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Call number: LD2668 .T4 1965 H78 / Master of Science

Chow, Cheung-ming. / Thesis submitted in: December 2002. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (leaves 149-155). / Abstracts in English and Chinese. / Thesis committee --- p.i / Statement --- p.ii / Abstract --- p.iii / Acknowledgement --- p.vi / General abbreviations --- p.viii / Abbreviations of chemicals --- p.ix / List of figures --- p.x / List of tables --- p.xiv / Table of contents --- p.xv / Chapter 1. --- Literature review --- p.1-26 / Chapter 1.1 --- General introduction of nitrogen metabolism in plants --- p.1 / Chapter 1.2 --- Interaction between nitrogen metabolism and other metabolic and developmental pathways in plants --- p.2 / Chapter 1.2.1 --- Carbon metabolism --- p.2 / Chapter 1.2.2 --- Development --- p.2 / Chapter 1.2.3 --- Flowering --- p.3 / Chapter 1.3 --- Metabolic signalling in plants --- p.5 / Chapter 1.3.1 --- Nitrogen signalling in plants --- p.5 / Chapter 1.3.1.1 --- Inorganic N signalling --- p.5 / Chapter 1.3.1.2 --- Organic N signalling --- p.6 / Chapter 1.3.2 --- Carbon signalling --- p.7 / Chapter 1.3.2.1 --- Signalling pathways --- p.7 / Chapter 1.3.2.2 --- Gene expression regulated by sugar --- p.8 / Chapter 1.3.2.3 --- Role of sugar signalling in growth and development --- p.9 / Chapter 1.4 --- Ways to elucidate a new signal transduction pathway --- p.10 / Chapter 1.4.1 --- Carbon signalling as a paradigm to provide hints for exploring nitrogen signalling in plants --- p.10 / Chapter 1.4.2 --- Designing long-term approach to tackle nitrogen signalling in plants --- p.14 / Chapter 1.5 --- "Molecular tools available to change the ""signal"" and the proposed ""sensor""" --- p.15 / Chapter 1.5.1 --- ASN1 overexpressing lines (35S-ASN1) --- p.15 / Chapter 1.5.2 --- PII overexpressing lines (PII ox) & PII truncated lines (PII trunc.) --- p.16 / Chapter 1.5.2.1 --- Plant PII and its possible role --- p.16 / Chapter 1.5.2.2 --- Nitrogen/carbon sensing as the proposed in vivo function of PII-like protein in Arabidopsis thaliana by the in planta bioassay of PII overexpressing (PII ox) lines --- p.17 / Chapter 1.5.2.3 --- Changes in physiological and transcriptional expression of nitrogen assimilatory genes in PII transgenic lines --- p.17 / Chapter 1.6 --- Review on nitrogen controls and sensing mechanism of microbial organism and higher plants --- p.21 / Chapter 1.6.1 --- Nitrogen sensing in enteric bacteria --- p.21 / Chapter 1.6.2 --- Nitrogen sensing in cyanobacteria --- p.21 / Chapter 1.6.3 --- Nitrogen sensing in fungi --- p.22 / Chapter 1.6.4 --- Implication of the nitrogen sensing mechanisms in microorganisms to nitrogen sensing in plants --- p.23 / Chapter 1.7 --- "Hypothesis, objectives and outlines of this thesis work" --- p.25 / Chapter 2 --- Materials and Methods --- p.27-50 / Chapter 2.1 --- Materials --- p.27 / Chapter 2.1.1 --- "Plants,bacterial strains and vectors" --- p.27 / Chapter 2.1.2 --- Chemicals and Regents --- p.28 / Chapter 2.1.3 --- "Buffer, solution and gel" --- p.28 / Chapter 2.1.4 --- Commercial kits --- p.28 / Chapter 2.1.5 --- Equipments and facilities used --- p.29 / Chapter 2.1.6 --- Growth medium --- p.29 / Chapter 2.1.7 --- Primers --- p.29 / Chapter 2.2 --- Methods --- p.29 / Chapter 2.2.1 --- Growth condition for plant materials --- p.29 / Chapter 2.2.1.1 --- General conditions --- p.29 / Chapter 2.2.1.2 --- Mature Arabidopsis for gene expression profile --- p.30 / Chapter 2.2.1.3 --- Arabidopsis seedlings for physiological experiment --- p.30 / Chapter 2.2.2 --- Molecular Techniques --- p.31 / Chapter 2.2.2.1 --- Bacterial cultures for recombinant DNA --- p.31 / Chapter 2.2.2.2 --- Preparation of pBluescript II KS(+) T-vector for cloning --- p.31 / Chapter 2.2.2.3 --- Cloning techniques --- p.32 / Chapter 2.2.2.4 --- Transformation of DH5a competent cell --- p.32 / Chapter 2.2.2.5 --- Gel electrophoresis --- p.33 / Chapter 2.2.2.6 --- DNA and RNA extractions from plant tissues --- p.34 / Chapter 2.2.2.7 --- First strand cDNA synthesis --- p.35 / Chapter 2.2.2.8 --- PCR techniques --- p.35 / Chapter 2.2.2.9 --- Sequencing --- p.37 / Chapter 2.2.3 --- Analysis of sequences and homology search --- p.37 / Chapter 2.2.4 --- Biochemical analysis --- p.41 / Chapter 2.2.4.1 --- Sugar content analysis --- p.41 / Chapter 2.2.4.2 --- Anthocyanin content analysis --- p.42 / Chapter 2.2.4.3 --- Fresh weight measurement --- p.43 / Chapter 2.2.4.4 --- Statistic analysis --- p.43 / Chapter 2.2.5 --- Generation of crossing progenies --- p.44 / Chapter 2.2.5.1 --- Artificial crossing of A. thaliana --- p.44 / Chapter 2.2.5.2 --- PCR screening for successful crossing --- p.44 / Chapter 2.2.6 --- Construction of subtractive libraries --- p.45 / Chapter 2.2.7 --- Reverse-dot blot screening --- p.45 / Chapter 2.2.7.1 --- in vitro transcription for making ampicillin cRNA --- p.46 / Chapter 2.2.7.2 --- PCR amplification --- p.47 / Chapter 2.2.7.3 --- Dot-blotting of PCR products on nylon membrane --- p.48 / Chapter 2.2.7.4 --- P probe preparation --- p.49 / Chapter 2.2.7.5 --- Hybridization --- p.49 / Chapter 2.2.7.6 --- Signal detection --- p.50 / Chapter 3 --- Results --- p.51-124 / Chapter 3.1 --- Differential growth behaviour and sugar content in 35S-ASNI lines --- p.51 / Chapter 3.1.1 --- Growth of the seedlings of 35S-ASN1 lines under different N and C supplementations --- p.51 / Chapter 3.1.2 --- Lowered reducing sugar content in 35S-ASN1 lines --- p.52 / Chapter 3.2 --- Development of markers for nitrogen signalling events related to altered N status in 35S-ASN1 lines --- p.60 / Chapter 3.2.1 --- Sugar-induced anthocyanin levels as common morphological marker shared by 35S-ASN1 lines and PII transgenic lines --- p.60 / Chapter 3.2.2 --- Expression markers related to altered N status in 35-ASN1 lines --- p.64 / Chapter 3.3 --- Generation of transgenic plants constitutively expressing ASN1 and GLB1 (or ASN1 and truncated GLB1) through crossing --- p.74 / Chapter 3.4 --- Search for homologs of well-known microbial nitrogen signalling components in Arabidopsis thaliana --- p.78 / Chapter 3.4.1 --- Homologs of yeast general amino acid control components --- p.80 / Chapter 3.4.1.1 --- Arabidopsis thaliana GCN2-like protein --- p.80 / Chapter 3.4.1.2 --- Arabidopsis thaliana GCN1 -like protein --- p.84 / Chapter 3.4.1.3 --- Arabidopsis thaliana GCN20-like protein --- p.84 / Chapter 3.4.1.4 --- Plant eIF2α --- p.86 / Chapter 3.4.1.5 --- Arabidopsis thaliana GCN4-CRE like sequences --- p.87 / Chapter 3.4.2 --- Homologs of fungi nitrogen sensing components: Globally acting factor in nitrogen control in fungi --- p.89 / Chapter 3.4.3 --- Homologs of cyanobacteria nitrogen control components: IF7 & IF 17 (Negative regulators of GS activity) --- p.89 / Chapter 4 --- Discussion --- p.125-147 / Chapter 4.1 --- Differential physiological and morphological behaviours found in the comparative studies between control lines and 35S-ASN1 lines --- p.125 / Chapter 4.1.1 --- in planta promotive effect of ASN1 overexpression on the seedlings growth under low nitrogen and in the absence of exogenous applied metabolizable sugar --- p.125 / Chapter 4.1.2 --- Modulation of sugar level in 35S-ASN1 lines --- p.126 / Chapter 4.2 --- Development of morphological marker and gene expression markers --- p.128 / Chapter 4.2.1 --- Anthocyanin accumulation as a morphological marker for epistatic analysis --- p.128 / Chapter 4.2.2 --- Differential expressed genes as candidates for gene expression markers of nitrogen signalling event --- p.131 / Chapter 4.3 --- Arabidopsis homolog search for well-known microbial signalling components --- p.132 / Chapter 4.3.1 --- "Possible amino acid sensing system in Arabidopsis constructed by homologs of yeast GCN2, GCN1, GCN20 and eIF2a" --- p.132 / Chapter 4.3.1.1 --- Arabidopsis GCN2-like (A. thaliana GCN2-like) protein --- p.132 / Chapter 4.3.1.2 --- Arabidopsis GCNl-like (A. thaliana GCNl-like) & GCN20-like (A. thaliana GCNl-like) proteins --- p.136 / Chapter 4.3.1.3 --- Plant eIF2a phosphorylation pathway --- p.139 / Chapter 4.3.1.4 --- GCN4 related transcriptional factors and GCN4-like motif (GLM) cis-element in plants --- p.140 / Chapter 4.3.1.5 --- Implication of the presence of plant homologs of fungi regulatory proteins involved in the general control of amino acid biosynthesis --- p.142 / Chapter 4.3.2 --- Failure in identifying homologs of nitrogen regulators responsible for switching of nitrogen source in Arabidopsis --- p.144 / Chapter 4.4 --- Overview of research platform construction --- p.146 / Chapter 5 --- Conclusion and Perspectives --- p.148 / Chapter 6 --- References --- p.149-155 / Chapter 7 --- Appendix --- p.156-167

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Ethanolamine was shown to replace vitamin B6 in the nutrition of excised tomato roots grown in sterile culture (Boll, 1954a, 1959a). This replacement cannot be explained as a precursor effect (Boll, 1954a; Nillernot, 1962). An alternative explanation of the replacement was made by Boll (1954a, 1959a). The explanation involved negative feedback control, by ethanolamine, of a postulated serine decarboxylase yielding ethanolamine, and requiring pyridoxal phosphate as coenzyme. In this way, ethanolsmine would exert a sparing action on the limited amount of vitamin B6 which, as shown by Boll (1954a), is synthesized by the roots. [...]

The abi3-1 mutant causes moderate perturbation of seed metabolism relative to wild-type seeds, so offering a distinct and discrete treatment for use in experiments investigating regulation of allocation between sources and sinks. abi3-1 plants continue to initiate new flowers, and hence siliques, for longer than wild-type plants. Total rates of carbon assimilation in the short-term were the same in both genotypes during early reproductive growth. This rate fell to 50-70 % in wild-type plants during later reproductive growth, but did not change in the mutant, consistent with delayed senescence of cauline leaves in abi3-1 plants. It was found that restriction of carbon and/or nitrogen availability restricted growth in both genotypes, and abolished the mutant phenotype. Specific leaf areas increased under shading and decreased when nitrate was limiting. Reduction in nitrate limitation from 90 to 80 % was considerably less limiting for wild-type plant growth, but remained grossly limiting for abi3-1 plants. In previously non-acclimatised plants of both genotypes, no difference was found in assimilation and allocation of ¹⁴C-radiolabel supplied at 200 ppm CO 2. 800 ppm CO 2 similarly had no effect on the wild-type, but caused abolition or inversion of normal source-sink relationships in abi3-1 plants. A significantly large amount of radiolabel was initially incorporated into starch in all tissues in abi3-1 plants, and later moved into the water soluble fraction in each tissue, most likely as sucrose. It is proposed that resource allocation is regulated by competition for resources between sinks maintaining sucrose concentration gradients in the phloem, and that sucrose is both the transport and signalling molecule in the mechanism described. No difference in concentration of sucrose in tissues was found between genotypes, but it was found that mutant siliques imported [U-¹⁴C]glucose into siliques significantly more slowly from the phloem than wild-type siliques. In conclusion, abi3-1 seeds may be seen as having reduced capacity for growth which causes stimulation of floral meristem development by feedback of sucrose in the phloem. Silique initiation is thereby prolonged, creating a demand for assimilates that delays cauline leaf senescence.

1. The life cycle of this rosette aquatic plant was discussed in detail. 2. Anatomical differences between terrestrial and submerged forms of the rosettes were found. Terrestrial rosettes were induced very rapidly by exposure of previously submerged rosettes to air. Laminal development was greatly reduced in the leaves and roots of the terrestrial rosettes. 3. Two main aspects of the life cycle were considered in detail propagation of the rosettes; and leaf elongation. 4. Propagation: In nature the rosettes have the ability to propagate by (a) stolon formation or (b) by a less efficient means where the rosette elongates vertically. (a) Experimental evidence presented indicates that stolon initiation is controlled by gibberellic acid, although cytokinins may be involved. In nature, stolons from similar rosettes may be of varying lengths, and so the elongation phase of stolon development was considered. It was found that gibberellic acid may bring about a protraction of this elongation phase of growth of the stolon, at the expense of the development of the terminal plantlet. Ethylene supplied as ETHREL E inhibited further elongation of the stolons, and furthermore, promoted accumulation of starch. Starch grains in stolons of rosettes treated with gibberellic acid were limited to a central ring of cells. Some radial growth in response to supplied ETHREL E was noted. Stolons may persist in some cases, thus interconnecting many rosettes, but in some cases the stolons senesce. Evidence presented above suggests that gibbarellic acid and ethylene may control senescence. Where gibberellins levels are low in the stolons, accumulated ethylene will rapidly effect the senescence of the stolon, thus making the rosetts independent. (b) Where rosettes are partially and repeatedly buried by sediment, they have the ability to perennate themselves vertically by means of an elongated stem axis. Various rooting levels on the same rosette reflect the sequential nature of the deposition of the sediment. This was found experimentally where rosettes were grown under different conditions of deposition of sediment, and rosettes similar to those experimentally induced were found on a silt fan, near the inflow in Loch Drumore, near Glenshee, in Perthshire. The ability of the plant to propagate so rapidly by stolon formation on eroded shores, or by altering its rooting level and then forming stolons, explains to a great extent the ubiquity of this species throughout Great Britain, and thus implicitly explains the persistence of this species through many of the stages in hydrosere formation.

I. Previous studies on biogenesis of simple phenols in plants have been restricted to hydroquinone. Among the other simple phenols, catechol is of particular interest because of its potential role as a ring-cleavage substrate. Tracer studies on the biogenesis of catechol in Gaultheria leaf discs showed that it was formed from salicylic acid by oxidative decarboxylation. Salicylate decarboxylating activity could be detected in buffered extracts of very young leaves. II. Among the numerous caffeic acid esters presently known in plants, only 3-0-caffeoylquinic acid (chlorogenic acid) has been studied in detail. Rosmarinic acid (alpha-O-caffeoyl-3, 4-dihydroxyphenyllactic acid) has been reported to occur in a number of plants but nothing was known of its biosynthesis or metabolic role. Tracer studies demonstrated that in Mentha the caffeic acid moiety was formed from phenylalanine via cinnamic and para-coumaric acids. In contrast, the structurally similar 3, 4-dihydroxyphenyllactic acid moiety was formed from tyrosine and 3, 4-dihydroxyphenylalanine. There was no evidence of the participation of a para-coumaroyl ester intermediate. Time-course studies and use of labelled rosmarinic acid showed that endogenous rosmarinic acid was turning over slowly. The caffeoyl moiety, however, does not appear to be contributing to the formation of insoluble polymers, as has been suggested for chlorogenic acid in other plants. III. Bacteria and fungi readily degrade aromatic compounds to carbon dioxide. Despite the large quantities of aromatic compounds formed in plants, little attention has been paid to the ability of plant tissues to degrade aromatic rings. No reported studies have used completely sterile plants and techniques. This has left open the possibility that the microflora associated with the plant might be carrying out the observed reactions. The ability of sterile plant tissue cultures to degrade aromatic ring-¹⁴C compounds to carbon dioxide was studied. It was established that a number of tissues (Ruta, Triticum, Phaseolus, Melilotus) have the ability to cleave the aromatic ring of phenylalanine. Melilotus tissue could also degrade cinnamic acid-ring-¹⁴C suggesting that a dihydroxy phenolic acid may be the ring-cleavage substrate. Neither Ruta nor Melilotus tissues were able to degrade benzoic acid or salicylic acid-ring-¹⁴C. Tryptophan benzene ring-¹⁴C was shown to be degraded to carbon dioxide by both Ruta and Melilotus. In summary, the ability of plants to cleave the benzene ring of aromatic compounds when free of micro-organisms was thus established. / Science, Faculty of / Botany, Department of / Graduate

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