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The lys5 mutations of barley reveal the nature and importance of plastidial ADP-Glc transporters for starch synthesis in cereal endosperm.

Much of the ADP-Glc required for starch synthesis in the plastids of cereal endosperm is synthesized in the cytosol and transported across the plastid envelope. To provide information on the nature and role of the plastidial ADP-Glc transporter in barley (Hordeum vulgare), we screened a collection of low-starch mutants for lines with abnormally high levels of ADP-Glc in the developing endosperm. Three independent mutants were discovered, all of which carried mutations at the lys5 locus. Plastids isolated from the lys5 mutants were able to synthesize starch at normal rates from Glc-1-P but not from ADP-Glc, suggesting a specific lesion in the transport of ADP-Glc across the plastid envelope. The major plastidial envelope protein was purified, and its sequence showed it to be homologous to the maize (Zea mays) ADP-Glc transporter BRITTLE1. The gene encoding this protein in barley, Hv.Nst1, was cloned, sequenced, and mapped. Like lys5, Hv.Nst1 lies on chromosome 6(6H), and all three of the lys5 alleles that were examined were shown to carry lesions in Hv.Nst1. Two of the identified mutations in Hv.Nst1 lead to amino acid substitutions in a domain that is conserved in all members of the family of carrier proteins to which Hv.NST1 belongs. This strongly suggests that Hv.Nst1 lies at the Lys5 locus and encodes a plastidial ADP-Glc transporter. The low-starch phenotype of the lys5 mutants shows that the ADP-Glc transporter is required for normal rates of starch synthesis. This work on Hv.NST1, together with the earlier work on BRITTLE1, suggests that homologous transporters are probably present in the endosperm of all cereals.

Adenosine Diphosphate Glucose↗

Peroxygenase-Catalyzed Fatty Acid Epoxidation in Cereal Seeds (Sequential Oxidation of Linoleic Acid into 9(S),12(S),13(S)-Trihydroxy-10(E)-Octadecenoic Acid).

Peroxygenase-catalyzed epoxidation of oleic acid in preparations of cereal seeds was investigated. The 105,000g particle fraction of oat (Avena sativa) seed homogenate showed high peroxygenase activity, i.e. 3034 [plus or minus] 288 and 2441 [plus or minus] 168 nmol (10 min)-1 mg-1 protein in two cultivars, whereas the corresponding fraction obtained from barley (Hordeum vulgare and Hordeum distichum), rye (Secale cereale), and wheat (Triticum aestivum) showed only weak activity, i.e. 13 to 138 nmol (10 min)-1 mg-1 protein. In subcellular fractions of oat seed homogenate, peroxygenase specific activity was highest in the 105,000g particle fraction, whereas lipoxygenase activity was more evenly distributed and highest in the 105,000g supernatant fraction. Incubation of [1-14C]linoleic acid with the 105,000g supernatant of oat seed homogenate led to the formation of several metabolites, i.e. in order of decreasing abundance, 9(S)-hydroxy-10(E),12(Z)-octadecadienoic acid, 9(S),12(S),13(S)-trihydroxy-10(E)-octadecenoic acid, cis-9,10-epoxy-12(Z)-octadecenoic acid [mainly the 9(R),10(S) enantiomer], cis-12,13-epoxy-9(Z)-octadecenoic acid [mainly the 12(R),13(S) enantiomer], threo-12,13-dihydroxy-9(Z)-octadecenoic acid, and 12(R),13(S)-epoxy-9(S)-hydroxy-10(E)-octadecenoic acid. Incubation of linoleic acid with the 105,000g particle fraction gave a similar, but not identical, pattern of metabolites. Conversion of linoleic acid into 9(S),12(S),13(S)-trihydroxy-10(E)-octadecenoic acid, a naturally occurring oxylipin with antifungal properties, took place by a pathway involving sequential catalysis by lipoxygenase, peroxygenase, and epoxide hydrolase.

Journal Article↗

Chitinase genes responsive to cold encode antifreeze proteins in winter cereals.

Antifreeze proteins similar to two different chitinases accumulate during cold acclimation in winter rye (Secale cereale). To determine whether these cold-responsive chitinases require post-translational modification to bind to ice, cDNAs coding for two different full-length chitinases were isolated from a cDNA library produced from cold-acclimated winter rye leaves. CHT9 is a 1,193-bp clone that encodes a 31.7-kD class I chitinase and CHT46 is a 998-bp clone that codes for a 24.8-kD class II chitinase. Chitinase-antifreeze proteins purified from the plant were similar in mass to the predicted mature products of CHT9 and CHT46, thus indicating that there was little chemical modification of the amino acid sequences in planta. To confirm these results, the mature sequences of CHT9 and CHT46 were expressed in Escherichia coli and the products of both cDNAs modified the growth of ice. Transcripts of both genes accumulated late in cold acclimation in winter rye. Southern analysis of winter rye genomic DNA indicated the presence of a small gene family homologous to CHT46. In hexaploid wheat, CHT46 homologs mapped to the homeologous group 1 chromosomes and were expressed in response to cold and drought. We conclude that two novel cold-responsive genes encoding chitinases with ice-binding activity may have arisen in winter rye and other cereals through gene duplication.

Amino Acid Sequence↗

Homology among 3S and 7S Globulins from Cereals and Pea.

The globulins from wheat caryopses were found to consist primarily of protein sedimenting at approximately 3S and 7S. These proteins displayed a molecular weight distribution similar to that of the purified vicilin-like fractions from oat and pea, with variations occurring in the isoelectric points and relative quantities of their major subunits. concanavalin A Sepharose chromatography suggested that the major polypeptides of the wheat (3S + 7S) fraction are glycosylated. Western blot analysis using antioat (3S + 7S) globulin immunoglobulin G revealed the vicilins from pea and the globulin fractions of oat, wheat, barley, rye, corn, and rice to contain immunologically homologous polypeptides. Major groups of polypeptides were shared by all the cereals and pea, including subunits of approximately 75, 50, 40 kilodaltons and 20 to 25 kilodaltons. These results indicate that legume-like 3S and 7S globulins have been conserved and are being expressed in cereals.

Journal Article↗

Early and late heat shock proteins in wheats and other cereal species.

Coleoptiles and roots of 3-day-old seedlings from five cereal species (Triticum aestivum L., T. durum Desf., Hordeum vulgare L., Secale cereale L., and Triticale) respond to heat shock at 40 degrees C by synthesizing a new set of 13 strong bands (as revealed by one-dimensional sodium dodecyl sulfate gel electrophoresis) as well as some 20 degrees C proteins. Heat shock proteins (HSPs) belong to three different size groups: high molecular mass HSPs in the 103 to 70 kilodalton range, intermediate molecular mass HSPs in the 62 to 32 kilodalton range, and low molecular mass HSPs about 17 to 16 kilodalton in size. At the beginning of the heat shock coleoptiles show a reduced ability to synthesize intermediate molecular mass HSPs but after 4 hours at 40 degrees C they exhibit fully developed HSP patterns identical to that found in roots. Synthesis of early HSPs declines after 7 hours of treatment followed by the appearance of a new set of 12 protein bands (late HSPs) in the ranges 99 to 83, 69 to 35, and 15 to 14 kilodaltons. After 12 hours at 40 degrees C, three other late HSPs of 89, 45, and 38 kilodalton are induced. The induction of late HSPs after 7 hours at 40 degrees C appears to be associated with an enhancement of radioactive methionine incorporation into proteins.

Journal Article↗

Do starch statoliths act as the gravisensors in cereal grass pulvini?

To determine if starch statoliths do, in fact, act as gravisensors in cereal grass shoots, starch was removed from the starch statoliths by placing 45-day-old intact barley plants (Hordeum vulgare cv 'Larker') in the dark at 25 degrees C for 5 days. Evidence from staining with I2-KI, scanning electron microscopy, and transmission electron microscopy indicated that starch grains were no longer present in plastids in the pulvini of plants placed in the dark for 5 days. Furthermore, gravitropic curvature response in these pulvini was reduced to zero, even though pulvini from vertically oriented plants were still capable of elongating in response to applied auxin plus gibberellic acid. However, when 0.1 molar sucrose was fed to the dark pretreated, starch statolith-free pulvini during gravistimulation in the dark, they not only reformed starch grains in the starch-depleted plastids in the pulvini, but they also showed an upward bending response. Starch grain reformation appeared to precede reappearance of the graviresponse in these sucrose-fed pulvini. These results strongly support the view that starch statoliths do indeed serve as the gravisensors in cereal grass shoots.

Darkness↗

Low Temperature-Induced Decrease in trans-Delta-Hexadecenoic Acid Content Is Correlated with Freezing Tolerance in Cereals.

The effect of growth at 5 degrees C on the trans-Delta(3)-hexadecenoic acid content of phosphatidyl(d)glycerol was examined in a total of eight cultivars of rye (Secale cereale L.) and what (Triticum aestivum L.) of varying freezing tolerance. In these monocots, low temperature growth caused decreases in the trans-Delta(3)-hexadecenoic acid content of between 0 and 74% with concomitant increases in the palmitic acid content of phosphatidyl(d)glycerol. These trends were observed for whole leaf extracts as well as isolated thylakoids. The low growth temperature-induced decrease in the trans-Delta(3)-hexadecenoic acid content was shown to be a linear function (r(2) = 0.954) of freezing tolerance in these cultivars. Of the six cold tolerant dicotyledonous species examined, only Brassica and Arabidopsis thaliana L. cv Columbia exhibited a 42% and 65% decrease, respectively, in trans-Delta(3)-hexadecenoic acid content. Thus, the relationship between the change in trans-Delta(3)-hexadecenoic acid content of phosphatidyl(d)glycerol and freezing tolerance cannot be considered a general one for all cold tolerant plant species. However, species which exhibited a low growth temperature-induced decrease in trans-Delta(3)-hexadecenoic acid also exhibited a concomitant shift in the in vitro organization of the light harvesting complex II from a predominantly oligomeric form to the monomeric form. We conclude that the proposed role of phosphatidyl(d)glycerol in modulating the organization of light harvesting complex II as a function of growth temperature manifests itself to varying degrees in different plant species. A possible physiological role for this phenomenon with respect to low temperature acclimation and freezing tolerance in cereals is discussed.

Journal Article↗

Complement activation by commercial allergen extracts of cereal grains.

Commercial allergen extract preparations of whole cereal grains were examined in vitro for their reactivity with the human haemolytic complement cascade. Total haemolytic complement was consumed in a dose-dependent manner which did not correlate with the protein nitrogen content or bacterial endotoxin contamination of the extracts. Alternative pathway activation was shown by C3 conversion in the presence of EGTA while classical complement components C1, C4 and C2 were also much decreased in serum which was free of detectable specific antibody. Very small amounts of allergen extract initiated the formation of complement-dependent factors which were chemotactic for human polymorphonuclear leucocytes. These data suggest a potential for complement activation in vivo by ingestion, inhalation, or injection of cereal grain products.

Allergens↗

Ochratoxin A producing Penicillium verrucosum isolates from cereals reveal large AFLP fingerprinting variability.

AIMS: To examine if molecular amplified fragment length polymorphism (AFLP) fingerprinting of the only ochratoxin A-producing species in European cereals, Penicillium verrucosum, can be used as a method in hazard analysis using critical control points (HACCP). METHODS AND RESULTS: A total of 321 isolates of P. verrucosum were isolated from ochratoxin A-contaminated cereals from Denmark (oats), UK (wheat and barley) and Sweden (wheat). Of these, 236 produced ochratoxin A as determined by thin layer chromatography; 185 ochratoxin A-producing isolates were selected for AFLP fingerprinting. A total of 138 isolates had unique AFLP patterns, whereas 52 isolates could be allocated to small groups containing from two to four isolates with similar AFLP patterns. A total of 155 clones were found among the 185 P. verrucosum isolates, thus 84% of the isolates may represent different genets of P. verrucosum. As the few isolates that were grouped often came from the same farm, and those groups that contained AFLP-identical isolates from different countries were morphotypically different. On single farms up to 35 clones were found. The few groups of ramets from the same genet indicated that a HACCP approach based on clones may require a very large number of AFLP analysis to work in practice, we recommend basing the HACCP approach on the actual species P. verrucosum. A more detailed characterization should rather be based on the profile of species present at different control points, or analysis of the mycotoxins ochratoxin A and citrinin in the isolates. Examination of 86 isolates with HPLC and diode array detection of P. verrucosum showed that 66% produced ochratoxin A, 87% produced citrinin, 92% produced verrucin and 100% produced verrucolone. CONCLUSIONS: Among 184 ochratoxin A-producing Penicillium verrucosum, 155 clonal lineages were indicated by AFLP fingerprinting, indicating a high genetical diversity, yet the species P. verrucosum is phenotypically distinct and valid. SIGNIFICANCE AND IMPACT OF THE STUDY: AFLP fingerprinting of Penicillium verrucosum indicates that genetic recombination takes place in this fungus.

Avena↗

Induced disease resistance and gene expression in cereals.

Disease resistance strategies reduce chemical input into the environment and are therefore powerful approaches to sustainable agriculture. Induced resistance (IR) has emerged as a potential alternative, or a complementary strategy, for crop protection. IR signifies the control of pathogens and pests by prior activation of plant defence pathways. A molecular understanding of IR in cereals, including the most important global crops wheat and rice, has been largely missing. Evidence indicating that central elements of IR pathways are conserved among Di- and Monocotyledoneae has only recently been presented, although their regulation and interaction with other plant pathways may be quite divergent. We present here a synopsis of current molecular knowledge of cereal IR mechanisms.

Edible Grain↗

The rhizosphere signal molecule lumichrome alters seedling development in both legumes and cereals.

The stimulatory role of lumichrome, a rhizosphere metabolite, was assessed on the growth of legume and cereal seedlings. At a very low nanomolar concentration (5 nm), lumichrome elicited growth promotion in cowpea, soybean, sorghum, millet and maize, but not in common bean, Bambara groundnut and Sudan grass. In soybean and cowpea only, 5 nm lumichrome caused early initiation of trifoliate leaf development, expansion in unifoliate and trifoliate leaves, increased stem elongation and, as a result, an increase in shoot and plant total biomass relative to control. Lumichrome (5 nm) also increased leaf area in maize and sorghum, and thus raised shoot and total biomass but there was no effect on the leaf area of the other cereals. Root growth was also stimulated in sorghum and millet by the supply of 5 nm lumichrome. By contrast, the application of a higher dose of lumichrome (50 nm) depressed development of unifoliate leaves in soybean, the second trifoliate leaf in cowpea, and shoot biomass in soybean. The 50 nm concentration also consistently decreased root development in cowpea and millet, but had no effect on the other species. These data show that lumichrome is a rhizosphere signal molecule that affects seedling development in both monocots and dicots.

Fabaceae↗

Effect of cereal brans on Lentinula edodes growth and enzyme activities during cultivation on forestry waste.

AIMS: To develop strategies for increasing the growth of Lentinula edodes in eucalyptus residues. To this end, we have examined the effects of cereal brans additions on production of mycelial biomass and enzymes. METHODS AND RESULTS: Three isolates of the mushroom shiitake, L. edodes (Berk. Pegler), were evaluated for enzyme and ergosterol production on eucalyptus residue supplemented with 5, 10, 15 and 20% (w/w) of soya, wheat or rice brans. Nitrogen imput on eucalyptus residues accelerated mycelial growth by supplying the L. edodes with this limiting nutrient. High levels of enzymes activities were produced in eucalyptus residues supplemented by soya bran. Comparison of cellulose and xylanase production with manganese peroxidase (MnP) at 20% soya bran indicated that hydrolytic enzymes, but oxidative enzymes were reduced. CONCLUSIONS: Mycelial growth measurements revealed that eucalyptus residues supplemented with cereal brans supported fast growth of L. edodes, indicating that mycelium extension is related to the bioavailability of nitrogen. The type and concentration of nutrient supplement has a considerable effect both on substrate colonization and on the type of hydrolytic and oxidative enzymes produced. These characteristics may be useful for mushroom growing. SIGNIFICANCE AND IMPACT OF THE STUDY: Lentinula edodes is commercially important for edible mushroom production and supplements which enhance growth and enzymes production might also be beneficial for mushroom yields.

Biomass↗

Prospects for exploitation of disease resistance from Hordeum chilense in cultivated cereals.

Hordeum chilense is a South American wild barley with high potential for cereal breeding given its high crossability with other members of the Triticeae. In the present paper we consider the resistance of H. chilense to several fungal diseases and the prospects for its transference to cultivated cereals. All H. chilense accessions studied are resistant to the barley, wheat and rye brown rusts, the powdery mildews of wheat, barley, rye and oat, to Septoria leaf blotch, common bunt and to loose smuts, which suggests that H. chilense is a non-host of these diseases. There are also lines resistant to wheat and barley yellow rust, stem rust and to Agropyron leaf rust, as well as lines giving moderate levels of resistance to Septoria glume blotch, tan spot and Fusarium head blight. Some H. chilense lines display pre-appressorial avoidance to brown rust. Lines differ in the degree of haustorium formation by rust and mildew fungi they permit, and in the degree to which a hypersensitive response occurs after haustoria are formed. Unfortunately, resistance of H. chilense to rust fungi is not expressed in tritordeum hybrids, nor in chromosome addition lines in wheat. In tritordeum, H. chilense contributes quantitative resistance to wheat powdery mildew, tan spot and loose smut. The resistance to mildew, expressed as a reduced disease severity, is not associated with macroscopically visible necrosis. Hexaploid tritordeums are immune to Septoria leaf blotch and to common bunt although resistance to both is slightly diluted in octoploid tritordeums. Studies with addition lines in wheat indicate that the resistance of H. chilense to powdery mildew, Septoria leaf blotch and common bunt is of broad genetic basis, conferred by genes present on various chromosomes.

Fusarium↗

Isolation and characterization of dinitrogen-fixing bacteria from the rhizosphere of temperate cereals and forage grasses.

N(2)(C(2)H(2))-fixing bacteria were isolated from the rhizosphere of various cereals and forage grasses grown in the greenhouse and from the rhizosphere of field-grown wheat in Sweden. All 46 isolates from the greenhouse plants lost their nitrogenase activity during purification. By imposing a stronger selection pressure, we obtained pure isolates with nitrogenase activity from field-grown wheat. Some isolates were identified as Enterobacter agglomerans and Bacillus polymyxa, but several bacteria of uncertain taxonomy also occurred. One of the isolates grew and reduced acetylene only in the presence of other bacteria or certain vitamins. Species of Azotobacter or Azospirillum could not be isolated from the rhizosphere of any of the cereals and forage grasses studied.

Journal Article↗

Selective medium for isolation of Fusarium species and dematiaceous hyphomycetes from cereals.

A selective medium containing 2 micrograms of dichloran per ml, 200 micrograms of chloramphenicol per ml, and 1.5% bacteriological peptone was developed for the isolation of Fusarium species and dematiaceous hyphomycetes from cereals. The medium, designated DCPA, was shown to select against species of Aspergillus, Penicillium, Cladosporium, and mucoraceous fungi. DCPA was evaluated for use as an enumeration medium and compared satisfactorily with dichloran-rose bengal-chloramphenicol agar when both media were tested with a range of cereal samples. Fusarium species and dematiaceous hyphomycetes produced well-formed colonies with good conidial production on DCPA, permitting rapid identification of such isolates on this medium.

Aniline Compounds↗

Isolation, characterization, and avenacin sensitivity of a diverse collection of cereal-root-colonizing fungi.

A total of 161 fungal isolates were obtained from the surface-sterilized roots of field-grown oat and wheat plants in order to investigate the nature of the root-colonizing fungi supported by these two cereals. Fungi were initially grouped according to their colony morphologies and then were further characterized by ribosomal DNA sequence analysis. The collection contained a wide range of ascomycetes and also some basidiomycete fungi. The fungi were subsequently assessed for their abilities to tolerate and degrade the antifungal oat root saponin, avenacin A-1. Nearly all the fungi obtained from oat roots were avenacin A-1 resistant, while both avenacin-sensitive and avenacin-resistant fungi were isolated from the roots of the non-saponin-producing cereal, wheat. The majority of the avenacin-resistant fungi were able to degrade avenacin A-1. These experiments suggest that avenacin A-1 is likely to influence the development of fungal communities within (and possibly also around) oat roots.

Antifungal Agents↗

Cereal based oral rehydration solutions.

A total of 257 boys (age range 4-55 months), who had acute diarrhoea with moderate to severe dehydration, were randomly assigned to treatment with either the World Health Organisation/United Nations Childrens Fund (WHO/Unicef) recommended oral rehydration solution or cereal based oral rehydration solution made either of maize, millet, sorghum, or rice. After the initial rehydration was achieved patients were offered traditional weaning foods. Treatment with oral rehydration solution continued until diarrhoea stopped. Accurate intake and output was maintained throughout the study period. Efficacy of the treatment was compared between the different treatment groups in terms of intake of the solution, stool output, duration of diarrhoea after admission, and weight gain after 24, 48, and 72 hours, and after resolution of diarrhoea. Results suggest that all the cereal based solutions were as effective as glucose based standard oral rehydration solution in the treatment of diarrhoea.

Dehydration↗

Does the reticulin binding property of cereal proteins demonstrable in vitro have pathogenetic significance for coeliac disease?

We used an indirect immunofluorescence technique, using rabbit antisera against cereal protein extracts, to determine which cereal proteins bind to reticulin in tissue sections and which do not. Wheat albumin extracts and globulins and gliadin extracts from a range of different wheat varieties, and prolamine extracts of barley and rye each bound to reticulin in vitro, while prolamine extracts of maize and rice did not. Wheat gluten subfractions were also tested. Subfractions B and C and subfractions B2 and B3 did bind, but fraction A and subfraction B1 did not. The results suggest an association between in vitro reticulin binding and the ability to induce gluten sensitive enteropathy on feeding.

Animals↗