Effect of soil salinity on grain filling and grain development in barley

Experiments were conducted with five barley cultivars with the aim of ascertaining the effect of salt stress on grain filling, grain development and bioohemical composition of developing grains. Reduced grain yield under salt stress was found to be due to reduced efficiency per day to fill the grains and consequent more effective days and also due to disturbed starch-sugar balance.     

Proteome analysis of B-cell maturation

Proteins affected by anti-mIgM stimulation during B-cell maturation were identified using 2-DE-based proteomics. We investigated the proteome profiles of stimulated and nonstimulated Ramos B-cells at eight time points during 5 d and compared the obtained proteomic data to the corresponding data from DNA-microarray studies. Anti-mIgM stimulation of the cells resulted in significant differences (> or =twofold) in the protein abundance close to 100 proteins and differences in post-translational protein modifications. Forty-eight up- or down-regulated proteins were identified by mass spectrometric methods and database searches. The identities of a further nine proteins were revealed by comparing their positions to the known proteins in other lymphocyte 2-DE databases. Several of the proteins are directly related to the functional and morphological characteristics of B-cells, such as cytoskeleton rearrangement and intracellular signalling triggered by the crosslinking of B-cell receptors. In addition to proteins known to be involved in human B-cell maturation, we identified several proteins that were not previously linked to lymphocyte differentiation. The results provide deeper insights into the process of B-cell maturation and may lead to novel therapeutic strategies for immunodeficiencies. An interactive 2-DE reference map is available at http://bioinf.uta.fi/BcellProteome.     

Effects of powdery mildew on grain filling in spring barley in contrasting environments

Circumstantial evidence from field experiments at Rothamsted suggested that effects of powdery mildew on grain filling in spring barley may be determined partly by temperature during the grain-filling period. An experiment was, therefore, done which compared the effects of fungicides applied to control powdery mildew on grain filling in early- and late-sown spring barley plants kept either out-of-doors throughout their growth (‘cool’ environment) or under the same conditions until the start of grain filling and then transferred to a heated glasshouse (‘warm’ environment) until harvested. Fungicides that controlled mildew increased the total grain yield of the late-sown barley more than that of the early-sown and much more in the warm environment than in the cool. On average, the effect of the fungicides in the cool environment was to increase grain yield by 17·7%. Small increases in numbers of grains/ear (+ 3·4%) and thousand-grain weight (TGW) (+ 2·3%) contributed to this increase but it could be attributed principally to an average increase in numbers of ears/plant of 12·4%. Contrastingly, fungicides increased average grain yield in the warm environment by 58·2%. Effects of the fungicides on numbers of ears/plant (+ 27·8%) and on numbers of grains/ear (+ 4·5%) were not significantly different to those in the cool environment, and the much greater responses in the warm than in the cool environment could be attributed mostly to much larger effects on grain size (+ 19·2%) The greater benefits of the fungicides and, by implication, the greater damage done by powdery mildew in the warm than in the cool environment cannot, unequivocally, be attributed to differences in temperature during grain-filling because the two environments clearly differed in other ways and especially in light intensity. Nevertheless, the results obtained do illustrate the potential risks involved in using data obtained under one set of circumstances to predict what will happen in another, especially when environments differ as greatly as glasshouses and fields.     

Proteome analysis of Paenibacillus polymyxa E681 affected by barley

Paenibacillus polymyxa E681 is known to be able to suppress plant diseases by producing antimicrobial compounds and to promote plant growth by producing phytohormones, and secreting diverse degrading enzymes. In spite of these capabilities, little is known regarding the flow of information from the bacterial strain to the barley roots. In an attempt to determine the flow of information from the bacterial strain to barley roots, the train was grown in the presence and absence of barley, and two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and MALDI-TOF mass spectrometry were used. 2D-PAGE detected approximately 1000 spots in the cell and 1100 spots in the supernatant at a pH 4-10 gradient. Interestingly, about 80 spots from each sample showed quantitative variations. Fifty-three spots from these were analyzed by MALDI-TOF mass spectrometry and 28 proteins were identified. Most of the cytosolic proteins expressed at higher levels were found in P. polymyxa E681 cells grown in the presence of barley rather than in the absence of barley. Proteins detected at a lower level in the surpernatant of P. polymyxa E681 cells grown in the presence of barley were lipoprotein, glucose-6-phosphate 1-dehydrogenase, heat-shock protein HtpG spermidine synthase, OrfZ, ribonuclease PH, and coenzyme PQQ synthesis protein, and flagellar hook-associated protein 2 whereas proteins detected at a higher level in the surpernatant of P. polymyxa E681 cells grown in the presence of barley included D-alanyl-D-alanine ligase A, isopentenyldiphosphate delta-isomerase, ABC transporter ATP-binding protein Uup, lipase. Many of the proteins belonging to plant-induced stimulons are associated with biosynthetic metabolism and metabolites of proteins and transport. Some of these proteins would be expected to be induced by environmental changes resulting from the accumulation of plant-secreted substances.     

Changeability of protein fractions and their amino acid composition during maturation of barley grain

The formation of the protein complex during barley grain maturation is characterized by unequal synthesis of different protein fractions. In ten day-old grain the dominating protein is glutelin, followed by albumin and globulin. The content of prolamin is at this stage negligible. Particularly after the eighteenth day of maturation intensive synthesis of prolamins begins, which continues during the entire maturing period in almost the same ratio as total N accumulation in the grain. From the twenty-fourth day the amount of prolamins exceeds that of glutelins, and at full maturity 50% of the proteins are prolamins. The glutelin content increases absolutely, but relatively it decreases from 41 to 26%. The amino acid composition of flour from total grain as well as the amino acid composition of the individual proteins is significantly changed during maturation. Generally the changes are manifested in an increasing content of Glu and Pro and a decreasing content of Asp, Ala and Lys. These changes seem to occur as a result of an increasing representation of prolamin, which is characterized by large content of Glu and Pro and low content of Asp, Ala and Lys compared with other protein fractions.     

Growth characteristics, grain filling, and assimilate transport in a shrunken endosperm mutant of barley

The reported inheritance pattern of the seg1 shrunken endosperm mutant of barley (Hordeum vulgare L. cv Betzes) suggests that some defective process in the maternal plant tissues, and not in the endosperm, prevents normal grain filling in the mutant. To identify the physiological mechanism of the mutation, we compared growth, carbon exchange, and assimilate transport of Betzes and seg1 plants. Betzes and seg1 plants did not differ in mean relative growth rate, mean net assimilation rate, or carbon exchange rate. The rate and duration of grain growth of seg1 was lower than Betzes on intact plants and on detached, cultured spikes. Increasing the supply of sucrose in culture media up to 300 mm sucrose did not eliminate differences between normal and mutant grain growth. Translocation of ¹⁴C-labeled assimilates into seg1 grains ceased by 21 days after anthesis, and assimilates were diverted to lower plant parts. In contrast, assimilates were still entering Betzes grains at 29 days after anthesis. Evidence suggests that some maternal spike or grain tissue is affected by the mutation after the onset of grain filling. Identification of the specific seg1 defect may provide information about the cessation of normal grain filling.     

Synthesis of salt-soluble proteins in barley. Pulse-labeling study of grain filling in liquid-cultured detached spikes

The accumulation of salt-soluble proteins in the endosperm of developing barley (Hordeum vulgare L.) grains was examined. Detached spikes of barley were cultured at different levels of nitrogen nutrition and pulse-labeled with [¹⁴C] sucrose at specific times after anthesis. Proteins were extracted from isolated endosperms and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and crossed immunoelectrophoresis. Fluorography revealed an early, middle and late synthesis of specific proteins during grain filling. Synthesis of proteins appearing at the later stages responded to increased nitrogen nutrition. Two major components, -amylase and protein Z in particular, had a synthesis profile almost identical to that of the endosperm storage protein, hordein.Abbreviations CIE Crossed immunoelectrophoresis - SDSPAGE Sodium dodecyl sulphate polyacrylamide gel electrophoresis     

QTL dissection of the loss of green colour during post-anthesis grain maturation in two-rowed barley

Ability to genetically manipulate the loss of green colour during grain maturation has potentials for increasing productivity, disease resistance, and drought and heat tolerance in crop plants. Two doubled haploid, two-rowed barley populations (Vlamingh × Buloke and VB9524 × ND11231*12) were monitored over 2 years for loss of green colour during grain filling using a portable active sensor. The aims were to determine the genomic regions that control trait heritability by quantitative trait locus (QTL) analysis, and to examine patterns of QTL-environment interactions under different conditions of water stress. In the Vlamingh × Buloke cross, broad-sense heritability estimate for loss of green colour (measured as the difference in sensor readings taken at anthesis and maturity, ?SRI) was 0.68, and 0.78 for the VB9524 × ND11231*12 population. In the VB9524 × ND11231*12 population, rapid loss of green colour was positively associated with grain yield and percent plump grains, but in the Vlamingh × Buloke population, a slower loss of green colour (low ?SRI) was associated with increased grain plumpness. With the aid of a dense array of single nucleotide polymorphisms (SNPs) and EST-derived SSR markers, a total of nine QTLs were detected across the two populations. Of these, a single major locus on the short arm of barley chromosome 5H was consistently linked with trait variation across the populations and multiple environments. The QTL was independent of flowering time and explained between 5.4 and 15.4 % of the variation observed in both populations, depending on the environment, and although a QTL × E interaction was detected, it was largely due to a change in the magnitude of the effect, rather than a change in direction. The results suggest that loss of green colour during grain maturation may be under the control of a simple genetic architecture, but a careful study of target populations and environments would be required for breeding purposes.     

Genetic analysis of grain and malt quality in an elite barley population

Quantitative trait loci (QTLs) associated with grain weight, grain width, kernel hardness and malting quality were mapped in a doubled haploid population derived from two elite Australian malting barley varieties, Navigator and Admiral. A total of 30 QTLs for grain weight, grain width and kernel hardness were identified in three environments, and 63 QTLs were identified for ten malting quality traits in two environments. Three malting quality traits, namely β-amylase, diastatic power and apparent attenuation limit, were mainly controlled by a QTL linked to the Bmy1 gene at the distal end of chromosome 4H encoding a β-amylase enzyme. Six other malting quality traits, namely α-amylase, soluble protein, Kolbach index, free amino-acid nitrogen, wort β-glucan and viscosity, had coincident QTL clustered on chromosomes 1HS, 4HS, 7HS and 7HL, which demonstrated the interdependence of these traits. There was a strong association between these malt quality QTL clusters on chromosomes 1HS and 7HL and the major QTL for kernel hardness, suggesting that the use of this trait to enable early selection for malting quality in breeding programs would be feasible. In contrast, the majority of QTLs for hot-water extract were not coincident with those identified for other malt quality traits, which suggested differences in the mechanism controlling this trait. Novel QTLs have been identified for kernel hardness on chromosomes 2HL and 7HL, hot-water extract on 7HL and wort β-glucan on 6HL, and the resulting markers may be useful for marker-assisted selection in breeding programs.     

Increasing the abscisic acid level in maize grains induces precocious maturation by accelerating grain filling and dehydration

Elevated levels of abscisic acid (ABA) are closely associated with cereal grain filling under water deficit. However, grain dehydration during grain filling has received little attention. In this paper, three experiments with drought stress and exogenous ABA treatments were conducted to investigate the relationship between ABA and grain dehydration in maize (Zea mays L.) during the grain-filling period. The results indicated that exogenous ABA application and drought stress led to the same tendency of the grain ABA concentration, carbohydrate concentration and dehydration rate to increase but the moisture content to decrease. Moreover, the time to reach the maximum grain-filling rate was advanced, and the grain-filling period was shortened. In in vitro culture experiments, the sucrose-to-starch conversion was promoted, mainly influenced by sucrose synthase, ADP-glucose pyrophosphorylase (AGPase), and soluble starch synthase during the middle grain-filling stage, and the improvement in starch synthesis was possibly induced by AGPase. Correlation analysis showed that the ABA level was significantly negatively correlated with the moisture content and positively correlated with the starch level. A close and notably negative correlation was observed between the grain moisture content and starch level. In summary, adequate grain ABA promoted sucrose-to-starch conversion, shortened the duration of grain filling and accelerated grain dehydration, resulting in precocious grain maturation.     

Proteome reference maps of vegetative tissues in pea. An investigation of nitrogen mobilization from leaves during seed filling

A proteomic approach was used to analyze protein changes during nitrogen mobilization (N mobilization) from leaves to filling seeds in pea (Pisum sativum). First, proteome reference maps were established for mature leaves and stems. They displayed around 190 Coomassie Blue-stained spots with pIs from 4 to 7. A total of 130 spots were identified by mass spectrometry as corresponding to 80 different proteins implicated in a variety of cellular functions. Although the leaf proteome map contained more abundant spots, corresponding to proteins involved in energy/carbon metabolism, than the stem map, their comparison revealed a highly similar protein profile. Second, the leaf proteome map was used to analyze quantitative variations in leaf proteins during N mobilization. Forty percent of the spots showed significant changes in their relative abundance in the total protein extract. The results confirmed the importance of Rubisco as a source of mobilizable nitrogen, and suggested that in pea leaves the rate of degradation of Rubisco may vary throughout N mobilization. Correlated with the loss of Rubisco was an increase in relative abundance of chloroplastic protease regulatory subunits. Concomitantly, the relative abundance of some proteins related to the photosynthetic apparatus (Rubisco activase, Rubisco-binding proteins) and of several chaperones increased. A role for these proteins in the maintenance of a Rubisco activation state and in the PSII repair during the intense proteolytic activity within the chloroplasts was proposed. Finally, two 14-3-3-like proteins, with a potential regulatory role, displayed differential expression patterns during the massive remobilization of nitrogen.     

Brassinosteroids regulate grain filling in rice

Genes controlling hormone levels have been used to increase grain yields in wheat (Triticum aestivum) and rice (Oryza sativa). We created transgenic rice plants expressing maize (Zea mays), rice, or Arabidopsis thaliana genes encoding sterol C-22 hydroxylases that control brassinosteroid (BR) hormone levels using a promoter that is active in only the stems, leaves, and roots. The transgenic plants produced more tillers and more seed than wild-type plants. The seed were heavier as well, especially the seed at the bases of the spikes that fill the least. These phenotypic changes brought about 15 to 44% increases in grain yield per plant relative to wild-type plants in greenhouse and field trials. Expression of the Arabidopsis C-22 hydroxylase in the embryos or endosperms themselves had no apparent effect on seed weight. These results suggested that BRs stimulate the flow of assimilate from the source to the sink. Microarray and photosynthesis analysis of transgenic plants revealed evidence of enhanced CO₂ assimilation, enlarged glucose pools in the flag leaves, and increased assimilation of glucose to starch in the seed. These results further suggested that BRs stimulate the flow of assimilate. Plants have not been bred directly for seed filling traits, suggesting that genes that control seed filling could be used to further increase grain yield in crop plants.     

Phosphoproteomic analysis of seed maturation in Arabidopsis, rapeseed, and soybean

To characterize protein phosphorylation in developing seed, a large-scale, mass spectrometry-based phosphoproteomic study was performed on whole seeds at five sequential stages of development in soybean (Glycine max), rapeseed (Brassica napus), and Arabidopsis (Arabidopsis thaliana). Phosphopeptides were enriched from 0.5 mg of total peptides using a combined strategy of immobilized metal affinity and metal oxide affinity chromatography. Enriched phosphopeptides were analyzed by Orbitrap tandem mass spectrometry and mass spectra mined against cognate genome or cDNA databases in both forward and randomized orientations, the latter to calculate false discovery rate. We identified a total of 2,001 phosphopeptides containing 1,026 unambiguous phosphorylation sites from 956 proteins, with an average false discovery rate of 0.78% for the entire study. The entire data set was uploaded into the Plant Protein Phosphorylation Database (www.p3db.org), including all meta-data and annotated spectra. The Plant Protein Phosphorylation Database is a portal for all plant phosphorylation data and allows for homology-based querying of experimentally determined phosphosites. Comparisons with other large-scale phosphoproteomic studies determined that 652 of the phosphoproteins are novel to this study. The unique proteins fall into several Gene Ontology categories, some of which are overrepresented in our study as well as other large-scale phosphoproteomic studies, including metabolic process and RNA binding; other categories are only overrepresented in our study, like embryonic development. This investigation shows the importance of analyzing multiple plants and plant organs to comprehensively map the complete plant phosphoproteome.     

Transcriptional network analysis of the tryptophan-accumulating rice mutant during grain filling

In a previous study, we selected a high tryptophan (Trp)-accumulating rice (Oryza sativa L.) mutant line by in vitro mutagenesis using gamma rays. To obtain detailed information about the Trp biosynthetic pathway during the grain-filling in rice, we investigated the gene expression profiles in the wild-type (cv. Dongan) and the high-level Trp-accumulating mutant line (MRVII-33) at five different grain-filling stages using microarray analysis. The mutant line showed approximately 6.3-fold higher Trp content and 2.3-fold higher amino acids compared with the original cultivar at the final stage (stage V). The intensity of gene expression was analyzed and compared between the wild-type and mutant line at each of the five grain-filling stages using the Rice 4?×?44K oligo DNA microarray. Among the five stages, stage III showed the highest gene expression changes for both up- and down-regulated genes. Among the Trp biosynthesis-related genes, trpG showed high expression in the mutant line during stages I to IV and trpE showed higher at stage III. Gene clustering was performed based on the genes of KEGG's amino acid metabolism, and a total of 276 genes related to amino acid metabolism were placed into three clusters. The functional annotation enrichment analysis of the genes classified into the three clusters was also conducted using ClueGO. It was found that cluster 3 uniquely included biological processes related to aromatic amino acid metabolism. These results suggest that gene analysis based on microarray data is useful for elucidating the biological mechanisms of Trp accumulation in high Trp-accumulating mutants at each of the grain-filling stages.     

Formation of Fusarium metabolites in barley grain

Summary The influence of moisture content and temperature during storage of grain on the formation of Fusarium metabolites was studied. Naturally and artificially contaminated barley grain samples were stored at 15%, 25%, and 30% moisture contents, and at temperatures of + 5°C + 25°C, and + 30°C. Time of storage varied between one week and six months and the occurrence of Fusarium species and metabolites was analysed. The only Fusarium metabolite detected was zearalenone. The extent of Fusarium contamination decreased during storage whilst the concentration of zearalenone increased. To avoid the danger of mycotoxicoses, grains must be dryed immediately after harvest and then stored at a low temperature.     

Kin selection and conflict in seed maturation

There are four genetically distinct components in the developing seeds of flowering plants: maternal sporophyte, gametophyte, endosperm, and embryo. Each component can potentially influence the quantity or quality of nutrients provided to the embryo of its seed, thereby reducing the amount available to embryos in other seeds of that plant. The theory of kin selection predicts that each component will be selected to favor its own embryo over the other embryos to the extent that it is more closely related to its own. Under this criterion, an embryo should be selected to try to acquire more nutrients than the endosperm should be selected to provide, the endosperm should try to supply more than the gametophyte should, and the gametophyte more than the parent sporophyte. Evidence for this conflict of interests is found in the higher frequency of endopolyploidy, nutrient-absorbing haustoria, and food storage tissues in the embryo and endosperm than in the gametophyte of maternal tissues.This theory also suggests how the gametophyte, which is the nurse tissue of gymnosperm seeds, was displaced from this role in the flowering plants by an endosperm initiated by a secondary fertilization. “Neoteny” in the pro-angiosperms created conditions in which (1) an endosperm initiated by double fertilization would be more closely related to the embryo than is the gametophyte and (2) the endosperm would be formed early enough to be of significant aid to the embryo.If this theory is correct it (1) requires a different approach to the study of seed morphology and physiology, (2) increases the plausibility of arguments that flowering plants are a polyphyletic group, (3) provides evidence that parents cannot always control the outcome of conflict with their offspring, and (4) forges a conceptual link in our understanding of the evolution of social interactions in plants and animals.     

Genotypic variation in silicon concentration of barley grain

Soluble silicon (Si) in foods and drinks has been suggested to have a protective effect against neurotoxicity of Al. We investigated the genotypic variation in Si concentration of barley grain, which has many uses including in livestock feeds, malts for beer and whisky, and some foods for human consumption. Two collections of barley, grown in the same field, were subjected to analysis; 274 standard varieties selected at the Barley Germplasm Center of the Research Institute for Bioresources, Okayama University (SV), and 135 varieties from the Barley Core Collection of Americans (BCCAM). The Si concentration of barley grain showed large variation, ranging from 0 (under detection) to 3600 mg kg^–1 in SV and from 0 to 3800 mg kg^–1 in BCCAM barleys. The Si concentration was much lower in hull-less barley than in hulled barley. The Si concentration of two-row barley was similar to that of six-row barley, suggesting that Si concentration is not affected by the number of spike rows. Si concentration also did not differ with the origin of the barley variety. More than 80% of total Si was localized in the hull. The Si concentration of the hull was between 15343 and 27089 mg kg^–1 in the varieties tested. A close correlation was obtained between the Si concentrations of barley grains harvested in different years, suggesting that the variation in Si concentration of barley grain is controlled genetically. These results provide fundamental data for breeding Si-rich cultivars.     

Localization of carboxypeptidase I in germinating barley grain

Activity measurements and Northern blot hybridizations were used to study the temporal and spatial expression of carboxypeptidase I in germinating grains of barley (Hordeum vulgare L. cv Himalaya). In the resting grain no carboxypeptidase I activity was found in the aleurone layer, scutellum, or starchy endosperm. During germination high levels of enzyme activity appeared in the scutellum and in the starchy endosperm but only low activity was found in the aleurone layer. No mRNA for carboxypeptidase I was observed in the resting grain. By day 1 of germination the mRNA appeared in the scutellum where its level remained high for several days. In contrast, little mRNA was observed in the aleurone layer. These results indicate that the scutellum plays an important role in the production of carboxypeptidase I in germinating barley grain.     

Metabolism of gibberellins by immature barley grain

Gibberellins A₁, A₄, A₉, A₁₂-aldehyde, A₂₀ and A₅₁, each labelled with both a radioactive and stable isotope were fed to immature barley grain by injection into the endosperm. After 7 d, extensive metabolism of all substrates had occurred, and metabolites were identified by combined capillary gas chromatography-mass spectrometry. A proposed scheme of gibberellin metabolism in immature barley grain is presented.Abbreviations GA_n gibberellin A_n - GC-MS combined gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography