controlling the development of the embryo.     

High night temperatures during grain number determination reduce wheat and barley grain yield: a field study

Warm nights are a widespread predicted feature of climate change. This study investigated the impact of high night temperatures during the critical period for grain yield determination in wheat and barley crops under field conditions, assessing the effects on development, growth and partitioning crop‐level processes driving grain number per unit area (GN). Experiments combined: (i) two contrasting radiation and temperature environments: late sowing in 2011 and early sowing in 2013, (ii) two well‐adapted crops with similar phenology: bread wheat and two‐row malting barley and (iii) two temperature regimes: ambient and high night temperatures. The night temperature increase (ca. 3.9 °C in both crops and growing seasons) was achieved using purpose‐built heating chambers placed on the crop at 19:000 hours and removed at 7:00 hours every day from the third detectable stem node to 10 days post‐flowering. Across growing seasons and crops, the average minimum temperature during the critical period ranged from 11.2 to 17.2 °C. Wheat and barley grain yield were similarly reduced under warm nights (ca. 7% °C^?1), due to GN reductions (ca. 6% °C^?1) linked to a lower number of spikes per m². An accelerated development under high night temperatures led to a shorter critical period duration, reducing solar radiation capture with negative consequences for biomass production, GN and therefore, grain yield. The information generated could be used as a starting point to design management and/or breeding strategies to improve crop adaptation facing climate change.     

BiP, HSP70, NDK and PDI in wheat endosperm. I. Accumulation of mRNA and protein during grain development

Biosynthesis and accumulation of seed storage proteins such as the wheat glutens depend on the activity of a variety of other proteins, including chaperones and foldases. cDNA probes and antibodies to two chaperone proteins and a foldase were used to follow mRNA and protein accumulation in developing grains of wheat ( Triticum aestivum , cvs Cheyenne and Butte). Endosperm was separated from other grain components and protein accumulation was analyzed on a per mg fresh weight basis. The ER resident chaperone BiP (binding protein) and foldase PDI (protein disulfide isomerase) accumulated to maximal levels in the middle stage of endosperm development, a period of rapid cell expansion and storage protein accumulation, whereas levels of a cytosolic chaperone, HSP70, remained relatively constant throughout grain development. In contrast, nucleoside diphosphate kinase (NDK), a cytosolic enzyme needed for synthesis of nucleoside triphosphates, accumulated early in endosperm development during the period of nuclear division and cell formation. When analyzed as a fraction of total protein the relative abundance of all four proteins peaked early in grain development and then declined. Accumulation of mRNA for the four proteins also peaked early in grain development. Although BiP and PDI formed a declining percentage of total protein as storage protein accumulated, their pattern of accumulation was compatible with a proposed role as catalysts for storage protein folding and accumulation in the ER.     

有机无机肥长期配合施用对冬小麦籽粒品质的影响

提高籽粒品质和产量是当前国内小麦生产的核心。小麦品质和产量取决于基因型、生态环境 (如土壤肥力等 )和栽培技术(尤其是养分管理技术 )。长期肥料试验是研究养分管理对小麦产量和品质影响的有效手段 ,迄今 ,长期施肥对小麦产量的影响报道很多 ,但对籽粒品质的影响报道很少。在 2 0 a长期定位肥料试验的基础上 ,研究了有机无机肥长期配合施用对不同类型小麦籽粒品质性状的影响。结果表明 ,有机肥主效应对小麦籽粒产量有显著作用 ,而对大部分品质指标无影响 ;无机肥处理主效应及有机无机肥交互效应对籽粒产量和大部分品质性状均有显著影响。有机无机肥料配合施用与单施无机肥处理相比提高了小麦大部分品质性状 ,有利于强筋小麦籽粒产量和品质的同步提高 ,但不利于弱筋小麦品质的改善。进一步分析了土壤肥力及磷钾肥对小麦籽粒品质的影响     

Abscisic acid and ethylene interact in wheat grains in response to soil drying during grain filling

Grain filling is an intensive transportation process regulated by soil drying and plant hormones. This study investigated how the interaction between abscisic acid (ABA) and ethylene is involved in mediating the effects of soil drying on grain filling in wheat (Triticum aestivum). Two wheat cultivars, cv. Yangmai 6 and cv. Yangmai 11, were field-grown, and three irrigation treatments, well-watered, moderately soil-dried (MD) and severely soil-dried (SD), were imposed from 9 d post anthesis until maturity. A higher ABA concentration and lower concentrations of ethylene and 1-aminocylopropane-1-carboxylic acid (ACC) were found in superior grains (within a spike, those grains that were filled earlier and reached a greater size) than in inferior grains (within a spike, those grains that were filled later and were smaller), and were associated with a higher filling rate in the superior grains. An increase in ABA concentration and reductions in ethylene and ACC concentrations in grains under MD conditions increased the grain-filling rate, whereas much higher ethylene, ACC and ABA concentrations under SD conditions reduced the grain-filling rate. Application of chemical regulators gave similar results. The results did not differ between the two cultivars. The grain-filling rate in wheat is mediated by the balance between ABA and ethylene in the grains, and an increase in the ratio of ABA to ethylene increases the grain-filling rate.     

Long- and short-term effects of decreased sink demand on carbohydrate levels and photosynthesis in wheat leaves

Wheat (Triticum aestivum L.) ears were removed to investigate long-term regulation of photosynthesis by sink demand at ambient CO₂ and 22 °C. The CO₂ level was also increased to 660 μmol mol^?1 and temperature was lowered to 5 °C to examine short-term responses of photosynthesis to low sink demand. Sink removal inhibited photosynthesis and increased leaf levels of glucose, fructose and ribulose-1, 5-bisphosphate (RuBP), and the glucose-6-phosphate (G6P)/fructose-6-phosphate (F6P) and RuBP/3-phosphoglycerate (PGA) ratios under growth conditions, but had no effect on the activity and activation state of ribulose-1, 5-bisphosphate carboxylase oxygenase (Rubisco) either under growth or short-term conditions, suggesting an inhibition of photosynthesis by decreased in vivo catalysis of Rubisco. Photosynthesis increased similarly in eared and earless shoots after a rise in CO₂ concentration, and the ratio of triose-phosphates (glyceraldehyde 3-phosphate and dihydroxyacetone phosphate, TP) to PGA was similar or higher for removed than intact ears, suggesting that feedback inhibition of photosynthesis was not caused by a limitation of ATP synthesis in chloroplasts. Under short-term conditions (660 μmol mol^?1 CO₂, 5 °C), TP and RuBP levels and the TP/PGA and TP/RuBP ratios were increased by sink removal, indicating an additional limitation of photosynthesis by the rate of RuBP regeneration.     

基于生理指标与籽粒产量关系的小麦品种抗冻性分析

以20个冬小麦品种为材料进行盆栽试验,对其在低温胁迫条件下功能叶超氧化物歧化酶（SOD）、过氧化物酶（POD）、过氧化氢酶（CAT）活性,丙二醛（MDA）、可溶性蛋白、可溶性糖含量以及籽粒产量、千粒重和籽粒形态性状进行测定.结果表明：拔节初期麦苗经-4 ℃低温胁迫后,不同品种冬小麦籽粒形态性状和产量性状均发生变化,绝大多数小麦品种籽粒长宽比、圆度和不育小穗数均增加,籽粒等效直径和面积及千粒重和籽粒产量均下降.通径分析表明,拔节初期低温处理后,功能叶SOD活性和可溶性糖含量是影响籽粒产量的主导因素,其中SOD活性对籽粒产量的直接影响较大,直接通径系数为-0.578.以籽粒产量下降的百分数作为小麦抗冻性评价的标准,可将20个小麦品种划分为3类：强抗冻类型的济麦19、济麦20、良星99、山农1135、山农8355、泰山23、泰山9818、汶农6号和烟农21,弱抗冻类型的临麦2号、潍麦8号、烟农19和淄麦12号,而其余7个品种属中度抗冻类型.苗期综合评价值（D值）与籽粒产量下降的百分数之间呈显著负相关(r=-0.512*),说明小麦苗期抗冻性强有利于获得较高的籽粒产量.苗期是小麦抗冻性鉴别选择的重要时期.     

Changes in low-molecular-weight thiol-disulphide redox couples are part of bread wheat seed germination and early seedling growth

The tripeptide antioxidant glutathione (γ-l-glutamyl-l-cysteinyl-glycine; GSH) essentially contributes to thiol-disulphide conversions, which are involved in the control of seed development, germination, and seedling establishment. However, the relative contribution of GSH metabolism in different seed structures is not fully understood. We studied the GSH/glutathione disulphide (GSSG) redox couple and associated low-molecular-weight (LMW) thiols and disulphides related to GSH metabolism in bread wheat (Triticum aestivum L.) seeds, focussing on redox changes in the embryo and endosperm during germination. In dry seeds, GSH was the predominant LMW thiol and, 15?h after the onset of imbibition, embryos of non-germinated seeds contained 12 times more LMW thiols than the endosperm. In germinated seeds, the embryo contained 17 and 11 times more LMW thiols than the endosperm after 15 and 48?h, respectively. This resulted in the embryo having significantly more reducing half-cell reduction potentials of GSH/GSSG and cysteine (Cys)/cystine (CySS) redox couples (E_GSSG/2GSH and E_CySS/2Cys, respectively). Upon seed germination and early seedling growth, Cys and CySS concentrations significantly increased in both embryo and endosperm, progressively contributing to the cellular LMW thiol-disulphide redox environment (E_{thiol-disulphide}). The changes in E_CySS/2Cys could be related to the mobilisation of storage proteins in the endosperm during early seedling growth. We suggest that E_GSSG/2GSH and E_CySS/2Cys can be used as markers of the physiological and developmental stage of embryo and endosperm. We also present a model of interaction between LMW thiols and disulphides with hydrogen peroxide (H₂O₂) in redox regulation of bread wheat germination and early seedling growth.     

Accumulation and conversion of sugars by developing wheat grains. 4. Effects of phosphate and potassium ions in endosperm slices

Abstract. Transverse slices through developing grains of Triticum aestivum cv. SUN 9E 16 d after anthesis were incubated in simple defined media with various radioactive labels. In some enzymic assays slices were pretreated with 2.5% Triton X-100 or with 5% butanol to remove cellular membranes and endogenous substrates.
Endogenous potassium leaked from endosperm slices into 30mol m⁻³ sucrose while sucrose was converted partly into starch. Exogenous alkali-ions, except Li⁺, stimulated conversion of sucrose to insoluble matter, specifically to starch with K⁺. Starch synthetase activity of Triton-pretreated slices was stimulated by K⁺ at both high and low substrate ADPG concentration, but was not affected by phosphate (25 mol m⁻³).
Phosphate in the medium had no effect on incorporation of sucrose or glucose into alcohol-insoluble material or starch in fresh slices (internal inorganic phosphate (P,) concentration was about 11 mol m⁻³). Three- to four-fold contrasts in internal P_i level, achieved by prolonged preincubations in different media, did not show an inhibition of starch synthesis by P_i. However, phosphate (25mol m⁻³) inhibited starch synthesis, that was mediated by ADPG pyrophosphorylase in butanol-pretreated endosperm slices by 15–18%.
It is concluded that starch synthesis in wheat endosperm is not regulated directly by apoplastic P_i; level.     

Expression of fission yeast cdc25 driven by the wheat ADP-glucose pyrophosphorylase large subunit promoter reduces pollen viability and prevents transmission of the transgene in wheat

Cell number was to be measured in wheat (Triticum aestivum) endosperm expressing Spcdc25 (a fission yeast cell-cycle regulator) controlled by a supposedly endosperm-specific promoter, AGP2 (from the large subunit of ADP glucose pyrophosphorylase). Wheat was transformed by biolistics either with AGP2::GUS or AGP2::Spcdc25. PCR and RT-PCR checked integration and expression of the transgene, respectively. In cv. Chinese Spring, AGP2::GUS was unexpectedly expressed in carpels and pollen, as well as endosperm. In cv. Cadenza, three AGP2::Spcdc25 plants, AGP2::Spcdc25.1, .2 and .3, were generated. Spcdc25 expression was detected in mature leaves of AGP2::Spcdc25.1/.3 which exhibited abnormal spikes, 50% pollen viability and low seed set per plant; both were small compared with the nonexpressing and normal AGP2::Spcdc25.2. Spcdc25 was not transmitted to the T(1) in AGP2::Spcdc25.1 or .3, which developed normally. Spcdc25 was PCR-positive in AGP2::Spcdc25.2, using primers for a central portion, but not with primers for the 5' end, of the ORF, indicating a rearrangement; Spcdc25 was not expressed in either T(0) or T(1). The AGP2 promoter is not tissue-specific and Spcdc25 expression disrupted reproduction.     

Amino acid metabolism associated with N-mobilization from the flag leaf of wheat (Triticum aestivum L.) during grain development

Field-grown winter wheat (Triticum aestivum L. cv. Castell) was used to study changes in the free amino acid pools of different plant parts and related enzyme activities in the flag leaf throughout the grain-filling period in three consecutive growing seasons. Amino acid analysis data indicated that, during senescence, the nitrogen flow in the flag leaf was directed towards the synthesis of glutamine as a specific nitrogen transport form. Of the enzymes involved, total glutamine synthetase (GS; EC 6.3.1.2) and especially ferredoxin-dependent glutamate synthase (Fd-GOGAT; EC 1.4.7.1) activities declined continuously as senescence progressed. Unlike (chloroplastic) GS₂, (cytosolic) GS₁ was shown to be very persistent suggesting a special role for this isoenzyme in the N-reallocation process. Glutamate-oxaloacetate transaminase (GOT; EC 2.6.1.1), glutamate-pyruvate transaminase (GPT; EC 2.6.1.2) and isocitrate dehydrogenase (IDH; EC 1.1.1.42) showed a characteristic biphasic activity profile after anthesis. It is proposed that these enzymes, for each of which at least two isoenzymes were demonstrated, are involved in glutamate synthesis at the later stages of leaf senescence. Ammonium levels were fairly constant throughout the flag leafs life span, an ultimate rise often following peak values of glutamate dehydrogenase (GDH; EC 1.4.1.4) activity. The enzymology of flag leaf amino acid metabolism during grain development is further discussed in relation to observations of NH₃-volatilization from naturally senescing wheat plants.     

Carbon mobilization in shoot parts and roots of wheat during grain filling: assessment by 13C/12C steady-state labelling, growth analysis and balance sheets of reserves

cv, cultivar
δ, deviation of C isotope composition from a standard
Δ, C isotope discrimination
WSC, water soluble carbohydrates

Steady-state labelling of all post-anthesis photosynthate of wheat was performed to assess the mobilization of pre-anthesis C (C fixed prior to anthesis) in vegetative plant parts during grain filling. Results were compared with estimates obtained by indirect approaches to mobilization of pre-anthesis C: ‘classical’ growth analysis and balance sheets of water soluble carbohydrates (WSC) and protein. Experiments were performed with two spring wheat cultivars grown with differential nitrogen fertilizer supply in 1991 and 1992. The fraction of pre-anthesis C mobilized in above-ground vegetative biomass ranged between 24 and 34% of total C present at anthesis. Treatment effects on mobilization of pre-anthesis C in total above-ground vegetative biomass were closely related (r² = 0·89) to effects on mobilization of WSC-C plus protein-C (estimated as N mobilized × 3·15). On average, 81% of pre-anthesis C mobilization was attributable to the balance of pre-anthesis WSC (48%) and protein (33%) between anthesis and maturity. In roots, WSC and protein mobilization accounted for only 29% of the loss of pre-anthesis C. Notably, mobilization of pre-anthesis C was 1·4–2·6 times larger than the net loss of C from above-ground vegetative biomass between anthesis and maturity. This discrepancy was mainly due to post-anthesis C accumulation in glumes and stem. Post-anthesis C accumulation was related to continued synthesis of structural biomass after anthesis and accounted for a mean 15% of total C contained in above-ground vegetative plant parts at maturity. A close correspondence between net loss of C and mobilization of pre-anthesis C was only apparent in leaf blades and leaf sheaths. Although balance sheets of WSC and protein also underrated the mobilization of pre-anthesis C by ≈ 19%, they gave a much better estimate of pre-anthesis C mobilization than growth analysis.     

黄淮海平原玉米施氮量对后茬小麦土壤剖面硝态氮和产量的影响

在冬小麦-夏玉米一年两熟模式下,玉米品种“郑单958”（植株密度9株/m^2）和小麦品种“93-9”（基本苗704株/m^2）,冬小麦基施144kg N/hm^2,研究了玉米5个施N量（0、90、180、270和360kg/hm^2）对后茬小麦期间土壤剖面硝态氮含量、无机氮总量,以及小麦氮素吸收利用和产量的影响.结果表明：（1）与不施氮相比,玉米施氮显著增加小麦季0～200cm土壤硝态氮含量;自拔节起,0～40cm、0～130cm和0～200cm硝态氮含量均随施氮量增加而递增,在硝态氮含量较高的小区增幅也大.（2）轮作一周期后,不施氮和施氮360kg/hm^2显著影响0～130cm和0～200cm无机氮总量,但在90～270 kg/hm^2之间,施氮量的影响不明显.（3）施氮小于180kg/hm^2时,成熟期小麦植株氮素和籽粒氮素积累量、氮肥利用率均随施氮量增加而递增,但不明显.（4）与不施氮相比,施氮90kg/hm^2的小麦产量和麦玉轮作总产均增加但不明显,施氮180 kg/hm^2均显著增加,施氮270kg/hm^2与180kg/hm^2无明显差异.本试验条件下,夏玉米施氮90～180 kg/hm^2是适宜的.     

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。