Similarities between gibberellins and related compounds in inducing Acid phosphatase and reducing sugar release from barley endosperm

Barley endosperm halves release acid phosphatase in response to several gibberellins and gibberellin precursors. Seed halves incubated with 10⁻⁷m GA₃ at 29° begin to release phosphatase after 11 hr and release it for another 26 hr in response to GA₃. After 37 hr, the rate of release slows to that of seed halves incubated without GA₃. GA₃ is active at 10⁻¹⁰m and maximally active at 10⁻⁷m. Comparative activity of 12 gibberellins and gibberellin precursors is GA₁ = GA₃ > GA₂ > GA₄ = GA₇ > GA₅ = GA₁₃ > GA₁₄ > GA₈ = GA₉ > (−)kaurenoic acid > (−)-kaurene. These compounds show the same order of activity and approximately the same relative activity in inducing reducing sugar release as in inducing phosphatase activity. The activity of each compound increases with its presumed position in a biosynthetic pathway leading from kaurene to GA₃. This correlation suggests that activity may be a reflection of the efficiency of conversion to an active form within the seed half.     

Control of carbohydrase formation by gibberellic acid in barley endosperm

α-Amylase, limit dextrinase and α-glucosidase were induced by gibberellic acid in barley grain from which the embryos had been excised. The responses to different concentrations of gibberellic acid were similar for the three carbohydrases. However α-glucosidase activity increased before the other two enzymes, and a low level of α-glucosidase was found in ungerminated grain. Experiments with cycloheximide and density-labelling in deuterium oxide suggest that the observed increases in activity are the result of de novo protein synthesis. The induction of these enzymes was reduced by pre-incubation in actinomycin D.     

Glyceride metabolism and gluconeogenesis in barley endosperm

Endosperms of quiescent barley grains contained, on average, 54.5 μg of neutral glyceride-glycerol, equivalent to ca 480 μg glyceride. Of this probably 90% was located in the aleurone layer. During germination the level of glyceride-glycerol declined. It also declined in degermed grains and aleurone layers incubated in vitro. The fall was accelerated by GA₃, but indoleacetic acid, kinetin and glutamine were without effect. Increases in the levels of malate synthase and isocitrate lyase from very low initial values, and the results of incorporation studies with [¹⁴C]-labelled substrates, indicate that the glyoxylate cycle functions to convert glycerides to sucrose in germinating grains and degermed grains incubated with GA₃, but not in degermed grains without the hormone. In the absence of GA₃ the glyceride could be a respiratory substrate in degermed grains. The aleurone layers converted exogenous glucose to sucrose. Little label from [¹⁴C]-amino acids appeared in sucrose but in some cases considerable incorporation occurred into glutamine.     

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     

Characterization of shrunken endosperm mutants in barley

Despite numerous studies on shrunken endosperm mutants caused by either maternal tissues (seg) or kernel per se (sex) in barley, the molecular mechanism for all of the eight seg mutants (seg1–seg8) and some sex mutants is yet to be uncovered. In this study, we determined the amylose content, characterized granule-binding proteins, analyzed the expression of key genes involved in starch synthesis, and examined starch granule structure of both normal (Bowman and Morex) and shrunken endosperm (seg1, seg3, seg4a, seg4b, seg5, seg6, seg7, and sex1) barley accessions. Our results showed that amylose contents of shrunken endosperm mutants ranged from 8.9% (seg4a) to 25.8% (seg1). SDS-PAGE analysis revealed that 87 kDa proteins corresponding to the starch branching enzyme II (SBEII) and starch synthase II (SSII) were not present in seg1, seg3, seg6, and seg7 mutants. Real-time quantitative PCR (RT-qPCR) analysis indicated that waxy expression levels of seg1, seg3, seg6, and seg7 mutants decreased in varying degrees to lower levels until 27 days after anthesis (DAA) after reaching the peak at 15–21 DAA, which differed from the pattern of normal barley accessions. Further characterization of waxy alleles revealed 7 non-synonymous single nucleotide polymorphisms (SNPs) in the coding sequences and 16 SNPs and 8 indels in the promoter sequences of the mutants. Results from starch granule by scanning electron microscopy (SEM) indicated that, in comparison with normal barley accessions, seg4a, seg4b, and sex1 had fewer starch granules per grain; seg3 and seg6 had less small B-type granules; some large A-type granules in seg7 had a hollow surface. These results improve our understanding about effects of seg and sex mutants on starch biosynthesis and granule structure during endosperm development and provide information for identification of key genes responsible for these shrunken endosperm mutants.     

Nature, intracellular distribution and formation of terpenoid quinones in maize and barley shoots

1. Maize and barley shoots have been shown to contain phylloquinone, plastoquinone, α-tocopherol (and γ-tocopherol in maize), α-tocopherolquinone and ubiquinone-9. 2. No solanesol was detected in any tissue examined. 3. In maize shoots plastoquinone and α-tocopherolquinone were localized in the chloroplast; ubiquinone was in the mitochondria. 4. Etiolated (dark-grown) shoots contained smaller amounts of phylloquinone and plastoquinone; α-tocopherolquinone was entirely absent; ubiquinone and α-tocopherol concentrations were unaffected. 5. On illumination of etiolated shoots the chloroplastidic quinones phylloquinone, plastoquinone and α-tocopherolquinone were synthesized in step with chloroplast development. α-Tocopherolquinone was not formed at the immediate expense of α-tocopherol.     

The rate of nuclear gene transcription in barley endosperm syncytia increases sixfold before cell-wall formation

In seedlings of the Scots pine (Pinus sylvestris L.), alanine aminotransferase (AlAT EC 2.6.1.2.) is present in the shoot and in the primary root but most activity is found in the cotyledons. During the experimental period (from 6 to 12 d after sowing), AlAT activity increased steadily. Anion exchange chromatography and native polyacrylamide gel electrophoresis were used to show that AlAT activity in extracts from cotyledons is associated with two isoforms of the enzyme. One isoform (AlAT 1) dominated in the cotyledons of lightgrown seedlings, but was absent from primary roots. Its accumulation was strongly increased by light, and both phytochrome and cryptochrome were shown to be involved in this effect. Results of experiments using dichromatic irradiation indicate that cryptochrome acts indirectly by establishing responsiveness towards phytochrome. When plastids were damaged by photooxidation, the accumulation of AlAT 1 decreased; however, AlAT 1 which had accumulated before the onset of photooxidative treatment seemed to remain undamaged. Therefore, and because of the absence of AlAT 1 from primary roots, it is suggested that this isoform is localized in leaf peroxisomes. The isoform AlAT 2 is the only one found in primary roots, and the predominant one in the cotyledons of dark-grown seedlings. It is unaffected by light. Upon photodestruction of plastids, a pronounced increase of its activity was found. This is taken as evidence that AlAT 2 is a cytosolic enzyme. Total AlAT activity in cotyledons was unaffected by feeding nitrate to the seedlings; supplying exogenous ammonium led to a considerably slower accumulation of AlAT compared with water controls. In contrast, AlAT accumulation in the primary roots was augmented by up to 45% if nitrogenous ions were supplied, ammonium being more effective than nitrate.Abbreviations and Symbols AlAT alanine aminotransferase (EC 2.6.1.2.) - B blue light - c continuous - D darkness - Fd-GOGAT ferredoxin-dependent glutamate synthase (EC 1.4.7.1.) - FR far-red light - HPR hydroxypyruvate reductase (EC 1.1.1.81.) - FPLC fast protein liquid chromatography - PAGE polyacrylamide gel electrophoresis - R red light - RG9 long-wavelength far-red light defined by the properties of the Schott glass filter RG9 (_RG9 < 0.01) - =Pfr/Ptot far-red-absorbing form of phytochrome/total phtochrome, wavelength-dependent photoequilibrium of the phytochrome system This work was supported by Heidelberger Akademie der Wissenschaften (Forschungsstelle Nitratassimilation). We are very grateful to Ms. B. Seith for measuring the DNA contents of the seedlings.     

The effect of zinc on the biosynthesis of tryptophan,andol auxins and gibberellins in barley

The action of zinc on the growth of barley and the biosynthesis of indol compounds and gibberellin-like substances was investigated in a number of concentrations of zinc from doses stimulating growth to toxic doses. The seeds were soaked before sowing in solutions of zinc sulphate (5.10^?5 to 5.10^?1% Zn), and the plants cultivated for 7 days in water. Lower concentrations of zinc increased both plant growth and the biosynthesis of tryptophan and auxins. At the optimum concentration of 5.10^?3% Zn this increase in tryptophan amounted to 241% of the variant without zinc; in substances with an R_F corresponding to indolyacetic acid, the increase determined by the biological test, was 207% as against the variant without zinc. Higher concentrations of zinc inhibited growth, the tryptophan content was decreased to below that of the control without zinc and the auxin content also fell to below the control values. Zinc also influenced the content of gibberellin-like substances in the plants. At a concentration of 5.10^?3% Zn the increase in the growth activity in the gibberellic acid area of the chromatogram was 294% of the variant without zinc. At toxic concentrations of zinc, the content of gibberellin-like substances fell to below that of the controls. The finding that zinc acts simultaneously on the biosynthesis of auxins and gibberellins is also evidence for the common action of growth substances of various chemical types on plant growth.     

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.     

Uptake and metabolism of radioactive gibberellins by barley aleurone layers

Summary When aleurone layers were treated with labeled gibberellin A₁ (³H-GA₁), gibberellin A₅ (³H-GA₅) and the methyl ester of ³H-GA₅ (³H-GA₅-ME), radioactivity was accumulated by the tissue for a period of 20–30 h. After this time, radioactivity was released into the medium. Concomitantly, ribonuclease was also liberated by the tissue. The radioactivity accumulated by aleurone layers was associated with polar metabolites of the respective GAs, and the extent of extent of accumulation was a function of the degree of GA metabolism (GA₅-ME>GA₅>GA₁). Accumulation of radioactivity was inhibited in the cold and by the metabolic poisons NaF and dinitrophenol. This was thought to be due to inbition of GA metabolism. The accumulation of ³H-GA₁ in aleurone tissue did not reach saturation when unlabeled GA₃ up to 10^-2 M was added to the incubation medium.Abbreviations GA gibberellin - GA₅ ME, gibberellin A₅ methyl ester - RNase ribonuclease     

A novel shrunken endosperm mutant of barley

Although mutations affecting several enzymes of the starch synthetic pathway in developing cereal endosperm have been isolated, none has a major effect on soluble starch synthase We report a new recessive shrunken endosperm mutant in barley ( Hordeum vulgare L. cv. Bomi-like), shx , which has 25% of normal starch content. We have assayed the activity of sucrose synthase (EC 2.4.1.13), ADP and UDP-glucose pyrophosphorylases (EC 2.7.7.27 and 2.7.7.9), branching enzyme (EC.2.4.1.18), and granule-bound and soluble starch synthase (EC 2.4.1.21) in shx. Sucrose synthase activity is reduced by 49% and UDP-glucose pyrrphosphorylase is 80% of the normal level. Branching enzyme and starch-bound starch synthase activities are normal, but ADP-glucose pyrophosphorylase activity is reduced by 72%. The soluble starch synthase that is primer-independent in the presence of sodium citrate shows 14% of normal activity in shx. whereas the primer-dependent form is unaffected. This lower starch synthase activity in shx cannot be explained by inhibition, substrate destruction or lack of primer. Although several starch-synthetic enzymes are affected, it is suggested that the primer independent from of soluble starch synthase may be the primary-site of the mutation in shx.     

The promoter of barley trypsin-inhibitor BTI-CMe,discriminates between wheat and barley endosperm protoplasts in transient expression assays

Several promoter fragments from the barley gene coding for trypsin inhibitor, BTI-CMe, have been fused to the -glucuronidase (GUS) reporter gene and these chimeric constructs used for transient expression in protoplasts. Transfection of developing endosperm protoplasts from barley (cv Bomi) show a maximum GUS expression of about 50% of that driven by the cauliflower mosaic virus 35S promoter, while in wheat endosperm protoplasts expression is less than 10%. No significant expression is found in transfected leaf protoplasts from barley, wheat or tobacco (<2% of the 35S control). All the information required for endosperm and barley specificity is present in the 343 bp proximal to the translation initiation site.Abbreviations MS Murashige and Skoog medium - PEG polyethyleneglycol - GUS -glucuronidase - MU methylumbelliferone - MUG 4-methylumbelliferyl--D glucuronide - pp protoplasts     

Protein breakdown and protease properties of germinating maize endosperm

Protein breakdown during germination of maize at 28° is closely correlated with the appearance of protease activity. In the first 2 days of germination, a slight disaggregation of only G₃ glutelins into more simple elements (albumin-globulins) can be observed. Between 2 and 2.5 days, there is extensive breakdown of all protein fractions, the rate of which coincides with the rate of appearance of proteolytic activity. After 2.5 days these phenomena slow down and the bulk of the endosperm proteins disappears. Three acid proteases in endosperm extracts of germinated grain (P₁₁, P₂₁ and P₂₂) have been isolated by affinity chromatography and gel filtration, and partially characterized. P₁₁ (MW 40 000) which is present in the ungerminated grain, cannot hydrolyse prolamins and is insensitive to reducing agents. P₂₁ (MW 36 000) and P₂₂ (MW 12 000), which appear on day 3 of germination, can degrade prolamins in vitro. Reducing agents enhance their activity and prevent their aggregation or denaturation. Comparative assays with different substrates suggest our enzyme preparations are principally endotype proteases with little contaminating carboxypeptidase activity.     

Mobilization of proline in the starchy endosperm of germinating barley grain

In germinating grains of barley, Hordeum vulgare L. cv. Himalaya, free proline accumulated in the starchy endosperm during the period of rapid mobilization of reserve proteins. When starchy endosperms were separated from germinating grains and homogenized in a dilute buffer of pH 5 (the pH of the starchy endosperm), the liberation of proline continued in these suspensions. The process was completely inhibited by diisopropylfluorophosphate, indicating that it was totally dependent on serine carboxy-peptidases. The carboxypeptidases present in the starchy endosperms of germinating grains were fractionated by chromatography on DEAE-cellulose. Four peaks were obtained, all with different activity spectra on the seven carbobenzoxydipeptides (Z-dipeptides) tested. Two of the peaks corresponded to previously known barley carboxypeptidases; these as well as a third peak hydrolyzed substrates of the types Z-X-Y and Z-X-Pro (X and Y denote any amino acid residue except proline). The fourth peak corresponded to a proline carboxypeptidase specific for substrates of the Z-Pro-X type. Apparently, in the hydrolysis of longer proline-containing peptides there must be sequential cooperation between the two carboxypeptidase types. The carboxypeptidases in extracts of starchy endosperms also liberated proline from the peptides Ala-Ala-Ala-Pro and Ala-Ala-Pro while Ala-Pro and Pro-Ala were not attacked. The dipeptides, however, were rapidly hydrolyzed around pH 7 by extracts prepared from the scutella of germinating grains. It is concluded that one part of the proline residues of the reserve proteins is liberated in situ in the starchy endosperm through the combined action of acid proteinases and carboxypeptidases, while another part is taken up in the form of small peptides by the scutellum, where proline is liberated by amino- and/or dipeptidases in some neutral compartment.Abbreviations DFP diisopropylfluorophosphate - DTT dithiothreitol - TNBS 2,4,6-trinitrobenzenesulphonic acid - Z N-carbobenzoxy - TLC thin layer chromatography A preliminary account of these results was given at the Meeting of the Federation of European Plant Physiological Societies in Edinburgh in July 1978. Abstract No. 181     

Morphology and ultrastructure of 11 barley shrunken endosperm mutants

Summary Eleven Na-azide induced barley shrunken endosperm mutants expressing xenia (sex) were characterized genetically and histologically. All mutants have reduced kernel size with kernel weights ranging from 11 to 57% of the wild type. With one exception, the mutant phenotypes are ascribable to single recessive mutant alleles, giving rise to a ratio of 31 of normal and shrunken kernels on heterozygous plants. One mutant (B10), also monofactorially inherited, shows a gene dosage dependent pattern of expression in the endosperm. Among the 8 mutants tested for allelism, no allelic mutant genes were discovered. By means of translocation mapping, the mutant gene of B10 was localized to the short arm of chromosome 7, and that of B9 to the short arm of chromosome 1. Based on microscopy studies, the mutant kernel phenotypes fall into three classes, viz. mutants with both endosperm and embryo affected and with a non-viable embryo, mutants with both endosperm and embryo affected and with a viable embryo giving rise to plants with a clearly mutant phenotype, and finally mutants with only the endosperm affected and with a normal embryo giving rise to plants with normal phenotype. The mutant collection covers mutations in genes participating in all of the developmental phases of the endosperm, i.e. the passage from syncytial to the cellular endosperm, total lack of aleurone cell formation and disturbance in the pattern of aleurone cell formation. In the starchy endosperm, varying degrees of cell differentiation occur, ranging from slight deviations from wild type to complete loss of starchy endosperm traits. In the embryo, blocks in the major developmental phases are represented in the mutant collection, including arrest at the proembryo stage, continued cell divisions but no differentiation, and embryos deviating only slightly from the wild type.     

Structural analysis and ensymic solubilization of barley endosperm cell-walls

Barley endosperm cell-walls were prepared and analysed. The-carbohydrate portion, which constituted most of the wall material, consisted of 10 % of l-arabinose, 13% Of d-xylose, 74% of d-glucose and 2.5% of d-mannose. Mixed-linkage β-d-glucan represented 70-72% of this material; the remaining 2-4% Of d-glucose maybe present as cellulose and glucomannan. Water and alkali-extracted β-d-glucans contained similar ratios of (1 → 3)- to (1 → 4)-Linkages, namely 3 to 7. The walls, which had a protein content of approximately 5 %, contained unidentfied, alkali-labile linkages. An endo-(1 → 3)β-d-glucanase from malted barley, and a fungal endo-(1 → 4)-β-d-glucanase, caused extensive solubilization of the wall polysaccharides.