In both plants and animals Polycomb group (PcG) genes act to maintain silencing of key developmental genes. A number of PcG genes have been identified in <i>Arabidopsis</i> through independent genetic screens. These can be placed in three groups based upon strong homology with the <i>Drosophila </i>proteins E(z), ESC and SU(Z)12. <i>Arabidopsis</i> contains multiple homologues of E(z) and these have evolved to act in different pathways (<i>CURLY LEAF</i> represses flowering in the immature plant; <i>MEDEA</i> prevents certain aspects of seed development in the absence of fertilisation; and <i>CURLY LEAF LIKE</i> has recently been shown to be partially redundant to <i>CLF</i>). Despite the high level of conservation between CLF and MEA proteins, I have shown that they are not equivalent. <i>MEA</i> is unable to rescue a <i>clf- </i>when expressed under either the <i>CLF</i> endogenous promoter or the constitutative 35S promoter. In <i>Drosophila</i> it has been shown that E(Z) acts in multimeric protein complexes with the PcG proteins ESC and SU(Z)12. Through yeast-two hybrid assays I have shown that CLF physically interacts with both the SU(Z)12 homologue EMF2 and the ESC homologue FIE, providing evidence of a similar complex acting to repress flowering in the immature plant. I have mapped the domains to which these interactions mediate. Of particular interest is the interaction between EMF2 and CLF; this region maps to the VEFS box of EMF2 and the C5 region of CLF. Embryos carrying a maternal inherited <i>fie</i> allele abort during deed development, making it impossible to observe the role of <i>FIE</i> post seed development. I have created a transgenic steroid-inducible <i>FIE </i>line, which I have used to rescue <i>fie</i>- embryos. The resulting plants show severe developmental abnormalities, with highly disorganised organ growth.

Activation tagging was employed in conjunction with the reporter gene line <i>PR-1::LUC </i>to uncover novel systemic acquired resistance (SAR) mutants. The mutant screen employed imaging for constitutive LUC expression or the absence of LUC expression after induction with the salicylic acid (SA) functional analogue, benzo (1,2,3) thiadiazole (BTH). A set of three mutants displaying constitutive LUC activity were selected for further characterisation. These mutants were subsequently named activated disease resistance 2,3 and 6 (<i>adr2, adr3 and adr6</i>), and showed constitutive <i>PRI </i>expression. Furthermore, all three mutants were resistant to the virulent oomycete pathogen <i>Peronospora parasitica </i>Noco2 and <i>adr3 </i>to the virulent bacterial pathogen <i>Pseudomonas syringae </i>pv. <i>Tomato </i>(DC3000). Genetic and molecular analysis indicated that two of these mutants were not tagged (<i>adr2, adr6</i>) and that plasmid rescue of <i>adr3 </i>gene was not a straightforward technique, therefore cloning was attempted using a map-based approach, <i>adr2 </i>maps to chromosome 5, whereas <i>adr3 </i>and <i>adr6 </i>map to chromosomes 4 and 1 respectively. Genetic analysis indicated that <i>adr2 </i>and <i>adr6 </i>are recessive mutations and define single genes. Although <i>adr3 </i>maps as a single mutation, its mode of inheritance is yet unidentified. Double mutants were produced between <i>adr </i>mutants and SA-, JA- and ET- insensitive mutants. Epistasis analysis and mapping data suggest that the <i>adr </i>mutants define three novel loci. Therefore they will provide new genetic tools for dissecting broad-spectrum resistance mechanisms.

Natural genetic variation found among accessions of <i>Arabidopsis thaliana </i>presents the opportunity of locating and identifying novel genes by means of quantitative trait locus (QTL) analysis. In this study, QTL analysis was used to identify loci involved in the genetic control of growth in <i>A. thaliana</i>. Non-destructive methods of analysis were developed and used for the measurements of growth rates in roots and leaves, whilst a simple size measurement of mature petals was used to assess growth in the floral organ. Two putative QTL were identified for primary root length, four for leaf number at day 32 and three for petal size in the Bay-0 x Shahdara recombinant inbred line (RIL) population. The Landsberg <i>erecta</i> x Columbia RIL population was also analysed, but no significant QTL were identified. The analysis suggested that, in all three organs, growth-rate is controlled by multiple small-effect QTL and is a highly plastic trait. Thus, minor environmental fluctuations during the course of experiments can lead to a large environmental variance in measurement of the traits, limiting the power of QTL analyses. Despite minimising these effects by adjusting growth techniques, the numbers and significance of QTL identified in each trait were lower then expected, and for the trait of relative growth rate in leaves no significant QTL were identified.

Expression of heat shock proteins (HSPs) is a ubiquitous plant response under heat stress and is mainly regulated at the transcriptional level by heat shock factors (HSFs) that when activated recognize the heat shock elements (HSE) in the promoter region of' the HSPs, activating their transcription. ASCORBATE PEROXIDASE2 (APX2) was the first gene that was not a HSP but was heat dependent and HSF de'pendent. The mutant altered APX2 e.\pressiollll -1 (alxll -1) when exposed to heat stress showed higher HSPs, HSFs, APX2 expression and hydrogen peroxide (H20 2) levels than the wild type (WT). Expression of APX2 and H202 levels were still visible in alx11-1 under heat stress when placed in the dark or treated with 3-(3,4-dichlorophenyl)-I, I-dimethyl urea (DCMU), which is a specific inhibitor of photosystem II (PSII) . In addition no differences in photosynthetic electron flux were observed under heat stress. H20 2 production and signalling from other sources was considered for induction of heat related genes, for example from NADPH oxidases in the plasma membrane. In alxl 1-1, abscisic acid (ABA) signalling was considered to be linked to H20 2 production and signalling in the induction of heat related genes. Expression of the Hsa32 encoding a novel HSP like 32 kDa protein, Hsa32 which is considered necessary for acquired but not for basal thermotolerance, was highly expressed in the WT compared to the mutant. Screening of the F2 population from the cross between alxl1-l and the Landsberg erecta showed that the alx11-l mutation responsible for the altered expression in APX2 is a single recessive locus. Therefore it was suggested that aLd 1 mutation in Arabidopsis negatively regulates ABA mediated production of H20 2 under heat stress which in turn could affect the expression of HSPs necessary for basal thermotolerance.

Ca<SUP>2+</SUP> is a frequently utilised second messenger in higher plants. Calmodulin is a small, highly conserved, multi-site receptor for Ca<SUP>2=</SUP>, which interacts with a large number of downstream effector proteins. Calmodulin binding proteins (CBPs) do not have a highly conserved binding domain, and interact with calmodulin in an unusual fashion involving both a hydrophobic surface and charged domains. A number of techniques to isolate CBPs from plants were used. Techniques based on DNA homology suffer from the extreme lack of conservation between calmodulin binding domains. Other, established techniques, utilise <I>in vitro </I>overlays of labelled calmodulin to screen cDNA libraries, and are necessarily artificial and thus potentially artifactual. Algorithmic modelling of the calmodulin binding interaction based on up to date information was carried out using rapid, quantitative computer profiles which utilised a novel double profile technique to combine both hydrophobic and charge related patterns. This model suggests that a large number of relatively weak yet biologically significant CBPs are to be expected. The consequences of the presence of these proteins is discussed. The yeast dihybrid system was utilised as an <I>in vivo</I> system to screen for calmodulin binding proteins, and a number of sequences isolated and partially sequenced. These sequences demonstrate the success of this technique, and show the existence of a partially conserved calmodulin binding domain within the plant kingdom, as well as a high number of CBPs of potential biological significance.

Recognition of avirulent microbial pathogens activates an oxidative burst, leading to the accumulation of reactive oxygen intermediates (ROIs), which are thought to integrate a diverse set of defence mechanisms resulting in the establishment of plant disease resistance. Two contrasting experimental strategies were devised to dissect genetic mechanisms governing these signal transduction pathways. Firstly, a novel transgenic <i>Arabidopsis</i> line containing a <i>gstl::luc</i> transgene was developed and employed to report the temporal and spatial dynamics of ROI accumulation and cognate redox signalling in response to attempted infection by avirulent strains of <i>Pseudomonas syringae </i>pv.<i> tomato (Pst).</i> Strong engagement of the oxidative burst was dependent on the presence of functional <i>Pst hrpS</i> and <i>hrpA</i> gene products. Experiments employing specific pharmacological agents suggested at least two distinct sources, including a NADPH oxidase and a peroxidase-type enzyme, contributed to the generation of redox cues. The analysis of <i>gst1::luc</i> gene expression in specific mutant backgrounds suggested engagement of the oxidative burst and cognate redox signalling functioned independently of ethylene, salicylic acid and methyl jasmonate in local <i>RPM1</i> mediated resistance. In contrast, studies using a panel of specific protein kinase and phosphatase inhibitors revealed mitogen activated protein kinase kinase (MAPKK) activity was required for the activation of the ROI-regulated genes <i>gstl</i> and <i>pall</i> in response to redox cues. Thus the engagement of a redox signalling network dependent on MAPKK activity may contribute to the establishment of plant disease resistance and the development of cellular protectant mechanisms. Secondly, Activation Tagging was employed in conjunction with the reporter gene line <i>Prla::luc,</i> to uncover a mutant with constitutive defence gene expression. This mutant subsequently, named activated disease resistance 1 (<i>adr1)</i>, was shown to have enhanced resistance to fungal and bacterial pathogens. <i>adr1</i> mutants were also shown to have enhanced drought tolerance, and as such are believed to be the first plants engineered with elevated resistance to both disease and drought stress.

The <i>Araucaria</i> conifers of New Caledonia are a globally significant group with 13 endemic species, eleven of which are threatened with extinction. The critically endangered conifer <i>Araucaria nemorosa</i> and common widespread congener <i>A. columnaris</i>, provide a comparative framework in which to empirically investigate the genetic consequences of fragmentation in understudied anemophilous group. A survey of the genetic diversity at nuclear microsatellite loci over the species’ total range suggests that adults of <i>A. nemorosa</i> are not genetically depauperate, maintaining equivalent levels of genetic diversity to <i>A. columnaris</i> (<i>A_e</i>=20.66; 14.7; <i>H_e</i>=0.715; 0.654 in <i>A. nemorosa</i> and <i>A. columnaris </i>respectively). However, quantifying genetic diversity and inbreeding coefficients in seedling populations revealed a significant loss of allelic richness and a two-fold increase in the inbreeding coefficients in <i>A. nemorosa </i> seedlings compared to adult populations (<i>F</i>_IS 0.195 vs 0.096). This indicates that genetic bottlenecks and elevated inbreeding are likely consequences of fragmentation in <i>A. nemorosa</i> populations. Stand structure, reproductive characteristics and population genetic structure within remnant populations of <i>A. nemorosa</i> were assessed in order to place the genetic consequences of fragmentation in an ecological context. This indicates that in severely fragmented populations ecological and genetic factors can interact to determine population persistence. The reproductive characteristics of <i>A. nemorosa</i> were evaluated, which revealed that seed set is generally very low (5% seed set per cone) with a high variance among individuals. The consequences of fertility variance for the effective population size were explored using both ecological and genetic methods. This suggests that fertility variance will have few consequences for population genetics of wild seedling cohorts. However, in a single season, the number of adults contributing to the seed crop may be small and this has implications for the sampling of germplasm for forest restoration.

<i>ASYMMETRIC LEAVES1 </i>(<i>AS1</i>) encodes a MYB transcription factor that is expressed in lateral organs of <i>Arabidopsis </i>where it excludes expression of meristem promoting <i>know</i> genes. This is unlikely to be the only function of <i>AS1</i> as reducing <i>knox </i>expression does not suppress the <i>as1</i> mutant phenotype. To identify additional targets of <i>AS1,</i> gain-of-function and loss-of-function mutant screens were used to identify modifiers of the <i>as1</i> mutant phenotype. A gain of function mutation in the <i>PTL</i> gene was isolated as a potential suppressor of the <i>as1</i> mutant phenotype in petals. Further genetic analysis suggested that <i>AS1 </i>and <i>PTL</i> are unlikely to act in same pathway. <i>symmetrica </i>(<i>sym</i>), a complete suppressor of <i>as1,</i> was isolated in loss-of-function screens. Expression analysis of <i>knox</i> expression in lateral organs of <i>sym</i> mutants suggests that <i>SYM</i> is required for <i>knox</i> upregulation in <i>as1</i> mutant lateral organs. <i>AS2,</i> which is thought to act in a similar position in development to <i>AS1</i> also appeared to interact with <i>SYM </i>as <i>as2</i> mutants were also suppressed in its absence. Triple mutant analysis indicated that <i>SYM</i> may work in a similar position in development to <i>SE</i>, a previously described partial suppresser of <i>as1.</i> In the absence of the <i>knox </i>gene <i>STM</i>, formation of a functional SAM during embryogenesis is prevented due to ectopic expression of <i>AS1 </i>and genes that specify lateral organ fate throughout the apex. However, <i>as1-1 stm-1 </i>double mutants form a functional SAM as lateral organ fate signals are reduced due to the lack of <i>AS1. </i>The SAM of <i>as1-1 sym stm-1 </i>triple mutants is arrested, indicating a redundant role for <i>SYM</i> in meristem function. <i>sym </i>also modified mutants at the <i>PNH</i> locus, suggesting an interaction with the post translational gene silencing machinery. <i>SYM </i>was mapped to an interval of 120 Mb linked to <i>AS1</i> on the long arm of chromosome 2.

To understand the molecular genetic basis of mitochondrial biogenesis during plant development, and the regulation of oxidative phosphorylation in particular, several nuclear genes encoding maize mitochondrial proteins had previously been cloned. Two of these genes termed <i>ANT1</i> and <i>ANT2</i> encode mitochondrial adenine nucleotide translocators. The work presented describes further characterisation of the ANT genes and isolation of a cDNA clone for <i>ANT2</i>. Steady state levels of <i>ANT</i> transcript have been analysed in different maize tissues and compared with other nuclear and mitochondrial genes encoding subunits at ATP synthase. The ANT genes have at least 5 exons termed exon 1 to exon 5 and at leat 4 introns termed intron A to intron D. The 5' end of the gene is more complex than originally concluded (Bathgate <i>et al</i>, 1985). Both ANT genes contain multiple transcript initiation sites, but the position and number of sites are not conserved between the two genes. The sizes of the 5' untranslated regions (5' UTRs) are between 124 bp and 356 bp as predicted from the various primer extension products. Both ANT genes have a minimum of two untranslated exons and two large introns (intron A and intron B) separate the untranslated exons. The 5' and 3' splice sites of intron A have not been accurately determined for <i>ANT1</i> or <i>ANT2</i> but the size of the intron in <i>ANT2</i> is in excess of 2.4 kb. Intron B is 1063 bp and 1021 bp in size in <i>ANT1</i> and <i>ANT2</i> respectively and is separated from intron A by exon 2 which is approximately 85 bp and 78 bp in <i>ANT1</i> and <i>ANT2</i> respectively. The start of translation is in exon 3 and is 21 bp 3' of the intron B 3' splice site. Sequence of the <i>ANT2</i> cDNA clone confirmed that the open reading frame includes a presequence which has been reported to be cleaved following import into mitochondria (Winning <i>et al</i>., 1992). <i>ANT</i> transcript steady state levels vary in a tissue specific manner. The highest steady state transcript levels were found in tissues known to exhibit mitochondrial oxidative phosphorylation and high metabolic rates. Tissues with lower metabolic rates or those which are photosynthetically active contain lower transcript levels, hence, a tentative correlation has been made between the transcript steady state level and metabolic activity. Probes which differentiate between the two ANT genes were used to investigate the possibility of differential regulation.

When barley, as female, was crossed with rye a high proportion of fertilised ovules developed. Cytological studies on developing ovules showed that the hybrid endosperm failed to develop normally, but contained polyploid nuclei, chromosome bridges, lagging chromosomes and micronuclei. Development of the endosperm from tetraploid crosses appeared to be more successful than from diploid crosses. In most ovules the endosperm degenerated by one week after pollination. Most hybrid embryos aborted about 8 days after pollination. Lagging chromosomes and aiorowclei, observed in developing embryos from day 3 after pollination appeared to be responsible for loss of viability. A small proportion of hybrid embryos which appeared to be free of cytological abnormalities could continue to develop as long as the fruits did not dry up. When crossed spikes were sprayed with specified growth substances, plump, liquid-filled fruits developed, from which a small proportion of developing hybrid embryos could be recovered at 18-20 days after pollination. These could be transferred to culture without much difficulty. When spikes were not sprayed, fruit development ceased before embryos reached a size suitable for culture. Most of the embryos transferred to culture continued to grow, although most ceased development before reaching a stage at which they could be transferred to soil. One embryo gave rise to callus which was subcultured on a medium free of growth substances. Green and albino hybrid plants regenerated from this callus. The green plants, which are sterile, grow vigorously. By means of Giemea C-banding techniques it was shown that the complement consisted of 8 rye and 6 barley chromosomes. It appears that the molecular chromasome of rye Me successfully substituted for a me leolar chromosome of barley.