Influence of exogenous abscisic acid on germination and plantlet conversion frequencies of hybrid larch somatic embryos (Larix × leptoeuropaea)

The effect of exogenous abscisic acid, provided to somatic embryos during the maturation step, on endogenous abscisic acid and its main conjugated form (abscisic acid glucose ester), germination and conversion frequencies is presented in this paper. Abscisic acid measurements were obtained after a methanolic extraction, a fractionation through high performance liquid chromatography, quantitation with an immunoassay and identification of the quantitated compound using gas chromatography-mass spectrometry. Results show that endogenous abscisic acid and abscisic acid glucose ester levels are clearly correlated with the exogenous abscisic acid concentration provided to the embryos. Maturation was clearly enhanced by exogenous abscisic acid, but no correlation was found between abscisic acid concentration and germination frequency. Conversely, development of the aerial part of the germinated somatic embryos was dependent upon the abscisic acid concentration in the culture medium and results suggest that this dependence could be related to the endogenous abscisic acid content.     

Gibberellins are not required for rice germination under anoxia

Production of -amylase during the germination of rice grains is thought to play an important role for tolerance to anoxia of these cereal grains. Under aerobic conditions -amylases production is enhanced in response to gibberellins produced by the embryos, but the role of these hormones is less clear under anoxia. In this paper we analysed -amylase gene expression in a rice mutant (Tan-ginbozu) severely impaired in gibberellin biosynthesis. Expression of -amylase genes others than the gibberellin-induced Amy1A gene is observed. The expression of the Amy3D gene, which does dot require gibberellins to be induced, is high under anoxia in the Tan-ginbozu mutant suggesting that germination under anoxia can proceed thanks to the activity of the -amylase isoform encoded by the Amy3D gene. Amy3D gene expression is repressed in the presence of high levels of soluble carbohydrates, indicating that the anaerobic expression of this gene can be triggered by a lower carbohydrate content of rice grains kept under anoxia. Germination under anoxia of Tan-ginbozu grains can proceed even in absence of exogenously-added gibberellic acid. Overall, results indicate that gibberellins are not required for the anaerobic germination of rice grains.     

The effect of intracellular pH on the regulation of the Rab 16A and the α-amylase 1/6-4 promoter by abscisic acid and gibberellia

Intracellular pH (pH_i) of barley aleurone cells is known to be affected by hormones and plant growth conditions. The possible mechanisms by which these pH_i shifts influence the actions of abscisic acid (ABA) or gibberellin (GA) is being investigated. Here we report an attempt to study the effect of pH_i on hormone-induced gene expression. We used weak acids and weak bases to artificially mimic the pH_i changes brought about by ABA and GA and found that chloramphenicol acetyltransferase (CAT) expression controlled by the Rab promoter was affected while the -amylase promoter seemed insensitive. CAT fused to the 35S promoter was used as a control which is not inducible by ABA or GA₃. The expression of this construct was not significantly affected by artificial pH_i changes.     

Cloning,characterization and expression of<Emphasis Type="Italic"> OsGLN2</Emphasis>, a rice endo-1,3-β-glucanase gene regulated developmentally in flowers and hormonally in germinating seeds

We report here the isolation and characterization of a new endo-1,3--glucanase (1,3--GLU) cDNA, OsGLN2, that is expressed both in flowers and in germinating seeds of rice (Oryza sativa L.). The isolated OsGLN2 gene encoded a protein which displayed 72%, 93% and 92% identity at the amino acid level with those encoded by barley GII, rice Gns4 and glu1 1,3--GLU genes, respectively. A GST-OsGLN2 recombinant protein expressed in Escherichia coli preferentially hydrolyzed Laminaria digitata 1,3;1,6--glucan and liberated only oligosaccharides, suggesting that the enzyme can be classified as a 1,3--GLU. Northern analysis with a 3-UTR gene-specific probe revealed that OsGLN2 is expressed exclusively in the paleae and lemmas during flowering, and no expression of OsGLN2 was detected in other tissues such as leaf blades, leaf sheaths, stems, nodes and roots in mature rice plants. The OsGLN2 gene is also expressed in germinating seeds, where its expression is predominant in endosperms rather than embryos. In de-embryonated rice half-seeds, addition of gibberellin A₃ (GA) greatly enhanced expression of the OsGLN2 gene, while the GA-induced gene expression was suppressed strongly by abscisic acid (ABA). This is the first report, to our knowledge, that OsGLN2 encodes a 1,3--GLU and is expressed specifically in paleae and lemmas during flowering and in germinating seeds, where its expression is enhanced by GA and suppressed by ABA.Abbreviations ABA Abscisic acid - GA Gibberellin A₃ - 1,3--GLU Endo-1,3--glucanase - GST Glutathione S-transferase - IPTG Isopropyl-1-thio--galactopyranoside - 3-UTR 3-Untranslated region     

Cloning and expression of a sorghum gene with homology to maize vp1. Its potential involvement in pre-harvest sprouting resistance

Pre-harvest sprouting (PHS) in sorghum is related to the lack of a normal dormancy level during seed development and maturation. Based on previous evidence that seed dormancy in maize is controlled by the vp1 gene, we used a PCR-based approach to isolate two Sorghum bicolor genomic and cDNA clones from two genotypes exhibiting different PHS behaviour and sensitivity to abscisic acid (ABA). The two 699 amino acid predicted protein sequences differ in two residues at positions 341 (Gly or Cys within the repression domain) and 448 (Pro or Ser) and show over 80, 70 and 60% homology to maize, rice and oat VP1 proteins respectively.Expression analysis of the sorghum vp1 gene in the two lines shows a slightly higher level of vp1 mRNA in the embryos susceptible to PHS than in those resistant to PHS during embryogenesis. However, timing of expression was different between these genotypes during this developmental process. Whereas for the former the main peak of expression was observed at 20 days after pollination (DAP), the peak in the latter was found at later developmental stages when seed maturation was almost complete.Under favourable germination conditions and in the presence of fluridone (an inhibitor of ABA biosynthesis), sorghum vp1 mRNA showed to be consistently correlated with sensitivity to ABA but not with ABA content and dormancy.     

Spatial and temporal expression patterns directed by theAgrobacterium tumefaciens T-DNA gene 5 promoter during somatic embryogenesis in carrot

We have analysed the patterns of expression of a gene encoding -glucuronidase (GUS) fused to the promoter of theAgrobacterium tumefaciens T-DNA gene 5 during embryogenesis in carrot,Daucus carota L. Gene expression was monitored by a histochemical assay of -glucuronidase activity. The gene 5 promoter, although of bacterial origin, conferred expression upon the marker gene in all stages of embryo development. The patterns of expression however, differed between embryos in different stages of development. In the globular stage expression was confined to the basal part of the embryo, suggesting that the promoter is sensitive to regulatory functions active in the primary establishment of polarity in the radially symmetric globular embryo. In the heart and torpedo stages of development GUS expression was high in the entire embryonic axis, but not in the cotyledons. During germination expression was reduced in the elongating hypocotyl and radicle, and high levels of expression were detected only in the shoot and root apices. Among the transformed cell lines analysed, one was found that showed an aberrant pattern of GUS expression during embryogenesis, in that expression in the upper part of the embryo was undetectable, and expression was restricted to the root apex in later stages of development. This difference in organ specificity of expression is likely due to a large deletion of the promoter.     

Construction of a fusion gene comprising the Taka-amylase A promoter and the Escherichia coli β-glucuronidase gene and analysis of its expression in Aspergillus oryzae

Summary Northern blot analysis of glucose-grown and starch-grown mycelia of Aspergillus oryzae R11340 was conducted using the cloned Taka-amylase A (TAA) gene as a probe. The amount of mRNA homologous to the TAA gene was increased when this fungus was grown with starch as a sole carbon source. In order to analyze the induction mechanism, we inserted the Escherichia coli uidA gene encoding -glucuronidase (GUS) downstream of the TAA promoter and introduced the resultant fusion gene into the A. oryzae genome. Production of a functional GUS protein was induced by starch, but not by glucose. When the effects of various sugars on expression of the fusion gene were examined, the results suggested that the expression of the fusion gene was under control of the TAA gene promoter.     

The Effect of Polyethylene Glycol on Proline Accumulation in Rice Leaves

The regulation of proline accumulation in polyethylene glycol (PEG, –1.5 MPa) treated rice leaves was investigated. PEG treatment resulted in a decrease in relative water content, indicating that PEG treatment caused water stress in rice leaves. Proline accumulation caused by PEG was related to protein hydrolysis, an increase in ornithine--amino- transferase activity, an increase in the content of ammonia, and an increase in the contents of the precursors of proline biosynthesis, glutamic acid, ornithine, and arginine. Results also show that abscisic acid accumulation is not required for proline accumulation in PEG-treated rice leaves.     

Calluses initiated from thin mature embryo fragments are suitable targets for wheat transformation as assessed by long-term GUS expression studies

Microprojectile- or Agrobacterium-mediated DNA delivery into calluses initiated from immature embryos has proven to be effective in transforming wheat. Yet, obtaining a large number of high quality immature embryos throughout the year is a laborious and delicate process. To circumvent these limitations, we propose an alternative technique applying the particle bombardment technology to calluses derived from fragmented mature embryos rather than immature tissues. The phosphinothricin acetyl transferase (bar) and -glucuronidase (gus) genes were used as selectable and screenable marker genes, respectively, to assess and optimise the performance of the proposed technique. Primary requirement for genetic transformation method development, the regeneration capacity of bombarded calluses was established. A preculture duration of 6 days was identified as optimal for DNA uptake and -glucuronidase (GUS) expression. The highest activity was recorded when calluses were selected. Long-term GUS expression studies (1–7weeks subsequent to bombardment), showed differentiated behaviours for tissues obtained from mature versus immature embryos. Notably, mature embryos exhibited the greatest number of cells stably expressing the reporter gene, thus providing an excellent source material for developing a stable transformation procedure.     

Production of fructooligosaccharides in high yield using a mixed enzyme system of β-fructofuranosidase and glucose oxidase

A mixed enzyme system, with -fructofuranosidase (obtained from Aspergillus japonicus) and commercial glucose oxidase (Gluzyme, Novo Nordisk), produced fructooligosaccharides (FOS) in high yield from sucrose. The reaction was performed in an aerated stirred tank reactor controlled at pH 5.5 by a slurry of CaCO₃. Glucose, an inhibitor of -fructofuranosidase, produced in the reaction was converted by glucose oxidase to gluconic acid, which was then precipitated to calcium gluconate in solution. The system produced more than 90% (w/w) FOS on a dry weight basis, the remainder was glucose, sucrose and a small amount of calcium gluconate. Most of the FOS and sucrose was hydrolyzed to fructose in the mixed enzyme system with glucose oxidase and -fructofuranosidase from Asp. niger.     

Temporally uncontrolled expression of linearized plasmid DNA which carries bacterial chloramphenicol acetyltransferase gene withXenopus cardiacα-actin promoter after injection intoXenopus fertilized eggs

Summary Circular and linearized plasmid DNA which contained bacterial chloramphenicol acetyltransferase (CAT) gene connected toXenopus cardiac-actin promoter was injected intoXenopus fertilized eggs to study their expression in the course of early embryonic development. While circular DNA was slightly replicated and expressed only after embryos reached neurula stage, linearized DNA formed a large amount of concatemers, and was expressed as early as at blastula stage, or about 14 hr earlier than the time of circular DNA expression. Similarly earlier expression of linearized DNA occurred slightly more strongly when the DNA was injected into presumptive dorsal than in ventral blastomeres at 4-cell stage, and the expression was not affected when embryos were dissociated at blastula stage and their cells were cultured under reaggregating or nonreaggregating conditions. These results show that although circular actin-CAT fusion gene is expressed during development according to endogenous temporal control, the expression of linearized DNA deviates from such developmental control even though it contains intact promoter of-actin gene. It is then recommended that study of the control of the expression of exogenously-introduced DNA inXenopus fertilized eggs should be carried out with circular but not linearized plasmids.     

Agrobacterium-mediated production of transgenic rice plants expressing a chimeric α-amylase promoter/β-glucuronidase gene

We have successfully transferred and expressed a reporter gene driven by an -amylase promoter in a japonica type of rice (Oryza sativa L. cv. Tainung 62) using the Agrobacterium-mediated gene transfer system. Immature rice embryos (10–12 days after anthesis) were infected with an Agrobacterium strain carrying a plasmid containing chimeric genes of -glucuronidase (uidA) and neomycin phosphotransferase (nptII). Co-incubation of potato suspension culture (PSC) with the Agrobacterium inoculum significantly improved the transformation efficiency of rice. The uidA and nptII genes, which are under the control of promoters of a rice -amylase gene (Amy8) and Agrobacterium nopaline synthase gene (nos), respectively, were both expressed in G418-resistant calli and transgenic plants. Integration of foreign genes into the genomes of transgenic plants was confirmed by Southern blot analysis. Histochemical localization of GUS activity in one transgenic plant (R0) revealed that the rice -amylase promoter functions in all cell types of the mature leaves, stems, sheaths and roots, but not in the very young leaves. This transgenic plant grew more slowly and produced less seeds than the wild-type plant, but its R1 and R2 progenies grew normally and produced as much seeds as the wild-type plant. Inheritance of foreign genes to the progenies was also confirmed by Southern blot analysis. These data demonstrate successful gene transfer and sexual inheritance of the chimeric genes.