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Tsai Sau-Na. / Thesis submitted in: 2003. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2004. / Includes bibliographical references (leaves 157-170). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Acknowledgements --- p.vii / General Abbreviations --- p.ix / Abbreviations of Chemicals --- p.xii / Table of contents --- p.xiv / List of figures --- p.xx / List of tables --- p.xxii / Chapter 1. --- Literature Review --- p.1 / Chapter 1.1 --- Salinization is a global problem --- p.1 / Chapter 1.2 --- Causes of soil salinization in agricultural lands --- p.2 / Chapter 1.3 --- Toxicity of salinity in plants --- p.4 / Chapter 1.3.1. --- Physiological drought --- p.4 / Chapter 1.3.2. --- Nutritional imbalance --- p.5 / Chapter 1.3.3 --- Specific ion toxicity --- p.6 / Chapter 1.4 --- Plant adaptation to salinity --- p.7 / Chapter 1.5 --- Ion transport proteins in plant --- p.10 / Chapter 1.5.1 --- "Pump, channel and carrier" --- p.10 / Chapter 1.5.2 --- Pumps --- p.11 / Chapter 1.5.2.1 --- P-ATPase --- p.11 / Chapter 1.5.2.2 --- V-ATPase --- p.12 / Chapter 1.5.2.3 --- PPiase --- p.12 / Chapter 1.5.3 --- Cation channels --- p.13 / Chapter 1.5.3.1 --- K+ channels --- p.13 / Chapter 1.5.3.1.1 --- Shaker family --- p.14 / Chapter 1.5.3.1.1.1 --- KIRCs --- p.16 / Chapter 1.5.3.1.1.2 --- KORCs --- p.17 / Chapter 1.5.3.1.1.3 --- VICs --- p.18 / Chapter 1.5.3.1.2 --- Kir family --- p.18 / Chapter 1.5.3.1.2 --- KCO family --- p.19 / Chapter 1.5.3.2 --- Ca2+ channels --- p.20 / Chapter 1.5.3.2.1 --- TPC family --- p.20 / Chapter 1.5.3.2.2 --- CNGC family --- p.21 / Chapter 1.5.4 --- Anion Channels --- p.22 / Chapter 1.5.5 --- Carriers --- p.23 / Chapter 1.5.5.1 --- High affinity K+ carriers --- p.23 / Chapter 1.5.5.1.1 --- HKT transporter --- p.24 / Chapter 1.5.5.1.2 --- HAK/KUP transporter --- p.25 / Chapter 1.5.5.2 --- Cation/H+ antiporters --- p.26 / Chapter 1.5.5.2.1 --- Na+/H+ antiporter --- p.27 / Chapter 1.5.5.2.2 --- Ca2+/H+ antiporters --- p.30 / Chapter 1.6 --- Ion homeostasis and salt tolerance --- p.31 / Chapter 1.6.1 --- Ion transporters involved in ion homeostasis during salt stress --- p.31 / Chapter 1.6.2 --- Sodium uptake under salt stress --- p.32 / Chapter 1.6.4 --- Sodium extrusion --- p.36 / Chapter 1.6.5 --- Intracellular compartmentation --- p.37 / Chapter 1.6.6 --- Genetic engineering of ion transporter for improvement of salt tolerance --- p.40 / Chapter 1.7 --- Soybean as a target for studies of salt tolerance --- p.41 / Chapter 1.7.1 --- Economic importance of soybean --- p.41 / Chapter 1.7.2 --- Salt tolerant soybean in China --- p.43 / Chapter 1.7.3 --- Previous studies of Wenfeng7 and Union in our laboratory --- p.43 / Chapter 1.7.4 --- Hypothesis and research strategy of my project --- p.46 / Chapter 2. --- Materials and methods --- p.49 / Chapter 2.1 --- Materials --- p.49 / Chapter 2.1.1. --- Plant materials --- p.49 / Chapter 2.1.2. --- Bacteria strains and plasmid vectors --- p.50 / Chapter 2.1.3. --- Growth media for soybeans and A. thaliana --- p.50 / Chapter 2.1.4. --- Chemicals and reagents used --- p.50 / Chapter 2.1.5. --- Solutions used --- p.51 / Chapter 2.1.6. --- Commercial kits used --- p.51 / Chapter 2.1.7. --- Equipment and facilities used --- p.51 / Chapter 2.1.8. --- Primers used --- p.51 / Chapter 2.2 --- Methods --- p.52 / Chapter 2.2.1 --- Cloning of ion transporters --- p.52 / Chapter 2.2.1.1. --- Sample preparation --- p.52 / Chapter 2.2.1.2 --- Total RNA extraction --- p.52 / Chapter 2.2.1.3 --- Primer design for RACE --- p.53 / Chapter 2.2.1.4 --- 5´ة& 3´ة RACE of ion transporters --- p.54 / Chapter 2.2.1.5 --- Subcloning of RACE cDNA fragments --- p.56 / Chapter 2.2.1.6 --- PCR screening of white colonies --- p.57 / Chapter 2.2.1.8 --- Preparation of recombinant plasmid for sequencing --- p.57 / Chapter 2.2.1.9 --- Sequencing and homology search --- p.58 / Chapter 2.2.1.10 --- Cloning of full length coding regions of ion transporters --- p.58 / Chapter 2.2.1.11 --- "Sequence comparison, analysis and multialignment" --- p.62 / Chapter 2.2.2 --- Gene expression profiles --- p.62 / Chapter 2.2.2.1 --- Sample stepwise treatment with different concentration of NaCl --- p.62 / Chapter 2.2.2.2 --- Sample treatment with different Hoagland's solution supplement with 1.2% NaCl --- p.63 / Chapter 2.2.2.3 --- Preparation of single-stranded DIG-labeled PCR probes --- p.64 / Chapter 2.2.2.4 --- Testing the concentration of DIG-labeled probes --- p.65 / Chapter 2.2.2.5 --- Northern blot technique --- p.66 / Chapter 2.2.2.6 --- RT-PCR (Reverse-transcription polymerase chain reaction) --- p.67 / Chapter 2.2.3 --- Functional test using transgenic plants --- p.68 / Chapter 2.2.3.1 --- Preparation of chimeric gene constructs and recombinant plasmids --- p.68 / Chapter 2.2.3.2 --- "Eletroporation of Agrobacterium, tumefaciens" --- p.69 / Chapter 2.2.3.3 --- Seed sterilization and plant growth --- p.70 / Chapter 2.2.3.4 --- Vacuum infiltration transformation of Arabidopsis thaliana --- p.71 / Chapter 2.2.3.5 --- Selection of hemizygous and homozygous transgenic plants --- p.72 / Chapter 2.2.3.6 --- Genomic DNA extraction and PCR screening --- p.72 / Chapter 2.2.3.7 --- RT-PCR and Northern Blot of transgenic plants --- p.73 / Chapter 2.2.3.8 --- Functional test on MS plate supplemented with NaCl --- p.73 / Chapter 2.2.3.9 --- Functional test on sand supplemented with Hoagland's solution and NaCl --- p.74 / Chapter 3. --- Results --- p.76 / Chapter 3.1 --- "Cloning of Nhx, AKT1 and CLC from Wenfeng7 and Union" --- p.76 / Chapter 3.1.1 --- "Cloning of 5'- & 3'- RACE cDNA fragments of Nhx, AKT1 and CLC" --- p.76 / Chapter 3.1.2 --- "Cloning of full length coding regions of Nhx, AKT1 and CLC from Wenfeng7 and Union" --- p.77 / Chapter 3.1.3 --- "Sequence comparison, analysis and multialignment" --- p.82 / Chapter 3.1.3.1 --- Sequence analysis and multialignment of GmNhx1 and GmNhx2 --- p.82 / Chapter 3.1.3.2 --- Sequence analysis and multialignment of GmAKTl --- p.92 / Chapter 3.1.3.3 --- Sequence analysis and multialignment of GmCLC --- p.101 / Chapter 3.2 --- "Gene expression profiles of GmNhx, GmCLC and GmAKTl" --- p.111 / Chapter 3.2.1 --- Induction of GmNhx and GmCLC gene expression by NaCl in different Hoagland's solution --- p.111 / Chapter 3.2.2 --- RT-PCR using gene specific primers to distinguish the gene expression of GmNhx1 and GmNhx2 --- p.116 / Chapter 3.2.3 --- RT-PCR analysis of the transcripts of GmAKTl in Wenfeng7 and Union --- p.118 / Chapter 3.3 --- Functional analysis of transgenic plants in salt tress --- p.120 / Chapter 3.3.1 --- "Construction of chimeric gene of GmNhx1´ة GmNhx2, GmCLC and GmAKT1 into V7 vector" --- p.120 / Chapter 3.3.2 --- Transformation of chimeric gene constructs into A. tumefaciens --- p.122 / Chapter 3.3.3 --- Vacuum infiltration transformation of Arabidopsis thaliana and selection of transgenic plants --- p.123 / Chapter 3.3.4 --- PCR screening of transgene from transgenic plants --- p.130 / Chapter 3.3.5 --- PT-PCR and Northern blot analysis of the transgene transcripts --- p.133 / Chapter 3.3.6 --- Functional test of transgenic plants under salt stress --- p.135 / Chapter 4. --- Discussion --- p.139 / Chapter 4.1 --- "Isolation of GmNhx, GmAKTl and GmCLC from Wenfeng7 and Union" --- p.139 / Chapter 4.1.1. --- GmNhx1 and GmNhx2 are putative vacuolar Na+/H+ antiporters from Wenfeng7 and Union --- p.139 / Chapter 4.1.2. --- GmAKT1 is an inward-rectifying K+ channel from Wenfeng7 and Union --- p.141 / Chapter 4.1.3 --- GmCLC is a putative vacuolar voltage-dependent chloride channel from Wenfeng7 and Union --- p.144 / Chapter 4.2 --- "Gene expression profiles of GmNhx, GmAKT1 and GmCLC from Wenfeng7 and Union" --- p.146 / Chapter 4.2.1 --- Differential expression between GmNhx1 and GmNhx2 in Wenfeng7 and Union --- p.146 / Chapter 4.2.2 --- Coordinated expression of GmNhx and GmCLC in wenfeng7 and Union --- p.147 / Chapter 4.2.3 --- GmAKT1 is preferentially expressed in roots of wenfeng7 and Union and presented in low abundance --- p.148 / Chapter 4.3 --- Functional tests of transgenic Arabidopsis plants --- p.150 / Chapter 4.3.1 --- Screening of heterozygous and homozygous transgenic plant --- p.150 / Chapter 4.3.2 --- Function tests of heterozygous and homozygous transgenic plants under salt stress --- p.151 / Chapter 4.3.3 --- Gene silencing in transgenic plants --- p.152 / Chapter 5. --- Conclusion and perspectives --- p.155 / References --- p.157 / "Appendix I: Buffer, restriction and modifying enzymes" --- p.171 / Appendix II: Major chemicals and reagents used in this research --- p.171 / Appendix III: Major common solutions used in this research --- p.174 / Appendix IV: Commercial kits used in this research --- p.177 / Appendix V: Major equipment and facilities used --- p.177

BURP域家族是植物特有的一個蛋白家族，結構多樣，共同點是在羧基端都有一個保守的BURP域。迄今為止，關於BURP域家族成員的功能及細胞定位的研究非常有限。部分RD22-like的亞家族成員由於顯示出受非生物脅迫而誘導表達的特性，因此被認為功能可能與非生物脅迫回應相關。本研究對克隆到的一個受非生物脅迫誘導表達的大豆基因(GmRD22)進行了生物進化分析，并詳細分析了其在不同的大豆品種以及不同非生物脅迫下的表達模式，揭示了其表達豐度與大豆的抗非生物脅迫能力有關，並且使用不同的轉基因系統(細胞水準跟植物水準)揭示了其過量表達有助於減輕非生物脅迫對植物造成的影響。研究利用GFP融合蛋白追蹤技術和免疫電鏡技術揭示GmRD22蛋白定位於細胞壁，其中BURP域對於GmRD22定位于細胞壁起到關鍵作用。研究也揭示了GmRD22能夠與細胞外的一種過氧化物酶GmPer1相互作用，GmRD22在轉基因擬南芥和轉基因水稻中的過量表達能夠顯著提高脅迫條件下轉基因植株中木質素的含量。我們認為GmRD22通過與細胞壁過氧化物酶的相互作用來提高植物在脅迫條件下細胞壁的完整性從而增強植株的抗性。 / The BURP-domain protein family comprises a diverse group of plant-specific proteins that share a conserved BURP domain at the C terminus. However, there have been only limited studies on the functions and subcellular localization of these proteins. Members of the RD22-like subfamily are postulated to associate with stress responses due to the stress-inducible nature of some RD22-like genes. In this report, different expression patterns of a stress-inducible RD22-like protein from soybean (GmRD22) either in different soybean species or under different osmotic stress conditions were analyzed, different transgenic systems (cells and in planta) were used to show that the ectopic expression of GmRD22 can alleviate salinity and osmotic stress. The detailed microscopic studies were also performed using both fusion proteins and immuno-electron microscopic techniques to demonstrate the apoplast localization of GmRD22, for which the BURP domain is a critical determinant of the subcellular localization. The apoplastic GmRD22 interacts with a cell wall peroxidase and the ectopic expression of GmRD22 in both transgenic A. thaliana and transgenic rice resulted in increased lignin production when subjected to salinity stress. It is possible that GmRD22 regulates cell wall peroxidase and hence strengthens cell wall integrity under osmotic stress conditions. / Detailed summary in vernacular field only. / Wang, Hongmei. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2012. / Includes bibliographical references (leaves 124-136). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese. / Abstract --- p.i / Acknowledgements --- p.iii / Table of contents --- p.v / List of tables --- p.x / List of figures --- p.xi / General abbreviations --- p.xiii / Chemical abbreviations --- p.xv / Chapter Chapter 1 --- Introduction --- p.1 / Chapter 1.1 --- Abiotic stress in the world --- p.2 / Chapter 1.2 --- The advances of plant abiotic stress resistance mechanisms --- p.5 / Chapter 1.2.1 --- Sensor of salt stress --- p.7 / Chapter 1.2.2 --- Reestablishment of ionic homeostasis --- p.9 / Chapter 1.2.3 --- Osmoregulation by compatible osmolytes --- p.11 / Chapter 1.2.4 --- Oxidative stress management --- p.12 / Chapter 1.2.5 --- Transcription regulation of gene expression in osmotic stress --- p.14 / Chapter 1.3 --- The BURP-domain protein family --- p.19 / Chapter 1.3.1 --- Introduction of BURP-domain protein family --- p.19 / Chapter 1.3.2 --- Advances of BURP-domain protein studies --- p.20 / Chapter 1.3.3 --- The BURP-domain protein and osmotic stress --- p.21 / Chapter 1.4 --- Background information of this project --- p.22 / Chapter 1.5 --- Hypothesis and objectives --- p.25 / Chapter Chapter 2 --- Materials and Methods --- p.26 / Chapter 2.1 --- Bacterial strains, vectors and plasmids, cell lines and plant materials --- p.27 / Chapter 2.2 --- Chemicals and reagents --- p.32 / Chapter 2.3 --- Primers used in this study --- p.35 / Chapter 2.4 --- Molecular cloning of GmRD22 --- p.38 / Chapter 2.5 --- DNA and RNA extraction and Northern blot --- p.40 / Chapter 2.5.1 --- DNA and plasmid extraction --- p.40 / Chapter 2.5.2 --- RNA extraction from plant --- p.41 / Chapter 2.5.3 --- Generation of DIG-labeled PCR probe --- p.41 / Chapter 2.5.4 --- Northern blot --- p.43 / Chapter 2.6 --- Reverse transcription and Real-time analysis --- p.44 / Chapter 2.7 --- Phylogenetic analysis --- p.45 / Chapter 2.8 --- Basic molecular techniques --- p.46 / Chapter 2.8.1 --- Recombinant DNA --- p.46 / Chapter 2.8.2 --- Transformation of E. coli competent cells --- p.46 / Chapter 2.8.3 --- Transformation of A. tumefacien competent cells --- p.47 / Chapter 2.8.4 --- Gel electrophoresis --- p.48 / Chapter 2.8.5 --- Sequencing --- p.48 / Chapter 2.9 --- Establishment of transgenic models --- p.49 / Chapter 2.9.1 --- Establishment of transgenic BY-2 cell --- p.49 / Chapter 2.9.2 --- Establishment of transgenic A. thaliana --- p.50 / Chapter 2.9.3 --- Establishment of transgenic rice --- p.51 / Chapter 2.10 --- Cell viability assay under osmotic stress treatment --- p.51 / Chapter 2.11 --- Root elongation assay of transgenic A. thaliana --- p.52 / Chapter 2.12 --- Osmotic stresses treatment of transgenic rice lines --- p.52 / Chapter 2.13 --- Protein expression, production of antibodies and Western blot --- p.53 / Chapter 2.14 --- Subcellular localization of fusion protein by confocal microscopic study --- p.55 / Chapter 2.15 --- Electron microscopic study --- p.56 / Chapter 2.16 --- Immunoprecipitation and mass spectrometry --- p.57 / Chapter 2.17 --- Cell wall components analysis --- p.60 / Chapter 2.18 --- Statistical analysis --- p.61 / Chapter Chapter 3 --- Results --- p.62 / Chapter 3.1 --- GmRD22 gene --- p.63 / Chapter 3.1.1 --- GmRD22 encodes a BURP-domain protein in soybean --- p.63 / Chapter 3.1.2 --- Phylogenetic analysis of GmRD22 --- p.65 / Chapter 3.2 --- GmRD22 gene expression --- p.73 / Chapter 3.2.1 --- GmRD22 shows a biphasic induction by salinity stress and ABA treatment --- p.73 / Chapter 3.2.2 --- GmRD22 is also inducible by osmotic stress --- p.76 / Chapter 3.2.3 --- GmRD22 shows stronger and faster induction in WF 7 than Union --- p.76 / Chapter 3.3 --- Functional study --- p.78 / Chapter 3.3.1 --- Construction of GmRD22 transformants --- p.78 / Chapter 3.3.2 --- Ectopic expression of GmRD22 improve osmotic stresses tolerance in transgenic BY-2 cells --- p.80 / Chapter 3.3.3 --- Ectopic expression of GmRD22 alleviates osmotic stresses in transgenic A. thaliana --- p.83 / Chapter 3.3.4 --- Ectopic expression of GmRD22 alleviates osmotic stresses in transgenic rice --- p.86 / Chapter 3.4 --- GmRD22 is an apoplastic protein --- p.90 / Chapter 3.4.1 --- Western blot analysis in different soybean extracts --- p.90 / Chapter 3.4.2 --- Subcellular localization of GmRD22-GFP fusion protein in onion epidermal and A. thaliana root system --- p.93 / Chapter 3.4.3 --- GmRD22 localization in native soybean --- p.96 / Chapter 3.5 --- BURP domain is essential for the subcellular localization --- p.99 / Chapter 3.6 --- GmRD22 interacts with a putative apoplastic peroxidase --- p.103 / Chapter 3.6.1 --- Identification of GmRD22 interacting protein --- p.103 / Chapter 3.6.2 --- GmPer1 is a putative extracellular class III peroxidase --- p.107 / Chapter 3.6.3 --- Overexpression of GmRD22 affected lignin metabolism in transgenic rice and A. thaliana under salinity stress --- p.109 / Chapter 3.6.4 --- GmPer1 homologues increased expression under salinity stress --- p.113 / Chapter Chapter 4 --- Discussion and Conclusion --- p.115 / Chapter 4.1 --- GmRD22 as a member of RD22-like subfamily --- p.116 / Chapter 4.2 --- Induction mechanism of GmRD22 expression is related to ABA --- p.117 / Chapter 4.3 --- Biological function of GmRD22 providing protective effect under osmotic stress --- p.118 / Chapter 4.4 --- The BURP domain of GmRD22 plays a key role in its apoplastic targeting --- p.119 / Chapter 4.5 --- The interaction between GmRD22 and apoplastic peroxidase provides the clue for the mechanism of enhanced osmotic stress tolerance in GmRD22 transgenic plants --- p.120 / Chapter 4.6 --- Conclusion --- p.123 / References --- p.124 / Chapter Appendix I --- Restriction and modifying enzymes --- p.137 / Chapter Appendix II --- Chemicals --- p.138 / Chapter Appendix III --- Buffer, solution, gel and medium formulation --- p.143 / Chapter Appendix IV --- Equipment and facilities --- p.146

Thesis (Ph.D.)--Virginia Commonwealth University, 2009. / Prepared for: Dept. of Chemistry. Title from resource description page. Includes bibliographical references.

Protein co-precipitates were prepared from whey powder and soybean flour using various extraction and co-precipitation techniques. The effect of extraction and co-precipitation on co-precipitate yield was investigated. Native and sodium dodecyl sulfate polyacrylamide gel electrophoresis (Native-PAGE, SDS-PAGE) and light compound microscopy (LCM) were used to study the structure of the co-precipitates. The rheological and gelation properties of the co-precipitates were determined. Highest yield (45%) for NaOH/Isoelectric Point IEP-Heating-Cooling, co-precipitate was obtained using the following conditions of extraction; extraction temperature, 40°C; temperature of precipitation 95°C, and pH of precipitation was 4.5. The yield of co-precipitates was affected by chelating agents and pH of precipitation and temperature of precipitation. Native-PAGE showed that 2 new protein bands result from the interactions between whey and soybean proteins during preparation of the co-precipitate. SDS-PAGE showed that the new proteins dissociated to give the protein subunits of whey and soybean proteins. LCM results showed differences in microscopic structure between the whey and soybean protein precipitates and the protein co-precipitates. Gels were characterized by measurement of water holding capacity (WHC), gelation start temperature (GST) and denaturation start temperature (DST) and gel strength (GS). Gels (16%) from a protein co-precipitate Mixed Powder MP:NaOH/IEP-Cooling had higher WHC and GS than gels from whey protein precipitate, soybean protein precipitate and protein co-precipitates Mixed Extract ME:NaOH/IEP-Cooling and co-precipitates MP: and ME:NaOH/IEP-Heating-Cooling. The DST of protein co-precipitates was dependent on protein concentration and pH, while GST was relatively dependent on protein concentration.

The biochemical changes taking place during soybean seed development and gennination, and some aspects of desiccation tolerance were assessed with reference to lipid peroxidation and antioxidant systems. During nonnal seed development, fresh weight and dry weight increased between 20 and 50 days after flowering (DAF), concomitant with the accumulation of triacylglycerols and sugar reserves, after which dry weight remained almost unchanged, and fresh weight decreased. Seed moisture content decreased rapidly during the last stages of development. High levels of lipid peroxidation were evident between 20 and 45 DAF, and decreased thereafter. An examination of antioxidant systems revealed that whereas total glutathione levels accumulated continuously throughout the 80 days of seed development, both dehydroascorbic acid (DHA) reductase and ascorbate free radical (AFR) reductase increased concurrently with the increase in total ascorbate content, and the overall levels did not decrease markedly during maturation drying. Ascorbate peroxidase (ASC POD) activity was high during the period ofgreatest ascorbate accumulation. Both catalase (CAn and superoxide dismutase (SOD) activities increased progressively during early seed development (20-40 DAF), but showed variable patterns of change during maturational drying, in marked contrast to ASC POD which declined from 40 DAF to undetectable levels at 70 DAF. An assessment of the relationship between the antioxidant systems and lipid peroxidation was made during imbibition and gennination, as it has been suggested that controlling free radicals was a critical event in early imbibition. Unexpectedly, lipid peroxidation increased progressively in both seeds and isolated axes, and were eight-fold higher at 48 hours of imbibition compared to dry tissues. A progressive, and co-ordinated, increase in CAT, total glutathione, total ascorbate pool, guaiacol POD, ASC POD, and SOD appeared to parallel the rise in lipid peroxidation in both whole seeds and axes. Variable responses were evident between seeds and axes for the enzymes AFR reductase and DHA reductase In order to gain a further insight into the dynamics of desiccation-tolerance and desiccationsensitivity, imbibing seeds were subjected to an unscheduled dehydration treatment, and then rehydrated for up to 24 hours. During these hydration-dehydration-rehydration (H-D-R) treatments, changes in lipid peroxidation and antioxidant systems were measured. Concurrent with the loss of viability in the axes of seeds dehydrated after 24 and 36 hours of imbibition, there were increases in both lipid peroxidation and solute leakage. Unscheduled drying was seen to be a critical stage, as intolerant axes showed four- to eightfold increases in lipid peroxidation, which were only partially reduced on subsequent rehydration. Tolerant axes, on the other hand, were able to maintain low, basal levels of lipid hydroperoxides on drying. The relationship between these observations and the antioxidant systems showed that the antioxidant enzymes CAT, ASC POD, AFR reductase, DHA reductase, guaiacol POD and SOD declined markedly during the unscheduled drying, whereas GSH and ASC declined only slightly. On rehydration, most of the enzymes, total glutathione, and total ascorbate pool increased, the only exception being the loss of ASC POD activity. ORA reductase, which was seen to decrease as a part of nonnal gennination, declined progressively also in H-D-R treatments. These results suggested that loss of viability was not attributable to a decline of the antioxidant systems but rather to the combined deleterious effects of increased lipid peroxidation, and a generalized and moderately compromised antioxidant system. These studies have indicated that the occurrence of lipid peroxidation can be seen as a nonnal part of seed development and gennination. The H-D-R studies, on the other hand, supported the concept that the balance between peroxidation reactions and the protective systems was critical to the development of desiccation tolerance. / Thesis (Ph.D.)-University of Natal, Durban, 2003.

Infection of legume roots with Rhizobium species results in the development of a root nodule structure in which the bacteria form an intracellular symbiosis with the plant. It is reported here that the infection of soybean (Glycine max L.) roots with Rhizobium japonicum results in the synthesis by the plant of at least 18-20 polypeptides other than leghemoglobin during the development of root nodules. Identification of these "nodule-specific" host polypeptides (referred to as nodulins) was accomplished by two-dimensional gel analysis of the immunoprecipitates formed by a "nodule-specific" antiserum with in vitro translation products of root nodule polysomes that are free of bacteroidal contaminations. Nodulins account for 7-11% of the total ('35)S-methionine-labeled protein synthesized in the host cell cytoplasm, and the majority of them are of 12,000-20,000 molecular weight. These proteins are absent from the uninfected roots, bacteroids and free-living Rhizobium, and appear to be coded for by the plant genes that may be obligatory for the development of symbiosis in the legume root nodules. Analysis of nodulins in ineffective (unable to fix nitrogen) nodules developed due to Rhizobium strains SM5 and 61A24 showed that their synthesis is reduced and their expression differentially influenced by mutations in rhizobia. / Apart from the low molecular weight nodulins, a 35,000 MW polypeptide present in the nodule cytoplasm was also identified as "nodule-specific". This protein, referred to as nodulin-35, represents about 4% of the total cytoplasmic protein in root nodules, and its appearance is not affected by mutations in several nodulating strains of Rhizobium. Nodulin-35 was not detected in uninfected soybean, bacteroids or free-living Rhizobium, and it appears to be synthesized by the plant during the formation of root nodules. / Whereas the transformation of free-living Rhizobium into bacteroids is accompanied by substantial changes within the population of cytoplasmic proteins, the majority of plant polypeptides from nodules are also present in uninfected (non-nodulated) roots. Hence, to further identify and isolate the "nodule-specific" proteins, it was essential to develop several immunological procedures, including a preparative adsorption of antibodies with antigens, the multiple immunoreplica technique, and isolation of a single-copy mRNA by immunoprecipitation of the nascent peptide-polysome complex, which are described in this thesis. / In addition, two polypeptides of bacterial origin were found to be cross-reactive with the "nodule-specific" antiserum, suggesting that they are secreted into the host cell cytoplasm during symbiotic nitrogen fixation.

Review of the literature suggested possible deficiencies of micronutrients in soils of North Western Zambia. Soil analysis, pot and field experiments were employed to investigate possible deficiencies. The pot experiments investigated how raising soil pH through liming influenced extractable micronutrients and their uptake by plants. Plant Mo and Ca were positively correlated with soil pH, while Mn and Zn were inversely correlated, aggravating the zinc inadequacy on all soils and that for Mn on arenosols. Effects of liming on plant uptakes of micronutrients generally followed the same trends as those on soil extraction. Incubating the soil under grass house conditions was found to influence amounts of extractable micronutrients, increasing most times above their levels before the soil was incubated. Field experiments generally showed that applying micronutrients were beneficial to crop yield only at some sites. Grain yield variables responded variously and were most significantly correlated with overall grain yield. Soil analysis usefully predicted deficiencies of Zn for both maize and soybean. However, predictions for B and Mo were ideal for soybean than maize. Cu also seemed to have been wrongly predicted for soybean. However, plant nutrient concentration was better at predicting nutrient status in relation to grain yield, but the lower limits of the suggested optimal concentration ranges may need to be worked out again. Soybean was found to have more micronutrient latent deficiencies at majority of the sites than maize. One of the characteristics of applied micronutrients was their beneficial residual effects of crop yield. The residual benefit was also noticed on maize when the fertilisers were directly applied to soybeans a season before, suggesting a possibility of crop rotation, thus spreading the costs. Results would suggest changing the current fertiliser recommendations in the region.

The organization and structure of the leghemoglobin (Lb) genes in soybean have been investigated. Using molecular cloning techniques, a Lb-cDNA recombinant molecule, pLb1, was prepared and characterized. Hybridization of this clone of genomic DNA revealed the presence of at least seven Lb genes in the soybean genome. The arrangement of these genes appeared to be the same in DNA isolated from infected or uninfected tissues. The plasmid pLb1 was also used to isolate three Lb sequences from a genomic library constructed in (lamda) Charon4 vector. Nucleotide sequence analysis showed that one Lb gene, present on an 11.5 kilobases (kb) Eco RI genomic fragment, spans about 1 200 nucleotides and appears to code for Lbc(,3). Its coding sequence is interrupted at amino acid positions 32 to 33, 68 to 69 and 103 to 104. The intervening sequences, as well as the 5' and 3' flanking regions of this gene, contain some consensus sequences found in other eukaryotic genes. The length of the 5'-untranslated region is 49 bases as determined by nuclease S1 mapping. R-loop analysis of the recombinant phage containing the 11.5 kb Eco RI fragment showed that another Lb gene is located 2.5 kb away. The nucleotide sequences of the second gene showed that this gene is incomplete, containing only two exons. The deduced amino acid sequence of this gene, although showing 78% homology with the corresponding region of the other Lb gene, is not represented in any of the known Lb proteins. Both genes are oriented in the same direction with respect to the coding strand. Analysis of the sequence present in a second genomic clone containing a 4.2 kb Eco RI fragment revealed a truncated Lb gene showing homology with the last exon and the non-coding region at the 3' end of the two other Lb genes. A very high homology is found among the nucleotide sequence of the Lbc(,3), Lba and Lbc(,1) genes. Comparison of the intervening sequences of these genes indicated that they diverged mainly through the creatio

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