Judith Strommer

The plant ADH gene family

The structures, evolution and functions of alcohol dehydrogenase gene families and their products have been scrutinized for half a century. Our understanding of the enzyme structure and catalytic activity of plant alcohol dehydrogenase (ADH-P) is based on the vast amount of information available for its animal counterpart. The probable origins of the enzyme from a simple β-coil and eventual emergence from a glutathione-dependent formaldehyde dehydrogenase have been well described. There is compelling evidence that the small ADH gene families found in plants today are the survivors of multiple rounds of gene expansion and contraction. To the probable original function of their products in the terminal reaction of anaerobic fermentation have been added roles in yeast-like aerobic fermentation and the production of characteristic scents that act to attract animals that serve as pollinators or agents of seed dispersal and to protect against herbivores.

Towards Development of an Edible Vaccine against Bovine Pneumonic Pasteurellosis Using Transgenic White Clover Expressing a Mannheimia haemolytica A1 Leukotoxin 50 Fusion Protein

ABSTRACT Development of vaccines against bovine pneumonia pasteurellosis, or shipping fever, has focused mainly on Mannheimia haemolytica A1 leukotoxin (Lkt). In this study, the feasibility of expressing Lkt in a forage plant for use as an edible vaccine was investigated. Derivatives of the M. haemolytica Lkt in which the hydrophobic transmembrane domains were removed were made. Lkt66 retained its immunogenicity and was capable of eliciting an antibody response in rabbits that recognized and neutralized authentic Lkt. Genes encoding a shorter Lkt derivative, Lkt50, fused to a modified green fluorescent protein (mGFP5), were constructed for plant transformation. Constructs were screened by Western immunoblot analysis for their ability to express the fusion protein after agroinfiltration in tobacco. The fusion construct pBlkt50-mgfp5, which employs the cauliflower mosaic virus 35S promoter for transcription, was selected and introduced into white clover byAgrobacterium tumefaciens-mediated transformation. Transgenic lines of white clover were recovered, and expression of Lkt50-GFP was monitored and confirmed by laser confocal microscopy and Western immunoblot analysis. Lkt50-GFP was found to be stable in clover tissue after drying of the plant material at room temperature for 4 days. An extract containing Lkt50-GFP from white clover was able to induce an immune response in rabbits (via injection), and rabbit antisera recognized and neutralized authentic Lkt. This is the first demonstration of the expression of anM. haemolytica antigen in plants and paves the way for the development of transgenic plants expressing M. haemolytica antigens as an edible vaccine against bovine pneumonic pasteurellosis.

Rapid plant regeneration of pea using thidiazuron

A simple and rapid pea regeneration procedure was developed. An average of up to 20 shoots formed from hypocotyl explants of cvs. Sugar Ann and Patriot cultured on Murashige and Skoog basal medium supplemented with 0.5 or 1.0 μM thidiazuron (N-phenyl-N′-1,2,3-thiadiazol-5′-ylurea). Hypocotyls of Puget and Sugar Daddy did not respond. Regenerated shoots rooted rapidly when cultured on Murashige and Skoog basal medium containing either 2.0 μM α-naphthaleneacetic acid or 1.0–2.0 μM indole-3-butyric acid. Seeds were harvested from regenerated plants after only 9–11 weeks.

AFLP maps of Petunia hybrida: building maps when markers cluster.

Abstract.AFLP mapping in Petunia hybrida was undertaken with the intention of building a high-density genetic map suitable for applications such as map-based gene cloning. In total five maps were constructed from two mapping populations, with placement of more than 800 markers. Despite the large number of markers the resulting map is roughly ten-fold smaller than those of other plant species, including the closely related tomato. Low levels of recombination are reflected in clusters of tightly linked markers, both AFLPs and RFLPs, in all the maps. Clustering patterns vary between mapping populations, however, such that loci tightly linked in one population may be separable in another. Combined with earlier reports of aberrant meiotic pairing and recombination, our results suggest that, for species like petunia, map-based cloning may be more complex than in model species such as arabidopsis and tomato.

Structure, expression, chromosomal location and product of the gene encoding ADH1 in Petunia

In most higher plants the genes encoding alcohol dehydrogenase comprise a small gene family, usually with two members. The Adh1 gene of Petunia has been cloned and analyzed, but a second identifiable gene was not recovered from any of three genomic libraries. We have therefore employed the polymerase chain reaction to obtain the major portion of a second Adh gene. From sequence, mapping and northern data we conclude this gene encodes ADH2, the major anaerobically inducible Adh gene of Petunia. The availability of both Adh1 and Adh2 from Petunia has permitted us to compare their structures and patterns of expression to those of the well-studied Adh genes of maize, of which one is highly expressed developmentally, while both are induced in response to hypoxia. Despite their evolutionary distance, evidenced by deduced amino acid sequence as well as taxonomic classification, the pairs of genes are regulated in strikingly similar ways in maize and Petunia. Our findings suggest a significant biological basis for the regulatory strategy employed by these distant species for differential expression of multiple Adh genes.

A gene-based RFLP map of petunia

Abstract Due in large part to the data accumulated from years of classic genetic analysis, petunia (Petunia hybrida Vilm) has remained a useful model system, particularly for studies of gene regulation and genome structure. We have used three segregating populations of petunia, including those serving as the source of an earlier actin gene RFLP map, for RFLP mapping of several additional genes. Twenty-seven loci have been merged with 11 previously mapped morphological and biochemical markers. Our results contribute additional evidence to reports of a high degree of genome plasticity and segregation distortion in this species and suggest that petunia may be a useful plant system for detailed analysis of plant genome organization, activity and evolution.

Anthocyanin regulatory mutations in pea: effects on gene expression and complementation by R-like genes of maize

Abstract Anthocyanin production in higher plants is a function of the tissue considered and its developmental stage, and is modulated by environmental factors. In maize, the best characterized system, regulation of the pathway is achieved largely through the action of proteins with homology to the transcriptional factors encoded by myc and myb proto-oncogenes of animals; these homologues control the expression of structural genes and thus regulate the availability of anthocyanin biosynthetic enzymes. We have studied anthocyanin biosynthesis and its regulation in flowers of pea (Pisum sativum). Our results demonstrate a correlation between anthocyanin accumulation and steady-state mRNA levels for genes encoding chalcone synthase, flavanone 3β-hydroxylase, and dihydroflavonol 4-reductase in developing flowers. Patterns of expression for these biosynthetic genes in both a and a2 mutants confirm the regulatory roles of the two a loci. The reduced expression of all three biosynthetic genes in mutant lines suggests that genes acting both early and late in the anthocyanin biosynthetic pathway are controlled by a and a2. Particle bombardment of floral tissue demonstrates the ability of two maize R-like genes, Lc and R-S, but neither myb-like genes nor R-like genes from snapdragon or petunia, functionally to complement a and a2 mutations. We cannot distinguish whether a and a2 act coordinately or sequentially in anthocyanin regulation, but the epistatic action of maize R-like genes suggests that they mimic the action of a gene that normally functions downstream of both a and a2 in the regulatory cascade.

Expression of a modified Mannheimia haemolytica GS60 outer membrane lipoprotein in transgenic alfalfa for the development of an edible vaccine against bovine pneumonic pasteurellosis.

The GS60 antigen is one of the protective antigens of Mannheimia haemolytica A1. GS60 contains conserved domains belonging to the LppC family of bacterial outer membrane lipoproteins. A high antibody titer to GS60 has been shown to be significantly correlated with resistance to pneumonic pasteurellosis. Calves vaccinated with a commercial vaccine (Presponse) and demonstrating protection against M. haemolytica A1 produced antibodies directed against GS60. Alfalfa was chosen as the platform for an edible vaccine. Agrobacterium tumefaciens was used to mediate the transformation of alfalfa with sequences encoding a slightly shortened derivative of the GS60 antigen (GS60(54)). Stable transgenic alfalfa lines were recovered and production of GS60(54) was examined by Western immunoblot analysis. The antigen is stable in dried transgenic plant material stored at ambient temperature for more than a year. The plant-produced GS60(54) protein was shown to be immunogenic when injected into rabbits. Feeding of the dried transgenic alfalfa expressing the GS60(54) to rabbits is capable of inducing seroconversion, suggesting that GS60(54) could be an effective oral antigen for stimulating mucosal immune responses.

Selective recruitment of Adh genes for distinct enzymatic functions in Petunia hybrida

Alcohol dehydrogenase (ADH) activity in plants is generally associated with glycolytic fermentation, which facilitates cell survival during episodes of low-oxygen stress in water-logged roots as well as chronically hypoxic regions surrounding the vascular core. Work with tobacco and potato has implicated ADH activity in additional metabolic roles, including aerobic fermentation, acetaldehyde detoxification and carbon reutilization. Here a combination of approaches has been used to examine tissue-specific patterns of Adh gene expression in order to provide insight into the potential roles of alcohol dehydrogenases, using Petunia hybrida, a solanaceous species with well-characterized genetics. A reporter-gene study, relying on the promoters of Adh1 and Adh2 to drive expression of the gene for a green fluorescent protein derivative, mgfp5, revealed unexpectedly complex patterns of GFP fluorescence in floral tissues, particularly the stigma, style and nectary. Results of GC-MS analysis suggest the association of ADH with production of aromatic compounds in the nectary. Overall the results demonstrate selective recruitment of Adh gene family members in tissues and organs associated with diverse ADH functions.

Transformation of alfalfa with a bacterial fusion gene, Mannheimia haemolytica A1 leukotoxin50-gfp: Response with Agrobacterium tumefaciens strains LBA4404 and C58

Alfalfa transformed with a portion of the leukotoxin gene from Mannheimia haemolytica was produced to test the feasibility of developing an edible vaccine capable of protecting cattle from pneumonic pasteurellosis. Leukotoxin (Lkt), has been identified as an important protective antigen of M. haemolytica, and a fragment, Lkt50, was shown to produce toxin-neutralizing antibodies in rabbits. The construct chosen for introduction into alfalfa carried lkt50 fused to a green fluorescent protein reporter gene, mgfp5-ER. The fusion gene was driven by either the cauliflower mosaic virus 35S promoter (35S) or the promoter from a rubisco small subunit (rbcS-3A) gene of pea. The constructs were introduced into alfalfa RSY27 germplasm using two Agrobacterium tumefaciens strains, LBA4404 and C58, producing a number of transformed lines with both A. strains. Although strain C58 had a slower initial response and produced less callus than strain LBA4404, it resulted in higher numbers of transformed embryos and plants. In total, 30 alfalfa lines (91% of those analyzed), each derived from a separate transformation event, produced detectable levels of Lkt50-GFP. Western analysis with anti-Lkt+66 antiserum revealed the presence of both full-length and truncated polypeptides in plants kept in magenta boxes, while plants transferred to the greenhouse produced only the full-length product. Immunoblotting with anti-GFP antiserum provided evidence that part of the GFP moiety was lost in the truncated protein. Southern blot analysis indicated a low number of insertion sites per event.