Thesis (Ph. D.)--University of Hong Kong, 2007. / Title proper from title frame. Also available in printed format.

Thesis (M.S.)--Northern Michigan University, 2007. / Bibliography: leaves 120-130.

Thesis (Ph. D.)--University of Washington, 2006. / Vita. Includes bibliographical references (leaves 111-123).

The object of this study was to isolate and characterise barley mutants that lacked a functional nitrite reductase activity. This work should complement previous studies on nitrate reductase to develop a fuller understanding of nitrate assimilation in barley. 30 nitrite reduction-deficient M2 barley plants (azide-treated in the M1) were identified as nitrite accumulators after treatment with nitrate. Biochemical analysis M2 selections revealed that leaf tissue from 9 selected plants lacked detectable nitrite reductase protein. Progeny from 4 of the selected plants (Golden Promise 2406, Tweed 3999, Klaxon 1010 and Klaxon 2760) inherited the phenotype of a lack of leaf nitrite reductase protein. These plants also lacked significant in vitro nitrite reductase activity. However, in vitro nitrate reductase and nitrate accumulation were comparable with wild type controls. Root tissue from nitrate-treated progeny of Golden Promise 2406, Tweed 3999, Klaxon 1010 and Klaxon 2760 also lacked nitrite reductase protein. Loss of nitrite reductase protein in selection Tweed 3999 was caused by a single recessive nuclear gene mutation. Thus, these plants are defective in nitrite reduction due to the inherited loss of nitrite reductase protein molecules in both leaf and root after treatment with nitrate in the light. This defect is caused by a mutation within a single nuclear gene in selection Tweed 3999. Analysis of wild type barley cv. Golden Promise revealed that increases in nitrite reductase activity in response to treatment with nitrate and light in leaf tissue and with nitrate in root tissue are due to de novo synthesis of enzyme molecules. In situ immunogold labelling of barley leaf sections with nitrite reductase antiserum demonstrates that the majority of labelling occurs within the chloroplasts.

The object of this research was to isolate nitrate reductase-minus cell-lines of a higher plant species in order to gain information on the genetic control of nitrate reductase production in higher plants. This demanded a culture system consisting of isolated cells or protoplasts, ideally carrying single-copy genetic information. To this end, haploid Nicotiana plants were raised by another culture and abortive attempts were made to culture haploid sylvestris protoplasts. Callus and cell suspension cultures of haploid N. sylvestris also proved unsuitable but eventually dihaploid M. tabacum suspension cultures were obtained which, it was decided, would be suitable for this work. After treatment of these cultures with EMS, 59 cell-lines were selected for resistance to chlorate, four of these were unable to grow on nitrate medium and were later shown to possess an assembled but Inactive nitrate reductase. None of the foul possessed xanthine dehydrogenase activity indicating that they were molybdenum co-factor (Mo-co) defective lines (cnx variants). These cnx lines are different from other Mo-co defective tabacum lines thus far described and are therefore new types, providing evidence that more than one gene locus is involved in the formation of functional Mo-co in N. tabacum nitrate reductase.

No description available.

Note:

published_or_final_version / abstract / Biological Sciences / Doctoral / Doctor of Philosophy

The effects of haem deficiency, as produced by treatment with laevulinic acid, on synthesis/assembly of NR in barley leaves was investigated. Interpretation of results was complicated by instability of NR in vitro therefore the age-dependent stability of NR was investigated. The half-time of loss of NR activity in 4, 5 and 6 day old leaves of barley was found to be 358, 107 and 70 min respectively. BSA, PMSF, and 1,10-phenanthroline stabilised NR in extracts from 5 and 6 day old primary leaves, but BSA was most effective. The increased instability of NR with age of leaf correlated with increased conversion of the MW 203 000 NR complex to small NADH-CR species of MW l63 000, 6l 000 and 4-0 000. The MW 163 000 CR species also possessed NADH-NR activity. BSA prevented and PMSF and 1,10-phenanthroline retarded the conversion of NR to the smaller OR species. The ability of BSA and the proteinase inhibitors to stabilise NR and inhibit conversion of NR to the small CR species indicates that the age-dependent in vitro stability of NR may be due to proteolytic degradation of NR. This suggestion is supported by the observation that trypsin cleaves purified NR into CR species sedimenting with the same coefficients as those observed in crude extracts. In addition, tryptic cleavage was retarded by the presence of BSA. Semi-purified preparations of maize root and barley leaf inactivating factors failed, however, to generate 3-4-S CR species, the maize root inactivator appearing to be active against the 3-4-S CR species in addition to NR. It was concluded that neither factor was responsible for generating the small CR species observed in crude extracts. The 4-0 000 MW CR species was purified and its MW confirmed using the method of Siegal and Monty (I966). The species was shown to possess NET reductase activity but attempts to characterise the species with respect to haem content proved unsuccessful. Antibodies raised against the 40 000 MW CR species were found to inhibit all partial activities of NR and it was therefore concluded that this species, at least, was likely to be derived from NR. Antibodies raised against purified barley NR were found to inhibit all NR activities to a similar extent. Pre-immune serum was found to stimulate NR activity and this was found not to be due to the presence of serum albumin. A protection of inhibition assay was developed for estimating NR-CRM. Negligible NR-CRM was detected in nitrate-less leaf extracts while substantial NR-CRM was found in ammonium-grown plant extracts. Pre-treatment of purified NR with NADH, but not with nitrate, was shown to preserve the enzyme, to some extent, from antibody inhibition. In the General Discussion, evidence available regarding the in vitro breakdown of NR is reviewed and a model for the structure of higher plant NR is presented. In addition the probable route of genetic evolution of NRs is discussed. Evidence for the role of proteolysis in regulation of NR activity in vivo is also analysed.

No description available.