The synthesis of isoflavonoid phytoalexins in legumes is relatively well understood, but far less is known about how these phytotoxic compounds are metabolised by the plant when no longer required. In this project medicarpin, the major isoflavonoid phytoalexin in alfalfa, was prepared in radiolabelled form and fed to cell cultures and seedlings of alfalfa. The metabolism of the radioactive phytoalexin was then studied by characterising the radiolabelled metabolites formed. Uptake of radiolabelled phytoalexin by cells was faster in elicitor-treated cultures than in untreated cultures. However, there was little difference in pattern or speed of metabolism in treated or untreated cultures. Labelled medicarpin was rapidly metabolised to a complex range of extractable medicarpin products (MPs). A very small proportion of the dose was broken down to (^14)C02. A total of 8 MPs could be resolved as distinct metabolites by HPLC and TLC. However, as incubation time increased the radioactivity became associated with multiple minor components which could not be identified. The 8 MPs were characterised by UV and mass-spectrometry and where possible by co- chromatography with authentic standards by TLC and HPLC. Four MPs were unambiguously identified as medicarpin-3-0-glucoside-6"-O-malonate (MGM), the isoflavans vestitol and sativan and the pterocarpan 6a-hydroxy-3,4'- dimethoxypterocarpan (variabilin). In addition a hydroxylated derivative of medicarpin, termed pseudomedicarpin was also tentatively identified. Of the four remaining metabolites MPl had a relative molecular mass of 166 but remained unidentified. MP2 was formed from pseudomedicarpin, but could not be characterised due to its labile nature. Similarly MP3 and MP6 remained unidentified, though the evidence suggested that MPS was a demethylated product of medicarpin. The metabolism of medicarpin in seedlings resembled that of cell cultures with the exception that rather more of the medicarpin was conjugated to form MGM.

No description available.

No description available.

No description available.

Nitric oxide (NO) is a key small molecule that orchestrates plant growth, development and immune function. The chief mechanism for the transfer of NO bioactivity is thought to be S-nitrosylation, the addition of an NO moiety to a protein cysteine thiol to form an S-nitrosothiol (SNO). The enzyme S-nitrosoglutathione reductase (GSNOR) indirectly controls the total levels of cellular S-nitrosylation, by turning over S-nitrosoglutathione (GSNO), the major cellular NO donor. In tomato (Solanum lycopersicum. L) a decrease in GSNOR expression, which is expected to increase the extent of cellular SNO formation, resulted in morphological phenotypes and disabled disease resistance. In contrast, increased GSNOR activity enhanced protection against an ordinarily virulent bacterial pathogen. Collectively, these results are similar to previous findings using the reference plant, Arabidopsis thaliana. Thus, the role of GSNOR may be highly conserved across the plant kingdom and manipulating the function of this protein may control important agricultural traits in crop plants.

Through a binding partner the CARD15 gene activates NF-kB, a molecule with a role in the initiation of the inflammatory immune response. The gene is highly conserved in both structure and function in human and mouse and has recently been implicated as a disease resistance gene in Crohn's disease and Blau Syndrome in human. The gene's relationship to disease and its conservation between species suggests that it may also have a conserved role in bovine disease resistance. To elucidate the potential role of bovine CARD15 in disease resistance, the gene was characterized in cattle. Bovine CARD15 is located 4.2 cR5000 telomeric to ADCY7 on chromosome 18. It spans ~30 kb and is comprised of 12 exons, 11 of which are coding. Bovine CARD15 is expressed in many tissues, but is most abundant in peripheral blood leukocytes. An extensive comparative analysis between the bovine, mouse and human CARD15 genes revealed high levels of inter-species conservation in sequence, genomic structure and protein domains. Conserved putative regulatory motifs were identified in the three species comparison of the 5'UTR, 3'UTR and the intronic sequences flanking exons. Additionally, diverse regulatory motifs were identified in each of the species indicating an evolutionary divergence in the mechanisms of regulation of gene expression. To assess the extent of genetic diversity within bovine CARD15, 41 individuals from nine breeds representing two subspecies were sequenced and screened for polymorphisms. Thirty-six single nucleotide polymorphisms (SNPs) were identified including 26 within the gene transcript. Haplotypes were estimated for each individual and parsimonious SNP sets were identified with which the multi-locus Bos taurus and Bos indicus haplotypes may be reconstructed. There was a significantly higher rate of substitutions within Bos indicus than in Bos taurus. A significantly higher rate of nonsynonymous to synonymous substitutions was found in Bos taurus indicating that positive Darwinian selection is acting on the gene within this subspecies. Association analyses were performed between these SNP loci and haplotypes with Johne's disease. No overwhelming evidence for a simple causal relationship was detected. Assays are provided to screen populations of cattle for variation in the CARD15 gene.

Alternative oxidase (AOX) is a non-energy conserving branch of the mitochondrial electron transport chain (ETC) which has been hypothesized to modulate the level of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in plant mitochondria. The aim of the research presented herein is to provide direct evidence in support of this hypothesis and to explore the implications of this during plant-pathogen interactions in Nicotiana tabacum. We observed leaf levels of ROS and RNS in wild-type (Wt) tobacco and transgenic tobacco with altered AOX levels and we found that plants lacking AOX have increased levels of both NO and mitochondrial O2 - compared Wt plants. Based on the results we suggest that AOX respiration acts to reduce the generation of ROS and RNS in plant mitochondria by dampening the leak of electrons from the ETC to O2 or nitrite. We characterized multiple responses of tobacco to different pathovars of the bacterial pathogen Pseudomonas syringae. These included a compatible response associated with necrosis (pv tabaci), an incompatible response that included the hypersensitive response (HR) (pv maculicola) and an incompatible response that induced defenses (pv phaseolicola). We show that the HR is accompanied by an early mitochondrial O2 - burst prior to cell death. Also, we found iii that the appearance of HR and the appearance of the mitochondrial O2 - burst are delayed in transgenic plants lacking AOX. A similar delay is seen in transgenic plants treated with the complex III inhibitor antimycin A. In Wt plants, expression of Aox1a is suppressed during the HR response to pv maculicola despite the accumulation of signaling molecules known to induce Aox1a transcription. Also, MnSOD activity declined during the HR. We suggest that the mitochondrial ROS burst controlled by AOX and MnSOD is an important component for the induction and coordination of the HR during plant-pathogen interactions.

Xanthomonas axonopodis pv. malvacearum(Smith), Pythium aphanidermatum(Edson) and Rhizoctonia solani(Kuhn) have all been shown to cause significant yield losses in cotton. Previous work has demonstrated that a set of high tannin cotton germplasm lines developed and released in 1989 by Texas A&M AgriLife Research may possess resistance to these three diseases. In this research, the usefulness of these high tannin lines in breeding for resistance to these pathogens as well as the role of tannin in conferring this resistance were examined. The high tannin lines were screened for their resistance to Xanthomonas axonopodis pv. malvacearum and five highly resistant lines were subjected to generation means and diallel analysis in order to determine the relative importance of different types of gene action in conferring resistance as well as which lines would be most useful in breeding for resistance. The effect of selection for R. solani resistance was measured and selected lines were subjected to diallel analysis. Lines showing elevated resistant to P. aphanidermatum were subjected to a single cycle of selection and the effect of selection was measured. Lines possessing high degrees of P. aphanidermatum resistance were subjected to generation means and diallel analysis. The importance of tannin content in conferring resistance to X. axonopodis and R. solani was also measured. Four high tannin lines were found to be not different from Tamcot Sphinx in their resistance to Xam. Generation means analysis for Xam resistance showed that in the five high tannin lines tested, additive gene effects were of the greatest importance. No correlation between tannin content and Xam resistance was detected. Under the conditions of this study tannin content was also shown to be unimportant in R. solani resistance. Two rounds of recurrent phenotypic selection were effective in increasing the resistance of selected HT lines to R. solani. One cycle of phenotypic selection for P. aphanidermatum resistance was found to produce significant improvements in seven of the most resistant HT lines. Generation means analysis for P. aphanidermatum resistance showed that in the HT lines tested, additive genetic effects were by far the most important.

Through a binding partner the CARD15 gene activates NF-kB, a molecule with a role in the initiation of the inflammatory immune response. The gene is highly conserved in both structure and function in human and mouse and has recently been implicated as a disease resistance gene in Crohn's disease and Blau Syndrome in human. The gene's relationship to disease and its conservation between species suggests that it may also have a conserved role in bovine disease resistance. To elucidate the potential role of bovine CARD15 in disease resistance, the gene was characterized in cattle. Bovine CARD15 is located 4.2 cR5000 telomeric to ADCY7 on chromosome 18. It spans ~30 kb and is comprised of 12 exons, 11 of which are coding. Bovine CARD15 is expressed in many tissues, but is most abundant in peripheral blood leukocytes. An extensive comparative analysis between the bovine, mouse and human CARD15 genes revealed high levels of inter-species conservation in sequence, genomic structure and protein domains. Conserved putative regulatory motifs were identified in the three species comparison of the 5'UTR, 3'UTR and the intronic sequences flanking exons. Additionally, diverse regulatory motifs were identified in each of the species indicating an evolutionary divergence in the mechanisms of regulation of gene expression. To assess the extent of genetic diversity within bovine CARD15, 41 individuals from nine breeds representing two subspecies were sequenced and screened for polymorphisms. Thirty-six single nucleotide polymorphisms (SNPs) were identified including 26 within the gene transcript. Haplotypes were estimated for each individual and parsimonious SNP sets were identified with which the multi-locus Bos taurus and Bos indicus haplotypes may be reconstructed. There was a significantly higher rate of substitutions within Bos indicus than in Bos taurus. A significantly higher rate of nonsynonymous to synonymous substitutions was found in Bos taurus indicating that positive Darwinian selection is acting on the gene within this subspecies. Association analyses were performed between these SNP loci and haplotypes with Johne's disease. No overwhelming evidence for a simple causal relationship was detected. Assays are provided to screen populations of cattle for variation in the CARD15 gene.

Alternative oxidase (AOX) is a non-energy conserving branch of the mitochondrial electron transport chain (ETC) which has been hypothesized to modulate the level of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in plant mitochondria. The aim of the research presented herein is to provide direct evidence in support of this hypothesis and to explore the implications of this during plant-pathogen interactions in Nicotiana tabacum. We observed leaf levels of ROS and RNS in wild-type (Wt) tobacco and transgenic tobacco with altered AOX levels and we found that plants lacking AOX have increased levels of both NO and mitochondrial O2 - compared Wt plants. Based on the results we suggest that AOX respiration acts to reduce the generation of ROS and RNS in plant mitochondria by dampening the leak of electrons from the ETC to O2 or nitrite. We characterized multiple responses of tobacco to different pathovars of the bacterial pathogen Pseudomonas syringae. These included a compatible response associated with necrosis (pv tabaci), an incompatible response that included the hypersensitive response (HR) (pv maculicola) and an incompatible response that induced defenses (pv phaseolicola). We show that the HR is accompanied by an early mitochondrial O2 - burst prior to cell death. Also, we found iii that the appearance of HR and the appearance of the mitochondrial O2 - burst are delayed in transgenic plants lacking AOX. A similar delay is seen in transgenic plants treated with the complex III inhibitor antimycin A. In Wt plants, expression of Aox1a is suppressed during the HR response to pv maculicola despite the accumulation of signaling molecules known to induce Aox1a transcription. Also, MnSOD activity declined during the HR. We suggest that the mitochondrial ROS burst controlled by AOX and MnSOD is an important component for the induction and coordination of the HR during plant-pathogen interactions.