Chan Yee-kwan. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 108-131). / Abstracts in English and Chinese. / Thesis Committee --- p.i / Abstract --- p.ii / Acknowledgments --- p.v / General Abbreviations --- p.vii / Abbreviation for Chemicals --- p.x / Table of Contents --- p.xi / List of Figures --- p.xiv / List of Tables --- p.xv / Chapter 1. --- Literature Review / Chapter 1.1 --- Salinity as a global problem --- p.1 / Chapter 1.2 --- Salinity and agriculture --- p.2 / Chapter 1.3 --- Plant adaptation to salinity --- p.4 / Chapter 1.3.1 --- Salt secretion --- p.6 / Chapter 1.3.2 --- Ion transport --- p.8 / Chapter 1.3.2.1 --- Role of H+-ATPase in salt tolerance --- p.8 / Chapter 1.3.2.2 --- Potassium and sodium uptake --- p.13 / Chapter 1.3.2.3 --- Sodium efflux --- p.15 / Chapter 1.3.3 --- Osmotic adjustment --- p.20 / Chapter 1.3.3.1 --- Accumulation of mannitol --- p.21 / Chapter 1.3.3.2 --- Accumulation of proline --- p.23 / Chapter 1.3.3.3 --- Accumulation of glycinebetaine --- p.23 / Chapter 2. --- Materials and Methods / Chapter 2.1 --- Plant materials and growth conditions --- p.26 / Chapter 2.1.1 --- Surface sterilization of Arabidopsis seeds --- p.26 / Chapter 2.1.2 --- Determination of sub-lethal inhibitory doses of sodium --- p.27 / Chapter 2.1.3 --- Growth conditions of Arabidopsis seeds for total RNA extraction --- p.27 / Chapter 2.1.4 --- NaCl dosage tests --- p.28 / Chapter 2.1.5 --- Expression kinetic tests --- p.28 / Chapter 2.2 --- Isolation of total RNAs --- p.28 / Chapter 2.3 --- Isolation of genes differentially expressed in NaCl concentration by RAP-PCR --- p.30 / Chapter 2.3.1 --- RNA fingerprinting by RAP-PCR --- p.30 / Chapter 2.3.2 --- PCR reamplificatin of RAP products --- p.31 / Chapter 2.3.3 --- Cloning of differentially expressed genes --- p.33 / Chapter 2.3.3.1 --- Ligation of inserts into pCR-Script vector and transformation --- p.33 / Chapter 2.3.3.2 --- Ligation of inserts into pBluescript II KS (+) T-vector and transformation --- p.36 / Chapter 2.3.3.3 --- Screening of recombinant plasmids --- p.37 / Chapter 2.4 --- Sequencing of differentially expressed genes --- p.39 / Chapter 2.4.1 --- DNA cycle sequencing --- p.39 / Chapter 2.5 --- Northern blot hybridization of NaCl inducible genes --- p.40 / Chapter 2.5.1 --- RNA fractionation by formaldehyde gel electrophoresis --- p.40 / Chapter 2.5.2 --- Northern blotting --- p.41 / Chapter 2.5.3 --- Preparation of single-stranded DIG-labeled PCR probes --- p.41 / Chapter 2.5.3.1 --- Isolation of Total RNA --- p.41 / Chapter 2.5.3.2 --- Primer design --- p.42 / Chapter 2.5.3.3 --- PCR amplification of single-stranded DIG PCR probes --- p.43 / Chapter 2.5.4 --- Hybridization --- p.45 / Chapter 2.5.5 --- Stringency washes --- p.46 / Chapter 2.5.6 --- Chemiluminescent detection --- p.46 / Chapter 3. --- Results / Chapter 3.1 --- Determination of sub-lethal inhibitory doses of sodium --- p.48 / Chapter 3.2 --- Isolation of total RNA from A. thaliana treated with sodium chloride --- p.48 / Chapter 3.3 --- Isolation of genes differentially expressed in sodium concentration by RNA arbitrarily primed polymerase chain reaction RAP-PCR --- p.52 / Chapter 3.3.1 --- Differential cDNA fragments identified by RAP-PCR --- p.52 / Chapter 3.3.2 --- PCR reamplification of RAP products --- p.52 / Chapter 3.3.3 --- Cloning of selected RAP-fragments --- p.62 / Chapter 3.4 --- Nucleotide sequence analysis of selected RAP PCR clones --- p.65 / Chapter 3.5 --- Expression pattern analysis of salt inducible genes by northern blot hybridization --- p.75 / Chapter 3.5.1 --- Preparation of single-stranded digoxigenin (DIG)-labeled probes --- p.75 / Chapter 3.5.2 --- Dosage response of NaCl inducible genes --- p.79 / Chapter 3.5.3 --- Expression kinetics of NaCl inducible genes --- p.80 / Chapter 4. --- Discussion / Chapter 4.1 --- Isolation of RAP-PCR targets --- p.93 / Chapter 4.2 --- Expression of NaCl inducible P450 genes --- p.94 / Chapter 4.2.1 --- Cytochrome P450 CYP73A5 --- p.97 / Chapter 4.2.2 --- Cytochrome P450 CYP83A1 --- p.98 / Chapter 4.3 --- NaCl induction gene related to post-transcriptional activities --- p.99 / Chapter 4.3.1 --- Glycine-rich RNA binding protein (BAC F3F19) --- p.100 / Chapter 4.3.2 --- Chloroplast signal recognition particle (54CP) --- p.103 / Chapter 4.4 --- Conclusion --- p.106 / References --- p.108

Chan Hiu-ki. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2000. / Includes bibliographical references (leaves 97-112). / Abstracts in English and Chinese. / Thesis Committee --- p.i / 摘要 --- p.ii / Abstract --- p.iii / Acknowledgements --- p.v / Abbreviations --- p.vi / Table of Contents --- p.vii / List of figures --- p.xi / List of tables --- p.xiii / Chapter Chapter 1 --- Literature Review --- p.1 / Chapter 1.1 --- Nitrogen assimilation in plants --- p.1 / Chapter 1.2 --- Importance of asparagine in plants --- p.5 / Chapter 1.3 --- Enzymatic reaction of asparagine synthetase (AS) --- p.8 / Chapter 1.4 --- Asparagine synthetase of non-plant organisms --- p.10 / Chapter 1.5 --- Biochemistry background of plant asparagine synthetases --- p.12 / Chapter 1.6 --- Molecular studies of asparagine synthetase genes in plants --- p.15 / Chapter 1.7 --- Arabidopsis thaliana as a model plant --- p.24 / Chapter 1.8 --- ASN studies in Arabidopsis thaliana --- p.24 / Chapter 1.9 --- Hypothesis --- p.27 / Chapter Chapter 2 --- Materials and Methods --- p.29 / Chapter 2.1 --- Chemicals --- p.29 / Chapter 2.2 --- Plant materials and growth conditions --- p.29 / Chapter 2.2.1 --- Surface sterilization of Arabidopsis seeds --- p.29 / Chapter 2.2.2 --- "Growth conditions of Arabidopsis seeds for total RNA extraction, enzyme assay, chlorophyll content measurement and dry weight measurement" --- p.30 / Chapter 2.3 --- Agrobacterium mediated transformation via vacuum infiltration method --- p.30 / Chapter 2.3.1 --- Principles --- p.30 / Chapter 2.3.2 --- Plant materials and bacterial strains of Agrobacterium mediated transformation --- p.31 / Chapter 2.3.2.1 --- Plant materials --- p.31 / Chapter 2.3.2.2 --- Gene constructs --- p.31 / Chapter 2.3.2.2 --- Bacterial strains --- p.32 / Chapter 2.3.3 --- Agrobacterium mediated transformation via vacuum infilitration --- p.32 / Chapter 2.4 --- Screening of transformants --- p.33 / Chapter 2.5 --- DNA and RNA manipulation --- p.34 / Chapter 2.5.1 --- DNA extraction and quantitation --- p.34 / Chapter 2.5.2 --- PCR amplification and detection of transgenes --- p.36 / Chapter 2.5.2.1 --- PCR amplification and detection of transgenes --- p.36 / Chapter 2.5.2.2 --- Primer sequence --- p.37 / Chapter 2.6 --- RNA analysis of transormants --- p.38 / Chapter 2.6.1 --- General introduction --- p.38 / Chapter 2.6.2 --- RNA extraction --- p.39 / Chapter 2.6.3 --- Making single-strand DIG PCR probes --- p.40 / Chapter 2.6.4 --- Quantitation of single-strand DIG-labeled probes --- p.42 / Chapter 2.7 --- Northern blot analysis --- p.42 / Chapter 2.7.1 --- Detection --- p.43 / Chapter 2.7.2 --- Film development --- p.43 / Chapter 2.8 --- "Amino acid, protein, dry weight and total nitrogen analysis" --- p.43 / Chapter 2.8.1 --- Extraction of free amino acids --- p.43 / Chapter 2.8.2 --- Protein assay --- p.44 / Chapter 2.8.3 --- Determination of nitrogen and carbon content in seeds --- p.45 / Chapter 2.8.4 --- Dry weight measurement --- p.45 / Chapter 2.8.5 --- Seed storage protein analyses --- p.45 / Chapter 2.8.6 --- Detection of chlorophyll content --- p.46 / Chapter 2.9 --- Asparagine synthetase activity analysis --- p.46 / Chapter 2.9.1 --- Crude extracts preparation --- p.46 / Chapter 2.9.2 --- AS enzyme assay --- p.47 / Chapter 2.9.3 --- Asparagine content measurement --- p.47 / Chapter 2.10 --- In situ hybridization --- p.48 / Chapter 2.10.1 --- Making cRNA probe --- p.48 / Chapter 2.10.2 --- In situ hybridization --- p.48 / Chapter Chapter 3 --- Results --- p.50 / Chapter 3.1 --- Construction of ASN1 overexpressing lines --- p.50 / Chapter 3.2 --- Changes in nitrogen status during vegetative growth of ASN1 overexpressing lines --- p.53 / Chapter 3.3 --- Changes in nitrogen status during seed development of ASN1 overexpressing lines --- p.55 / Chapter Chapter 4 --- Discussion --- p.76 / Chapter Chapter 5 --- Conclusion --- p.85 / Chapter Chapter 6 --- Perspective --- p.86 / Appendix --- p.87 / References --- p.97

[Truncated abstract] Under prolonged wet and damp conditions, barley grain with low dormancy can germinate precociously, a condition known as preharvest sprouting that causes a number of detrimental effects in grain quality. In particular, preharvest sprouting renders the grain unsuitable for malting. The aim of this study was to take a genomics approach to identify and characterise candidate genes that could be linked to the control of seed dormancy in barley. This thesis developed a bioinformatic strategy that exploited the availability of gene sequences with functional evidence in the model species of Arabidopsis and rice. The bioinformatic strategy integrated phenotypic data (QTL data) and comparative genomics for a targeted approach in identifying candidate genes with a high probability of having a conserved function in cereals. This bioinformatic study identified two candidate genes ERA1 and ABI2 with strong evidence for a role in seed dormancy based on their function in Arabidopsis in abscisic acid (ABA) signal transduction and their co-location to seed dormancy QTLs in Arabidopsis, rice and wheat. In order to establish whether the candidate genes mapped to seed dormancy QTLs in barley, QTL analyses were performed on a double haploid population, not previously studied, developed from a cross between Stirling, a major Australian malting cultivar, and Harrington, a major Canadian malting cultivar. This cross was specifically chosen for this study, as elucidation of chromosomal regions associated with seed dormancy in the background of a malting cultivar would make a significant contribution for the malting industry. '...' Identification of a seed dormancy QTL on the long arm of 3H, in a region syntenic to the wheat chromosome locations of ESTS aligning to the ERA1 and ABI2 genes, laid the foundation for physical and genetic mapping of the candidate genes to investigate whether the genes co-located to the QTL on 3H. Physical mapping of the genes in wheat barley addition lines confirmed their positions on the long arm of 3H. Genetic mapping of the ERA1 gene was performed using a CAPS marker developed in this thesis. The genetic mapping of the ERA1 gene did not place the gene within either of the minor QTLs on 3HL, although segregation distortion may have influenced the map position of this gene. Further investigation is required to resolve the positioning of the ERA1 and ABI2 genes in relation to the 3H seed dormancy QTL. The main outcomes of this study have been 1) identification of candidate genes for further study; 2) identification of QTLs on the long arm of 3H that were previously unknown; 3) demonstration of the potential differences in dormancy that can be achieved through the use of specific gene combinations, highlighting the importance of minor genes and the epistatic interactions that occur between them and; 4) the development of a CAPS marker for the ERA1 gene, which can be used to track the gene in barley breeding programs to observe its association with important agronomic traits. This thesis also pioneered the implementation of several new technologies including multiplex-ready PCR (Hayden et al. 2008) for fluorescence–based SSR genotyping and QTLNetwork (Yang et al. 2008) for statistical analysis of QTLs. Seed dormancy is a complex trait and is likely to involve the interplay of a number of genes that have a role in other developmental and regulatory processes.

Includes bibliographical references (leaves 192-211). Maps major genes or quantitative trait loci associated with zinc nutrition in the vegetative and reproductive tissues of barley (Hordeum vulgare)

1 v. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, Waite Agricultural Research Institute, 1993

Includes bibliographical references (leaves 192-211). / x, 211 leaves : ill. ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Maps major genes or quantitative trait loci associated with zinc nutrition in the vegetative and reproductive tissues of barley (Hordeum vulgare) / Thesis (Ph.D.)--University of Adelaide, Dept. of Plant Science, 2001

"August, 2003" / Includes bibliographical references (leaves 260-292) / v, 294 leaves : ill. (some col.), plates (col.), maps (col.) ; 30 cm. / Title page, contents and abstract only. The complete thesis in print form is available from the University Library. / Thesis (Ph.D.)--University of Adelaide, School of Agriculture and Wine, 2003

Resistance (R) genes, a key factor in determining the resistance of plants, have been shown often to be highly allelic entities existing in duplicated regions of the genome. This characteristic suggests that R-gene acquisition may have arisen through frequent genetic rearrangements as a result of transient, reduced genome stability. Tabacco plants transgenic for a recombination construct exhibited reduced genome stability upon infection with a virulent pathogen (tobacco mosaic virus). The reduced genome stability manifested as an increase in recombination events in the transgene. Such increases were observed following a virulent pathogen attack. This increase in recombination was shown to be systemic and was observed prior to systemic viral movement suggesting the presence of a systemic recombination signal. Further molecular analyses revealed that specific R-gene loci experience a large frequency of rearrangements following a virulent pathogen encounter. The possible targeting of instability to R-gene regions may be controlled through epigenetic processes, in particular, DNA methylation. / xiii, 119 leaves ; 29 cm.

Many plants accumulate organic osmolytes in response to the imposition of environmental stresses that cause cellular dehydration. Of these, proline is the most extensively studied. Conclusive demonstration that this imino acid acts as a compatible solute which mediates osmotic adjustment has yet to be achieved, although a causal relationship between increased proline synthesis and plant tolerance of hyperosmotic stresses has previously been demonstrated. It is proposed that in many plants, the metabolic implications of the regulated increase in proline synthesis and/or a decline in proline degradation during stress may play a more important role in acclimation to adverse conditions than the simple accumulation of the end-product of these adjustments. In particular, the stress-induced increase in the transfer of reducing equivalents into proline by Δ¹-pyrroline-5-carboxylate (P5C) synthetase (P5CS) and P5C reductase (P5CR) may be a protective mechanism whereby many species ameliorate shifts in cellular redox potential which accompany all biotic and abiotic stresses which cause proline accumulation, including those that do not cause cellular dehydration. The presence of several putative stress-regulated promoter elements in the AtP5CS1, AtP5CS2 and AtP5CR genes of Arabidopsis thaliana strongly implicates an adaptive role for stress-induced increases in proline synthesis in this species. Sequence homologies of several regions within the 5' untranslated regions of these genes to promoter elements which have been shown to participate in redox control of gene expression, the actions of phytochrome and hormones, and tissue-specific regulation of gene expression are also identified. These provide useful indicators both of the mechanisms by which proline synthesis is regulated and how these may relate to its importance in maintaining metabolic homeostasis. In an attempt to resolve the functionality of proline accumulation under stress, chimeric antisense genes comprising 1050 bp and 999 bp fragments of Arabidopsis cDNAs encoding AtP5CS1 and AtP5CR respectively were inserted in the reverse orientation between the CaMV 35S promoter and the GUS gene (encodes β-glucuronidase) in the plant transformation vector pBI121. These constructs were introduced separately into Arabidopsis by cocultivation with Agrobacterium tumefaciens strains carrying the pBI-P5CS1 (AS) and pBI-P5CR(AS) plasmids. Transgenic plants, which were selected on the basis of kanamycin resistance, regenerated at a low frequency in the presence of 1 mM proline. Transformation of 13 pBI-P5CS1(AS) and 7 pBI-P5CR(AS) lines was confirmed by PCR-mediated amplification of gene fragments within the introduced T-DNA. Segregation ratios for kanamycin resistance indicated that most of the lines have multiple T-DNA insertions. Transformants were characterised with respect to their growth rates and free proline content. In at least two pBI-P5CS1(AS) transformants and two pBI-P5CR(AS) transformants, a reduction in root growth rates in the presence of inhibitory concentrations of NaCI correlated with reduced β-glucuronidase activity relative to transgenic lines that were no more sensitive to NaCI than were controls. A reduction in root growth rate both in the absence and presence of hyperosmotic stress was noted in two pBI-P5CS1(AS) transformants, designated A5 and B12. In 14 day-old plants of the T₂ generation of both A5 and B12, free proline levels were significantly lower than in wild-type plants both in the absence of stress and following 24 h incubation in either 250 mM NaCI or 550 mM sorbitol or at 5 °C. In both lines, reduced growth rates in the absence of osmotic stress could be restored by exogenous proline, but not by exogenous glutamate. When used at isosmotic concentrations, sorbitol caused a larger reduction in free proline levels in both A5 and B12 than did NaCI. This observation may relate to an ABM-mediated post-transcriptional effect on AtP5CS1 gene expression which affects NaCI-, but not sorbitol-mediated proline accumulation in Arabidopsis. Post-transcriptional regulation of the expression of the genes involved in proline biosynthesis may account, at least partly, for the absence of dramatic phenotypic effects in any of the pBI-P5CS1(AS) or pBI-P5CR(AS) lines. Under the premise that regulation of shifts in proline metabolism regulate cellular redox potential under conditions of stress may be mirrored by the involvement of proline metabolism in modulating metabolism during normal growth and development, the effects of exogenous proline on Arabidopsis seed germination, seedling growth and in vitro shoot organogenesis were investigated. A dose-dependent inhibition of radicle emergence by millimolar concentrations of proline could be overcome by the artificial oxidants methylene blue and phenazine ethosulphate. Assays of the rate-limiting dehydrogenases of the oxidative pentose phosphate pathway (OPPP), as well as changes in the contributions of ¹⁴C₁ - and ¹⁴C₆ -labelled glucose to respired CO₂ during germination, are consistent with activation of the OPPP during Arabidopsis seed germination. An approximately four-fold increase in free proline, which peaked at the time of radical emergence, was not parallelled by changes in other amino acids and could not be ascribed to degradation of seed storage proteins. Delayed radical emergence in T₂ generation seeds of the pBI-P5CS1(AS) lines A5 and B12 correlated with an approximately 35% reduction in the maximal concentration of proline accumulated during germination. Millimolar concentrations of exogenous proline had a dose-dependent inhibitory effect on Arabidopsis seedling growth both in the light and in darkness. This reduction in growth arises at least in part from a decline in cell elongation. Accordingly, exogenous proline increased total extractable peroxidase activity in Arabidopsis seedlings through the selective induction of peroxidase isoforms. Histochemical analysis of the hypocotyls of plants grown in the presence of exogenous proline suggested that proline increased the levels of lignin and/or the phenolic precursors thereof. A dose-dependent decrease in extractable chlorophyll and damage to chloroplastic and mitochondrial ultrastructure was observed in 21 day-old Arabidopsis seedlings grown in the presence of millimolar concentrations of exogenous proline. In vitro shoot organogenesis from Arabidopsis hypocotyl explants was stimulated by 1 mM proline, and to a lesser extent by 5 mM proline, but inhibited by inclusion of 10 mM proline in the hormonallysupplemented regeneration media. The ability of low concentrations of proline analogues (azetidine-2-carboxylate and thioproline) to overcome the stimulatory effect of 1 mM proline, and a slight increase in the stimulative effects of 1 mM proline by D-proline, are consistent with an important role for the interconversions of proline and its precursors in regulating cell division and differentiation. Together, these data strongly support an important role for the interconversions of proline and its precursors in the regulation of intermediary metabolism under both normal and stressful conditions. These findings draw into question the widely accepted, although poorly investigated, hypothesis that proline is an inert compatible solute that can be accumulated to high levels with minimal effects on cellular metabolism. The novel proposal that stress-induced changes in proline metabolism exert a regulatory effect though an influence on the level of reduction of the cellular NADP pool is discussed in relation to recent evidence that a signal related to proline synthesis and/or degradation selectively increases the expression of stress-induced plant genes. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1998.

Content removed due to copyright: Young, C. A., Bryant, M. K., Christensen, M. J., Tapper, B. A., Bryan, G. T., & Scott, B. (2005). Molecular cloning and genetic analysis of a symbiosis-expressed gene cluster for lolitrem biosynthesis from a mutualistic endophyte of perennial ryegrass. Molecular Genetics and Genomics, 274(1), 13-29. / Lolitrems are indole-diterpene alkaloids produced by Epichloë and Neotyphodium endophytes in association with their host grass Lolium perenne. Some indole-diterpene (ID) alkaloids are proposed to have insecticidal properties, but lolitrem B is known as the causative agent of the animal syndrome ryegrass staggers. Lolitrems are preferentially synthesised in planta. which suggests that the genes required for lolitrem biosynthesis are symbiotically expressed. The lolitrem biosynthesis pathway has been proposed as a metabolic grid based on the identification of likely intermediates from endophyte-infected ryegrass. Closely related ID compounds are expected to serve as substrates for the same enzyme, but until recently these steps had not been validated. The identification and characterisation of a Petticillium paxilli gene cluster required for the synthesis of the ID paxilline has identified key enzymes required for the production of the ID backbone. Based on the similarity of lolitrem B to paxilline it was proposed that these two biosynthesis pathways would share orthologous early steps but later steps to convert paxilline to the more complex lolitrem B would require additional enzymes. The lolitrem biosynthesis genes (ltm) were isolated using degenerate PCR and from candidate genes identified as ESTs in cDNA libraries. Ten ltm genes were identified that had functions consistent with those required for lolitrem B biosynthesis. The 10 ltm genes were contained on three gene clusters that are separated by repetitive AT-rich sequences that contain remnants of retrotransposons. The ltm clusters 1 and 2 contain eight genes, seven of which are orthologues of the characterised P. paxilli paxilline biosynthesis gene cluster (pax). Functional characterisation of ltmM an FAD-dependent monooxygenase and ltmC a prenyl transferase confirmed these two genes were required for ID biosynthesis and were orthologues of paxM and paxC, respectively. All 10 ltm genes have similar expression profiles and were highly expressed in planta where the production of lolitrem B is most prevalent. The taxonomic distribution of the ltm genes has established which endophyte strains are likely to produce ID compounds. This work provides the basis for elucidation of the lolitrem biochemical pathway and opens the way for determining how the plant regulates the synthesis of this important group of bioprotective molecules.