Selective expression of a probable amylase/protease inhibitor in barley aleurone cells: Comparison to the barley amylase/subtilisin inhibitor

We have cloned and sequenced a 650-nucleotide cDNA from barley (Hordeum vulgare L.) aleurone layers encoding a protein that is closely related to a known -amylase inhibitor from Indian finger millet (Eleusine coracana Gaertn.), and that has homologies to certain plant trypsin inhibitors. mRNA for this probable amylase/protease inhibitor (PAPI) is expressed primarily in aleurone tissue during late development of the grain, as compared to that for the amylase/subtilisin inhibitor, which is expressed in endosperm during the peak of storage-protein synthesis. PAPI mRNA is present at high levels in aleurone tissue of desiccated, mature grain, and in incubated aleurone layers prepared from rehydrated mature seeds. Its expression in those layers is not affected by either abscisic acid or gibberellic acid, hormones that, respectively, increase and decrease the abundance of mRNA for the amylase/subtilisin inhibitor. PAPI mRNA is almost as abundant in gibberellic acid-treated aleurone layers as that for -amylase, and PAPI protein is synthesized in that tissue at levels that are comparable to -amylase. PAPI protein is secreted from aleurone layers into the incubation medium.Abbreviations ABA abscisic acid - ASI barley amylase/subtilisin inhibitor - bp nucleotide base pairs - Da dalton - dpa days post anthesis - GA₃ gibberellic acid - PAPI probable amylase/protease inhibitor - poly(A)RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.) somatic embryogenesis from isolated scutellum: Days post anthesis,days of spike storage,and sucrose concentration affect efficiency

Summary To improve the efficiency of somatic embryogenesis of isolated scutella from commercial wheat (Triticum aestivum L.) cultivars, two factorial experiments were conducted to examine effects of days post anthesis (DPA), days of spike storage (DSS) at 4°C, and sucrose concentrations (SC) on the percentage of scutella producing mature embryos and the number of mature embryos produced per responsive scutellum. In the first experiment, scutella isolated from spikes collected at 10, 11, 12, 13, 14, 15, and 16 DPA and stored at 4°C for 7, 10, 13, and 16d were placed on embryo induction medium [Murashige and Skoog plus 9.96 μM 2,4-dichlorophenoxyacetic acid (2,4-D) and 110 mg l⁻¹ casamino acids], incubated in darkness for 12–14 d and then under light for 2 wk. The interaction of DPA × DSS significantly affected the percentage of scutella producing mature embryos, while only DPA affected the number of mature embryos per responsive scutellum. In the second experiment, scutella isolated from spikes collected at 12 DPA and stored for 15, 16, 17, 18, and 19d were placed on embryo induction medium containing 2, 3, 4, and 5% sucrose. The interaction of DSS × SC significantly affected both the percentage of scutella producing mature embryos and the number of mature embryos per responsive scutellum. In general, DPA/DSS/SC combinations, 12/17/3, 12/18/3, and 12/19/2, yielded the numerically highest embryogenesis efficiencies.     

RNA complementary to α-amylase mRNA in barley

Two experimental approaches demonstrate that different types of RNA complementary to -amylase mRNA are present in barley. S1 nuclease assays identify an RNA that is complementary to essentially the full length of both the type A and type B -amylase mRNAs. Complementarity, however, is imperfect: the S1 nuclease-resistant products can only be identified if they are electrophoresed as RNA-DNA hybrids. This RNA is present in developing endosperm + aleurone tissue and in mature aleurone tissue cultured in the absence of hormonal treatment or in the presence of abscisic acid, but not in shoot or root tissue. In mature aleurone tissue treated with abscisic acid, its steady-state abundance is similar to that of -amylase mRNA. Northern blot analysis indicated the presence of a second type of antisense RNA. Under conditions of moderate stringency, antisense-specific probes detect discrete hybridizing species of 1.6, 1.4, and 1.0 kilobases in mature aleurone and shoot tissues that do not represent spurious hybridization to rRNA, -amylase mRNA, or the abundant, G+C-rich mRNA for a probable amylase/protease inhibitor. The different results are consistent with the fact that the hybridization assay can tolerate relatively short regions of complementarity separated by large, nonhomologous sequences, while the nuclease protection assay cannot.     

Localizing a-Amylase Gene Expression in Germinated Rice Grains

In situ hybridization was used to localize the sites of expressionfor two a-amylase genes (RAmy1A and RAmy3D) in rice (Oryza sativaL.) over five days of germination. Messenger RNAs from bothgenes were initially detected in the scutellar epithelium andappeared at later stages of germination in the aleurone layer.RAmy3D mRNA reached its peak of accumulation 2 days(d) earlierin the scutellar epithelium than RAmy1A mRNA. Both mRNAs continuedto accumulate in the aleurone layer up to 5 d of germination,although RAmy1A mRNA reached significantly higher levels thanRAmy3D mRNA. Overall, the aleurone layer was responsible forproducing the majority of the total grain a-amylase mRNA. (Received July 27, 1991; Accepted November 6, 1991)     

Secretion of basic and acidic chitinases from salt-adapted and -unadapted winged bean cells

Northern hybridizations were used to study the site of synthesis of three carboxypeptidases (Cpases I-III) which occur in the starchy endosperm of germinating barley grain ( Hordeum vulgare L.). Further evidence was obtained by studying secretion of these enzymes from scutella or aleurone layers separated from germinating grains. Messenger RNA for Cpase II was detected only in developing grain, and the bulk of the mRNA was localized in the starchy endosperm. This suggests that Cpase II is synthesized at the site of its accumulation, the starchy endosperm. In contrast, Cpase I is expressed during germination and the predominant site of synthesis is the scutellum, from which it is secreted into the starchy endosperm. Cpase III is also synthesized during germination, but the bulk of it is synthesized in and secreted from the aleurone layer. Thus, the three carboxypeptidases, all of which seem to play a role in hydrolysis of the reserve proteins in the starchy endosperm during germination, have different sites of synthesis.     

Partial purification and characterization of mRNAs encoding glycollate oxidase and catalase

Polyadenylated mRNA was prepared from etiolated and greening leaves of Lens culinaris and cotyledons of Cucumis sativus during the transition from etiolated to photoautotrophic stage. These mRNA preparations were used to identify, by translation in vitro, the precursor forms of glycollate oxidase and catalase, both enzymes being markers of microbodies. The level (per fresh weight) of translatable RNA coding for glycollate oxidase was found to increase ten fold during the first 3 d of illumination of etiolated leaves. For catalase mRNA activity, this increase was less pronounced. Characterizing the products of in-vitro translation directed by the mRNA prepared, we observed a 43-kDa species of glycollate oxidase and a 56-kDa species of apo-catalase. Limited proteolysis of the in-vitro-formed proteins and comparison with the respective mature enzymes present in vivo revealed differences between the cucumber and the lens protein but not between the monomeric precursor and the subunit of mature glycollate oxidase from Lens culinaris. Messenger RNA coding for glycollate oxidase was highly purified by electrophoresis on low-melting-point agarose in the presence of methylmercuric hydroxide. The size of the mRNA was determined to be 1.47 kb. By this procedure, the mRNA for glycollate oxidase in the subfraction could be enriched in such a way that the activity, assayed by translation in a reticulocyte lysate, amounted to 30% of the total translation activity.Abbreviations PAGE polyacrylamide gel electrophoresis - poly(A)⁺ RNA polyadenylated RNA - SDS sodium dodecyl sulfate     

Changes in legumin messenger RNAs throughout seed development in Pisum sativum L.

The patterns of accumulation of three classes of legumin mRNA from Pisum sativum have been followed through seed development by cell-free translation and hybridization to complementary DNAs. Maximum amounts of mRNA were found at 19 days after flowering (DAF) for two classes and at 24 DAF for the third class. The proportions of the three classes varied through development: the RNA species which was 40% of the total legumin mRNA at 14 DAF represented 15–20% of the total at 25–27 DAF, whereas a second mRNA species represented approx. 30% and 70% at the same stages, respectively. Based on these results, some deductions about the possible contribution of individual genes within classes are made.Abbreviations cDNA complementary DNA - DAF days after flowering - IgG immunoglobulin G - SSC standard saline citrate (0.15 M NaCl, 0.015 M trisodium citrate)     

Proteins and messenger RNAs of the transforming region of wild-type and mutant adenoviruses

We have studied the proteins encoded by the transforming region of the closely related human adenovirus serotypes 2 and 5. Messenger RNAs complementary to the two parts of this region, E1A and E1B, were prepared separately by hybridization to cloned DNA fragments encompassing 0.8 to 4.5 map units (for E1A) and 9.8 to 11.1 map units (for E1B). These RNAs were further fractionated by electrophoresis through agarose gels containing methylmercuric hydroxide, and then translated in vitro to identify the proteins encoded by each RNA species. E1A and E1B RNAs isolated at early and at late times after infection were compared. Three size classes of E1A mRNA direct the synthesis of at least five proteins: a28K³ protein encoded by a 0.6 kb mRNA, 42K and 54K proteins encoded by a 0.9 kb mRNA(s), and 48K and 58K proteins encoded by a 1.1 kb mRNA(s). The mRNA for the 28K protein accumulates preferentially at late times. Three size classes of early E1B mRNA direct the synthesis of three proteins: a 15K protein encoded by a 0.9 kb mRNA, an 18K protein encoded by a 1.2 kb mRNA, and a 57K protein encoded by a 2.6 kb mRNA. The mRNA for the 15K protein continues to accumulate at late times, and an additional 22K protein is made, while the 18K and 57K proteins are synthesized poorly, if at all, with late RNA.Substantially different E1A and E1B proteins are encoded by RNA from cells infected with the adenovirus type 5 mutants dl311, dl312, dl313, dl314 and hr1, which are all defective for replication on human cells and, except for dl311, for transformation. dl312, dl314 and hr1 are also defective for early viral gene expression. No viral mRNA could be detected in either dl312 or dl314-infected cells. hr1-infected cells contain a 0.9 kb mRNA encoding E1A 54K and 42K, but instead of 58K and 48K, the 1.1 kb hr1-E1A mRNA is translated into a 26K protein. The E1B mRNAs are present in substantially decreased amounts in hr1-infected cells. dl311-infected cells contain E1A mRNAs of 1.1 and 0.9 kb, encoding 38K and 34K proteins, respectively, and normal E1B mRNAs. The dl313 mRNAs of 1.1 and 0.9 kb contained fused E1A and E1B sequences and were translated into 40K and 36K proteins, respectively. These results are related to the mRNA structures and the biological activity of regions of the individual proteins.     

Sequence and expression of the mRNA encoding the chymotrypsin inhibitor in winged bean (Psophocarpus tetragonolobus (L.) DC.)

The accumulation of the Kunitz-type chymotrypsin inhibitor WCI-3 in winged bean seeds is controlled developmentally. In vitro translation experiments showed that the WCI-3 mRNA was present in 35- and 40-day-old immature seeds after flowering. The size of the in vitro translation product is about 2 000 Da larger than that of the mature WCI-3 protein. The WCI-3 cDNA clones were isolated from a gtll cDNA library of 35-day-old immature seeds by immunoscreening. A nearly full-length cDNA clone was obtained containing an open reading frame of 207 amino acid residues. The deduced sequence of the 183 carboxy terminal amino acids coincides precisely with the amino acid sequence determined for purified WCI-3. The amino terminal extension of 24 residues has the characteristics of a signal peptide. Northern hybridization analysis of total poly(A)⁺ RNA showed that the WCI-3 mRNA is approximately 900 nucleotides long and accumulates in 35- and 40-day-old but not in 30-day-old immature seeds.     

Differential synthesis in vitro of barley aleurone and starchy endosperm proteins

To widen the selection of proteins for gene expression studies in barley seeds, experiments were performed to identify proteins whose synthesis is differentially regulated in developing and germinating seed tissues. The in vitro synthesis of nine distinct barley proteins was compared using mRNAs from isolated endosperm and aleurone tissues (developing and mature grain) and from cultured (germinating) aleurone layers treated with abscisic acid (ABA) and GA₃. B and C hordein polypeptides and the salt-soluble proteins β-amylase, protein Z, protein C, the chymotrypsin inhibitors (CI-1 and 2), the α-amylase/subtilisin inhibitor (ASI) and the inhibitor of animal cell-free protein synthesis systems (PSI) were synthesized with mRNA from developing starchy endosperm tissue. Of these proteins, β-amylase, protein Z, and CI- 1 and 2 were also synthesized with mRNA from developing aleurone cells, but ASI, PSI, and protein C were not. CI-1 and also a probable amylase/protease inhibitor (PAPI) were synthesized at high levels with mRNAs from late developing and mature aleurone. These results show that mRNAs encoding PAPI and CI-1 survive seed dessication and are long-lived in aleurone cells. Thus, expression of genes encoding ASI, PSI, protein C, and PAPI is tissue and stage-specific during seed development. Only ASI, CI-1, and PAPI were synthesized in significant amounts with mRNA from cultured aleurone layers. The levels of synthesis of PAPI and CI-1 were independent of hormone treatment. In contrast, synthesis of α-amylase (included as control) and of ASI showed antagonistic hormonal control: while GA promotes and ABA reduces accumulation of mRNA for α-amylase, these hormones have the opposite effect on ASI mRNA levels.     

Developmental and organ-specific expression of an ABA- and stress-induced protein in barley

An mRNA species, HVA1, has been shown to be rapidly induced by abscisic acid (ABA) in barley aleurone layers (Hong, Uknes and Ho, Plant Mol Biol 11: 495–506, 1988). In the current work we have investigated the expression of HVA1 in other organs of barley plants. In developing seeds, HVA1 mRNA is not detected in starchy endosperm cells, yet it accumulates in aleurone layers and embryo starting 25 days after anthesis, and its level remains high in these organs in dry seeds. Although the levels of HVA1 mRNA are equivalent in the dry embryos of dormant and nondormant barley seeds, upon imbibition HVA1 mRNA declines much slower in the dormant than in the nondormant embryos. The HVA1 mRNA and protein levels are highly induced by ABA treatment in all organs of 3-day-old seedlings. However, the induction in the leaf of 7-day-old seedlings is less than one tenth the level observed in the leaf of 3-day-old seedlings. In the leaf, HVA1 mRNA and protein are induced mainly at the base. These observations indicate that the expression of HVA1 is under developmental regulation. Besides the HVA1 protein, a smaller protein (p20) of approximately 20 kDa cross-reacting with anti-HVA1 polyclonal antibodies, is induced by ABA in barley seedlings but not in seeds. HVA1 mRNA is induced by drought, NaCl, cold or heat treatment. Similar to ABA treatment, the drought induction of HVA1 occurs in all the tissues of 3-day-old seedling, but the induction decreases dramatically in the leaf of 7-day-old plants. The significance of organ-specific, developmentally regulated, and stress-induced expression of HVA1 is discussed.     

Expression of storage-protein genes during soybean seed development

Mature seeds of Glycine max (L.) Merr. contain two major storage proteins, a glycosylated 7S protein (conglycinin) and a non-glycosylated 11S protein (glycinin). Accumulation of these proteins and their mRNAs during seed development in cv. Provar was studied by SDS polyacrylamide gel electrophoresis and by Northern (DNA-RNA) hybridization. The 11S acidic and basic subunits and the 7S and subunits began to accumulate 18–20 d after pollination, shortly after the termination of cell division in developing cotyledons, whereas the 7S and 11S A-4 subunits were not detected until one to two weeks later, during the maturation phase of development. Messenger RNAs for 7S and 11S proteins were first detected 14–18 d after pollination, several days before the accumulation of storage proteins. Extracts from embryonic axes contained reduced levels of the 7S subunit, very little 11S protein, no detectable 7S or 11S A-4 subunits, and an additional 7S subunit not found in cotyledons. Soybean axes and cotyledons therefore differ in their synthesis of seed storage proteins.Abbreviations cDNA complimentary DNA - mRNA messenger RNA - SDS sodium dodecyl sulfate     

Development of aleurone and sub-aleurone layers in maize

Electron-microscope studies indicate that the aleurone tissue of maize (Zea mays L.) starts developing approximately 10–15 days after pollination in stocks that take ca. 40 days for the aleurone to mature completely. Development commences when specialized endosperm cells adjacent to the maternal nucellar layer start to differentiate. Differentiation is characterized by the formation of aleurone protein bodies and spherosomes. The protein bodies of the aleurone layer have a vacuolar origin whereas the protein bodies of the immediate underlying endosperm cells appear to develop from protrusions of the rough endoplasmic reticulum. Thus, two morphologically and developmentally distinct types of protein bodies are present in these adjacent tissues. The spherosomes of the aleurone layer form early in the development of this tissue and increase in number as the tissue matures. During the final stages of maturation, these spherosomes become closely apposed to the aleurone grains and the plasma membrane. No further changes are apparent in the structure of the aleurone cells after 40 days from pollination when the caryopsis begins to desiccate.