Ronald W. Skadsen

Transgenic oat plants via visual selection of cells expressing green fluorescent protein

Abstract New selectable markers and selection systems are needed to increase the efficiency and flexibility of plant transformation. The objective of this research was to determine if the green fluorescent protein (gfp) gene could be utilized as a visual selectable marker for transformation of oat (Avena sativa L.). A modified gfp gene was delivered into oat cells by microprojectile bombardment. Cell clusters expressing gfp were visually identified using fluorescence microscopy and physically isolated at each subculture. Eleven independent transgenic cell lines were obtained, and fertile plants regenerated from all lines. Transgene integration and expression were confirmed in transgenic plants and progeny. Transgene expression segregated in a 3 : 1 ratio in progeny of the majority of the transgenic lines.

Expression of thaumatin-like permatin PR-5 genes switches from the ovary wall to the aleurone in developing barley and oat seeds.

Permatins are antifungal thaumatin-like proteins (TLPs) of the PR-5 family of pathogenesis-related proteins. They occur in many cereals, but little is known of their expression and roles. Permatin cDNA clones were produced and used to study expression in developing barley and oat seeds. Actin and CDC48 mRNAs declined rapidly following inoculation of barley spikes with Fusarium graminearum. Despite this, permatin mRNA levels remained constant or increased slightly. Studies of permatin gene expression in healthy plants revealed that developing barley and oat seeds accumulate permatin mRNA in an unusual bimodal pattern. Permatin mRNA and protein are highly abundant around the time of pollination and then decrease rapidly to near-zero. A second peak occurs in the doughy stage of development. Antibody and DNA probe hybridization studies showed that expression initially occurs in the ovary wall and then switches to the aleurone and ventral furrow of developing seeds, reaching a peak in the doughy stage. Small amounts of permatin mRNAs also occur in certain vegetative tissues. The barley and oat permatin sequences provided sufficient comparisons between cereal TLPs to suggest that deletions or additions in specific elements could have led to the divergence of leaf- and seed-specific TLPs.

Spatial and Temporal Divergence of Expression in Duplicated Barley Germin-like Protein-encoding Genes

Subfunctionalization is the process by which a pair of duplicated genes, or paralogs, experiences a reduction of individual expression patterns or function while still reproducing the complete expression pattern and function of the ancestral gene. Two germin-like protein (GLP)-encoding genes, GerB and GerF, are paralogs that belong to a small gene family in barley (Hordeum vulgare). Both genes share high nucleotide sequence similarity in coding and noncoding regions and encode identical apoplastic proteins. The use of RNA gel blots, coupled with single-stranded conformation polymorphism (SSCP) analysis of RT–PCR products, elucidated the developmental and tissue-specific expression patterns of each gene. Individual expression patterns provided evidence of both overlapping redundancy and early subfunctionalization. GerB is predominantly expressed in developing shoots, while GerF is predominantly expressed in seedling roots, developing spikes, and pericarp/testa. GerF promoter deletion studies located a region (−356/−97) responsible for high promoter activity and showed the ability of GerB and GerF upstream regions to drive gfp expression in coleoptiles, epicarps, and lemma/palea of developing spikes. The observed expression patterns are consistent with proposed roles in plant development and defense mechanisms for this gene family. These roles may explain why redundancy has been selectively maintained in this duplicate gene pair.

Cloning of the promoter for a novel barley gene, Lem1, and its organ-specific promotion of Gfp expression in lemma and palea

The differential display method was used to identify a novel barley gene, Lem1, expressed primarily in the outer organs (lemma and palea) that enclose developing florets and seeds. The promoter was isolated from a BAC genomic clone and used in a translational fusion with a green fluorescent protein gene (Gfp) to produce a transient expression vector. After particle bombardment, Gfp was expressed only in lemmas, paleas and awns of developing spikelets. Lem1 did not promote Gfp expression in vegetative leaves or in mature spikes, although expression of co-bombarded uidA (GUS) occurred under the regulation of a ubiquitin promoter. This reproduced the developmentally regulated pattern of mRNA accumulation. Deletion studies showed that the promoter activity is confined to a cis element within 80 bp of the transcription start site. Upstream from this, the promoter contains putative auxin-, ethylene- and gibberellin-responsive elements or homologues. Lem1 was found to be a single intronless gene encoding an acidic 102 amino acid protein, possibly associated with membranes. In a two-rowed barley, Lem1 mRNA was absent in the lateral spikelets, which fail to develop, and present only in the developing median spikelets. This suggests that Lem1 may play a role in organ development.

Expression of enzymatically active, recombinant barley alpha-glucosidase in yeast and immunological detection of alpha-glucosidase from seed tissue.

An α-glucosidase cDNA clone derived from barley aleurone tissue was expressed in Pichia pastoris and Escherichia coli. The gene was fused with the N-terminal region of the Saccharomyces cerevisiae α-factor secretory peptide and placed under control of the Pichia AOX1 promoter in the vector pPIC9. Enzymatically active, recombinant α-glucosidase was synthesized and secreted from the yeast upon induction with methanol. The enzyme hydrolyzed maltose > trehalose > nigerose > isomaltose. Maltase activity occurred over the pH range 3.5–6.3 with an optimum at pH 4.3, classifying the enzyme as an acid α-glucosidase. The enzyme had a Km of 1.88 mM and Vmax of 0.054 µmol/min on maltose. The recombinant α-glucosidase expressed in E. coli was used to generate polyclonal antibodies. The antibodies detected 101 and 95 kDa forms of barley α-glucosidase early in seed germination. Their levels declined sharply later in germination, as an 81 kDa α-glucosidase became prominent. Synthesis of these proteins also occurred in isolated aleurones after treatment with gibberellin, and this was accompanied by a 14-fold increase in α-glucosidase enzyme activity.Abbreviations: AGL, barley seed α-glucosidase; rAGL, recombinant barley seed α-glucosidase; BMGY, buffered glycerol-complex medium; BMMY, buffered methanol-complex medium; GA, gibberellic acid; UTR, untranslated region.

Comparative transcriptional profiling established the awn as the major photosynthetic organ of the barley spike while the lemma and the palea primarily protect the seed.

The lemma, palea, and awn of a barley (Hordeum vulgare L.) spike are photosynthetic organs that supply the developing seed with carbohydrates. The lemma and palea also enclose the seed and protect it from pathogens and insects. Despite the important roles they play, little information exists on gene expression in these organs that identifies their function. In this study, we compared gene expression among the lemma, palea, awn, and developing seed of barley during grain filling using the Barley1 Genome Array to identify highly expressed genes involved in the primary function of these organs. Hierarchical clustering and mixed model analysis revealed that the lemma and palea have closely related gene expression patterns. In addition, the lemma and palea overexpressed defense‐related genes compared with the awn. The awn preferentially expressed genes for photosynthesis, the biosynthesis of chlorophyll and carotenoids, and reactive oxygen species scavenging. This suggests the lemma and palea are mainly protective organs whereas the awn is primarily a photosynthetic structure. The seed was enriched with genes for the biosynthesis of starch, storage proteins, enzyme inhibitors, and cell proliferation.

Biosynthesis of avenanthramides in suspension cultures of oat (Avena sativa)

Oats produce a group of secondary metabolites termed avenanthramides (avn). These compounds are biosynthesized through the action of the enzyme hydroxycinnamoyl CoA: hydroxyanthranilate N-hydroxycinnamoyl transferase (HHT) which catalyzes the condensation of one of several cinnamate CoA thioesters with the amine functionality of anthranilic acid, 4-hydroxy- or 5-hydroxy-anthranilic acid. In oat leaf tissue the biosynthesis of avenanthramides appears to result from elicitation by fungal infection. Here we demonstrate the biosynthesis of several avenanthramides in suspension cultures of oat apical meristem callus tissue. This phenomenon appears as a generalized pathogen response, evidenced by the production of PR-1 mRNA, in response to elicitation with chitin (poly-N-acetyl glucosamine). The suspension cultures also produce relatively large quantities of avnA and G in response to chitin elicitation. Under certain culture conditions avnB and C are also produced as well as three additional metabolites tentatively identified as avnH, O and R. These findings portend the utility of oat suspension culture as a tool for more detailed investigation of the mechanisms triggering their biosynthesis as well as the factors dictating the particular types of avenanthramides biosynthesized.

Expression of an Altered Antimicrobial Hordothionin Gene in Barley and Oat

Alpha-hordothionin (HTH) is specifically produced in barley endosperm. Purified HTH has antimicrobial activity against a wide range of pathogenic microbes. Native HTH does not protect barley from infection because HTH is confined to endosperm, and many pathogens infect seeds through outer tissues. Therefore, it would be desirable to express HTH gene in floral tissues. We describe transformation and expression analyses of an altered HTH gene driven by a constitutive promoter in transgenic barley and oat.