Molecular cloning, sequence analysis and expression studies of a novel GAmyb homologous gene, hvmyb

Using a knownGAmyb gene as the probe, two fully identical clones were isolated from a barley aleurone cDNA library. Sequence analysis showed that their 5′ termini are highly homoIogous to the 3′ termini ofGAmyb (97%) and their 3′ termini share no significant homology with any myb genes. Therefore, the deduced protein may hold intact putativeGAmyb activation domain but lack the normal DNA-binding domain. Northern blot reveals thathumyb expression in barley aleurone layers is strongly up-regulated by gibberellin (GA) and down-regulated by abscisic acid (APIA). The tissue-and developmental-stage-specificity ofhvmyb was also found, which was only expressed in barley aleurone cells and dropped to non-detectable level soon after germination. EMBL accession number Y14658.     

Barley embryo globulin 1 gene,Beg1: Characterization of cDNA,chromosome mapping and regulation of expression

We report identification of a 2189 by cDNA clone from barley corresponding to a single-copy gene, Beg1 (Barley embryo globulin), on chromosome 4, which encodes a storage globulin. In barley, the major protein reserve in the aleurone layer belongs to the 7S globulin class of proteins found in many seeds. Electrophoretically and antigenically similar proteins are present in the barley embryo. Accumulation of Beg1 mRNA was noted beginning 15–20 days post-anthesis in both the aleurone layer and embryo of the developing barley grain but not in the starchy endosperm. A high level of Beg1 mRNA is also present in the mature imbibed aleurones, which can be repressed by treatment with gibberellic acid. This repressive effect of gibberellin on the levels of Beg1 mRNA is confirmed in the gibberellin response-constitutive mutant, slender, whose aleurone layers do not accumulate Beg1 mRNA even in the absence of applied gibberellic acid. The deduced primary translation product of the Beg1 mRNA is a 637 amino acid (72 kDa) protein with homology to maize embryo globulin 1 (GLB1) and a partial sequence of a wheat 7S globulin. The internal amino acid sequence of BEG1 closely matches the N-terminal sequence of isolated barley aleurone globulin. Seven imperfect tandem repeats of 16 amino acids each are present near the N-terminus of BEG1, which conform to the consensus HGEGEREEEXGRGRGR, and contribute to the observed unusual amino acid composition of this protein. A second, distinct barley globulin gene, Beg2, which is homologous to maize Glb2, was detected by Northern and Southern analysis. Beg-2 and Beg1 are regulated differently which may indicate variation in storage or utilization properties among the barley globulins.     

Abscisic Acid Inhibition of Gibberellic Acid and Cyclic 3',5'-Adenosine Monophosphate Induced α-Amylase Synthesis

The induction of α-amylase synthesis in barley aleurone by cyclic 3′,5′-adenosine monophosphate or GA₃ was inhibited by abscisic acid. The concentration of ABA required to inhibit α-amylase induction by the cyclic nucleotide in the extract was one-fiftieth to one hundredth of that required for GA₃-induced α-amylase. It is concluded that the effects of ABA on GA₃ and cyclic nucleotide induced α-amylase synthesis in the aleurone are independent and indirect.     

Evidence of two isomers of phosphatidylinositol in plant tissue

The structure of phosphatidylinositol in barley (Hordeum vulgare) aleurone layers was investigated by chemical degradation. In vivo myo-[2-³H]inositol-labeled phosphatidylinositol was first converted to glycerophosphoinositol and, subsequently, after removal of the glycerol moiety, to inositol monophosphate. Here, we present data that show that, in addition to the commonly occurring 1,2-diacylglycero-3-(d-myo-inositol-1-phosphate), barley aleurone cells contain a novel second isomer of phosphatidylinositol that differs in structure of the head group.     

Uncovering the evolutionary origin of blue anthocyanins in cereal grains

Functional divergence after gene duplication plays a central role in plant evolution. Among cereals, only Hordeum vulgare (barley), Triticum aestivum (wheat) and Secale cereale (rye) accumulate delphinidin‐derived (blue) anthocyanins in the aleurone layer of grains, whereas Oryza sativa (rice), Zea mays (maize) and Sorghum bicolor (sorghum) do not. The underlying genetic basis for this natural occurrence remains elusive. Here, we mapped the barley Blx1 locus involved in blue aleurone to an approximately 1.13 Mb genetic interval on chromosome 4HL, thus identifying a trigenic cluster named MbHF35 (containing HvMYB4H, HvMYC4H and HvF35H). Sequence and expression data supported the role of these genes in conferring blue‐coloured (blue aleurone) grains. Synteny analyses across monocot species showed that MbHF35 has only evolved within distinct Triticeae lineages, as a result of dispersed gene duplication. Phylogeny analyses revealed a shared evolution pattern for MbHF35 in Triticeae, suggesting that these genes have co‐evolved together. We also identified a Pooideae‐specific flavonoid 3′,5′‐hydroxylase (F3′5′H) lineage, termed here Mo_F35H2, which has a higher amino acid similarity with eudicot F3′5′Hs, demonstrating a scenario of convergent evolution. Indeed, selection tests identified 13 amino acid residues in Mo_F35H2 that underwent positive selection, possibly driven by protein thermostablility selection. Furthermore, through the interrogation of barley germplasm there is evidence that HvMYB4H and HvMYC4H have undergone human selection. Collectively, our study favours blue aleurone as a recently evolved trait resulting from environmental adaptation. Our findings provide an evolutionary explanation for the absence of blue anthocyanins in other cereals and highlight the importance of gene functional divergence for plant diversity and environmental adaptation.     

Control of lactate dehydrogenase, lactate glycolysis, and alpha-amylase by o(2) deficit in barley aleurone layers

After 4 days in an atmosphere of N₂, aleurone layers of barley (Hordeum vulgare L. cv Himalaya) remained viable as judged by their ability to produce near normal amounts of α-amylases when incubated with gibberellic acid (GA₃) in air. However, layers did not produce α-amylase when GA₃ was supplied under N₂, apparently because α-amylase mRNA failed to accumulate.     

Gibberellic Acid enhancement of DNA turnover in barley aleurone cells

When imbibed, deembryonated halfseeds from barley (Hordeum vulgare L., var. Himalaya) are incubated in buffer, the DNA content of the aleurone layer increases 25 to 40% over a 24-hour period. In contrast, the DNA of isolated aleurone layers declines by 20% over the same time period. Gibberellic acid (GA) causes a reduction in DNA levels in both halfseed aleurone layers and isolated aleurone layers. GA also increases the specific radioactivity of [³H]thymidine-labeled halfseed aleurone layer DNA during the first 12 hours of treatment. Pulse-chase studies demonstrated that the newly synthesized DNA is metabolically labile.     

Dormancy of the barley grain is correlated with gibberellic Acid responsiveness of the isolated aleurone layer

The relationship between barley grain dormancy and gibberellic acid (GA₃) responsiveness of aleurone layers has been investigated. Barley (Hordeum distichum L. cvs Triumph and Kristina) grains were matured under defined conditions in a phytotron. Grains of Triumph plants grown under long-day/warm conditions had lower dormancy levels than grains of plants grown under short-day/cool conditions. Aleurone layers isolated from grains of long-day Triumph plants secreted more α-amylase and had a higher responsiveness to GA₃ as measured by α-amylase secretion. Storage of the grains increased both the percentage of germination and the responsiveness of the aleurone to GA₃. Use of different sterilization methods to break dormancy confirmed the correlation between germination percentage and aleurone layer GA₃ responsiveness. The response of embryoless Triumph grains to GA₃ was lower than that of the isolated aleurone layers, suggesting a role of the starchy endosperm in regulating the GA₃ response of the aleurone layer. Grains of the cultivar Kristina harvested from short day- and long day-grown plants lacked dormancy, and their isolated aleurone layers had a similar responsiveness to GA₃ as measured by α-amylase secretion. The data indicate that the physiological state of the aleurone layers contributes to the percentage germination of the grains.     

A fast and easy technique for the isolation of aleurone layers

Although the aleurone layer of ceral grain seeds has many advantages for the study of gibberellin action, it has the disadvantage that the hand-isolation of the aleurone layers is time-consuming. To overcome this disadvantage, a commercially available pasta machine was modified and used to remove aleurone layers from imbibed barley (Hordeum vulgare) seeds. This equipment allows isolation of a thousand layers in 5 minutes compared to the 3 to 4 hours required to hand-isolate them. The machine-made aleurone layers are gibberellic-acid responsive and the response is similar both qualitatively and quantitatively to that of hand-isolated layers.     

Density labeling of proteins with 13C-labeled amino acids: no accumulation of an inactive alpha-amylase precursor in barley aleurone cells.

Gibberellic acid enhances α-amylase (EC 3.2.1.1) production in isolated barley aleurone layers after a lag period of 4 to 8 h, and most of the enzyme is produced after 12 h of hormone treatment. Amino acids necessary for protein synthesis in barley aleurone layers are derived from the degradation of storage proteins in this tissue. Since bromate is an inhibitor of barley protease, in the presence of bromate the production of α-amylase in aleurone layers becomes dependent on exogenous amino acids. We have incubated aleurone layers with bromate plus ¹³C-labeled amino acids and [³H]leucine from 0 to 24, 0 to 12, and 12 to 24 h after the application of gibberellic acid. The chemical quantity of [³H]leucine was negligible in comparison to that of ¹³C-labeled amino acids. Therefore, any density shift of proteins observed must be due to the incorporation of ¹³C-labeled amino acids. The density shift of α-amylase and that of newly synthesized proteins (radioactivity profile) were determined by isopycnic centrifugation in CsCl density gradients. The density shift of α-amylase isolated from aleurone layers incubated with ¹³C-labeled amino acids from 12 to 24 h after the addition of hormone was much larger than that of α-amylase isolated from aleurone layers incubated with ¹³C-labeled amino acids from 0 to 12 h of hormone treatment. By comparing the density shift of α-amylase with that of newly synthesized proteins, it is apparent that essentially all the amylase molecules are de novo synthesized. We can conclude that there is little or no accumulation of an inactive α-amylase precursor in barley aleurone cells between the time of the application of gibberellic acid and the time of the rapid increase in α-amylase activity.     

A survey of the sequence of some effects of gibberellic Acid in the metabolism of cereal grains

The sequence in which a variety of enzymes and metabolites are affected by gibberellic acid after application of the hormone to aleurone layers of half seeds of barley (Hordeum vulgare var. Betzes) and half seeds of wheat (Triticum aestivum var. Gensee) was investigated. With barley aleurone layers the first hormonal effect observed was the increased secretion of soluble carbohydrate, some of which appears to be a glucan containing some β-1,3 linkages. This was followed by increased oxygen consumption and increased secretion of ATPase, GTPase, phytase, phosphomonoesterase, phosphodiesterase, inorganic phosphate, carbohydrates other than amylase, peroxidase and amylase. Similar sequential effects were seen in wheat half seeds. Increased activity of alcohol dehydrogenase in barley seeds was elicited by the hormone but there was no effect on glucose-6-phosphate isomerase.     

Regulated expression of three alcohol dehydrogenase genes in barley aleurone layers

Three genes specify alcohol dehydrogenase (EC 1.1.1.1.; ADH) enzymes in barley (Hordeum vulgare L.) (Adh 1, Adh 2, and Adh 3). Their polypeptide products (ADH 1, ADH 2, ADH 3) dimerize to give a total of six ADH isozymes which can be resolved by native gel electrophoresis and stained for enzyme activity.

Under fully aerobic conditions, aleurone layers of cv Himalaya had a high titer of a single isozyme, the homodimer containing ADH 1 monomers. This isozyme was accumulated by the aleurone tissue during the later part of seed development, and survived seed drying and rehydration. The five other possible ADH isozymes were induced by O₂ deficit. The staining of these five isozymes on electrophoretic gels increased progressively in intensity as O₂ levels were reduced below 5%, and were most intense at 0% O₂.

In vivo³⁵S labeling and specific immunoprecipitation of ADH peptides, followed by isoelectric focusing of the ADH peptides in the presence of 8 molar urea (urea-IEF) demonstrated the following. (a) Aleurone layers incubated in air synthesized ADH 1 and a trace of ADH 2; immature layers from developing seeds behaved similarly. (b) At 5% O₂, synthesis of ADH 2 increased and ADH 3 appeared. (c) At 2% and 0% O₂, the synthesis of all three ADH peptides increased markedly.

Cell-free translation of RNA isolated from aleurone layers, followed by immunoprecipitation and urea-IEF of in vitro synthesized ADH peptides, showed that levels of mRNA for all three ADH peptides rose sharply during 1 day of O₂ deprivation. Northern hybridizations with a maize Adh 2 cDNA clone established that the clone hybridized with barley mRNA comparable in size to maize Adh 2 mRNA, and that the level of this barley mRNA increased 15- to 20-fold after 1 day at 5% or 2% O₂, and about 100-fold after 1 day at 0% O₂.

We conclude that in aleurone layers, expression of the three barley Adh genes is maximal in the absence of O₂, that regulation of mRNA level is likely to be a major controlling factor, and that whereas the ADH system of barley has strong similarities to that of maize, it also has some distinctive features.

     

Hormonal regulation of the development of protease and carboxypeptidase activities in barley aleurone layers

Carboxypeptidase and protease activities of hormone-treated barley (Hordeum vulgare cv Himalaya) aleurone layers were investigated using the substrates N-carbobenzoxy-Ala-Phe and hemoglobin. A differential effect of gibberellic acid (GA₃) on these activities was observed. The carboxypeptidase activity develops in the aleurone layers during imbibition without the addition of hormone, while the release of this enzyme to the incubation medium is enhanced by GA₃. In contrast, GA₃ is required for both the production of protease activity in the aleurone layer and its secretion. The time course for development of protease activity in response to GA₃ is similar to that observed for α-amylase. Treating aleurone layers with both GA₃ and abscisic acid prevents all the GA₃ effects described above. Carboxypeptidase activity is maximal between pH 5 and 6, and is inhibited by diisopropylfluorophosphate and p-hydroxymercuribenzoate. We have observed three protease activities against hemoglobin which differ in charge but are all 37 kilodaltons in size on sodium dodecyl sulfate polyacrylamide gels. The activity of the proteases can be inhibited by sulfhydryl protease inhibitors, such as bromate and leupeptin, yet is enhanced by 2-fold with 2-mercaptoethanol. In addition, these enzymes appear to be active against the wheat and barley storage proteins, gliadin and hordein, respectively. On the basis of these characteristics and the time course of GA₃ response, it is concluded that the proteases represent the GA₃-induced, de novo synthesized proteases that are mainly responsible for the degradation of endosperm storage proteins.     

Isolation and Partial Characterization of a Factor from Barley Aleurone that Modifies alpha-Amylase in Vitro

Posttranslational modifications that give rise to multiple forms of α-amylase (EC 3.2.1.1) in barley (Hordeum vulgare L. cv Himalaya) were studied. When analyzed by denaturing polyacrylamide gel electrophoresis, barley α-amylase has a molecular mass of 43 to 44 kilodaltons, but isoelectric focusing resolves the enzyme into a large number of isoforms. To precisely identify these isoforms, we propose a system of classification based on their isoelectric points (pl). α-Amylases with pls of approximately 5, previously referred to as low pl or Amy1 isoforms, have been designated HAMY1, and α-amylases with pls of approximately 6, referred to as high pl or Amy2, are designated HAMY2. Individual isoforms of HAMY1 and HAMY2 are identified by their pls. For example, the most acidic α-amylase synthesized and secreted by barley aleurone layers is designated HAMY1(4.56). Some of the diversity in the pls of barley α-amylases arises from posttranslational modifications of the enzyme. We report the isolation of a factor from barley aleurone layers and incubation media that can modify HAMY1 isoforms in vitro. This factor has a molecular mass between 30 and 50 kilodaltons, and it can catalyze the conversion of HAMY1(4.90) and HAMY1(4.64) to isoforms 4.72 and 4.56, respectively. The in vitro conversion of HAMY1 isoforms by the factor is favored by pH values of approximately 5 and is inhibited at approximately pH 7. The level of this factor in aleurone layers and incubation media is not affected by treatment of the tissue with gibberellic acid. The amylase-modifying activity from barley will also modify α-amylases isolated from human saliva and porcine pancreas. An activity that can modify HAMY1 isoforms in vitro has also been isolated from Onozuka R10 cellulase. Because the activity isolated from barley lowers the pl of α-amylase from barley, human saliva, and porcine pancreas, we speculate that it is a deamidase.     

Uptake,distribution, and turnover rates of selenium in barley

The present communication elucidates initially the topographic distribution of selenium in barley grains. Then by the fluorimetric method the uptake of selenium (selenite) in 8–16 d old germinating barley was estimated. Finally by means of⁷⁵Se the anabolic and catabolic rates (turnover) of⁷⁵Se (selenite) was compared. The distribution of selenium in barley was evaluated after microdissection of barley grains. In dried grains the highest concentration was found in husk and pericarp with about 0.6 ppm Se. Then followed Scutellum with 0.4 and 0.3 ppm in embryon. The aleurone layer, embryonic leaves, and initial root did only have 0.2 ppm Se. In order to know more about the uptake and distribution of selenium in 8-d-old barley, the plants were cultivated for a further 8 d in the culture medium with variation in selenite concentration. In roots and leaves, the uptake did not arrive at saturation during the period studied since the dose-response curve increased up to 0.34 mM selenite in the medium, whereas the selenium levels were about 200 ppm in roots and 30 ppm in leaves. However, the uptake was linear, with concentration during 8 d of cultivation up to 0.84 μM selenite for grain and stem. At higher concentrations the dose-response curve diminished its slope. At 0.34 mM selenite the concentration in grain increased to 6.87 ppm and in the stem to 8.13 ppm. The uptake, distribution, and catabolic rate of selenium components in germinating barley were further evaluated by exposing the plants to 0.0492 μCi⁷⁵Se (12.6 μM selenite) for up to 4 d. Then the plants were moved to a selenium deficient medium for further 4 d. Then finally the medium was supplemented with high doses of cold selenite (0.126 mM selenite) for further 4 d. The first third period made it possible to estimate the rate of uptake. It was highest in roots (313 fmol/h/mg dw), i.e., about 10 times those of grains, stems, and leaves. The intermediate period where the barley was transferred to a selenium deficient medium made it possible to estimate the kinetics and eventual sparing mechanisms. The selenium losses were highest for leaves (39%), then followed by roots and stems (22 and 25%, respectively). The losses were lowest in grain with 9% Se losses. The losses were three times more pronounced during the first day than in the following 3 d. These data may argue that the selenium is distributed into different pools and that sparing mechanisms may be in function. The last period, i.e., the chase experiment, revealed the rate of elimination of selenium under conditions with surplus selenium. The catabolic rate was about 10 times faster in roots (169 fmol/h/mg dw) than in grains and about 8 times faster than in leaves.     

Phosphoinositides in barley aleurone layers and gibberellic Acid-induced changes in metabolism

Phospholipids of barley (Hordeum vulgare L. cv Himalaya) aleurone layers were labeled with myo-[2-³H]inositol or [³²Pi], extracted, and analyzed by physical (chromatography) and chemical (deacylation) techniques. Three phospholipids were found to incorporate both myo-[2-³H]inositol and [³²Pi]—phosphatidylinositol, phosphatidylinositol-monophosphate, and phosphatidylinositol-bisphosphate. Stimulation of [³H]inositol prelabeled aleurone layers with GA₃ showed enhanced incorporation of label into phosphatidylinositol within 30 seconds and subsequent rapid breakdown. Stimulation of phosphatidylinositol labeling observed in these studies is the earliest response of aleurone cells to gibberellic acid reported.