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The progamic phase of plant reproductive development involves events from pollen germination to gamete fusion. Physiological studies suggest that complex mechanisms are involved in this pathway. In order to identify gametophytic genes which function in this pathway, an insertional mutagenesis screen based on segregation ratio distortion was performed on a population of 4094 independent dSpm transposon insertion lines. This screen did not produce any mutants of interest, but two mutants, seth4 and seth7, were identified from a similar screen of Ds transposon insertion lines, seth4 and seth7 showed stably reduced segregation ratios arising from reduced gametophytic transmission and reciprocal crosses showed no (seth4) or severely reduced (seth7) transmission of the antibiotic resistance marker only through pollen. In both mutants, pollen morphology was normal but pollen germination was severely affected. In seth4, the insertion disrupted the coding region of a gene encoding an armadillo (ARM) repeat protein. In seth7, the transposon was inserted within the 3'-UTR of a gene encoding a putative serine/threonine protein kinase. A wild-type copy of SETH4 complemented the seth4 mutation and restored male transmission. SETH4 is the founding member of a discrete Arabidopsis gene family that contains two SETH FOUR- LIKE genes, SFL1 and SFL2, SETH4 was found to be preferentially expressed in the male gametophyte while SFL1 and SFL2 were expressed exclusively in the sporophyte. SETH4, SFL1 and SFL2 proteins, when fused to GFP, suggest cytoplasmic location in transient expression assays. In this work two mutants identified as essential for the male gametophyte during the progamic phase have been phenotypically characterised. SETH4 was analysed using genetic, molecular and bioinformatic analyses and is proposed to be part of a novel molecular pathway controlling cellular growth in the gametophyte.

During Arabidopsis development the shoot apical meristem (SAM) generates lateral primordia which display stage-specific traits. In long days, wild-type Arabidopsis generates leaves in an initial vegetative phase (V). Upon integration of environmental and endogenous signals, the SAM enters the reproductive phase. First it makes an 11 phase, which consists of 2-3 leaves (cauline) subtending secondary shoots (coflorescences). Next it enters the 12 phase and produces flowers on its flanks. The TFL 1 gene is a key component of the phase change machinery as mutations in TFL 1 affect the timing of phase switching. Also ffl1 mutants enter a novel phase whereby the SAM, after 12, is converted into a terminal flower, a phase normally absent in wild type. The molecular mechanism of how TFL 1 protein acts is unclear. In animal systems, TFL 1-like proteins have been shown to be components of signal transduction pathways. To understand the mechanism underlying TFL 1 function I aimed to identify proteins interacting with TFL 1 by introducing into Arabidopsis a functional TAP tag version of TFL 1 under the control of the 35S promoter. I set up conditions which allowed me to isolate and visualize by total protein staining TAPtag TFL 1. However, no obvious proteins appeared to co-purify with TFL 1. To understand how TFL 1 is modified, and to follow TFL 1 protein expression throughout development and in cell fractions, I developed polyclonal antibodies against TFL 1. These antibodies recognized TFL 1 in vivo and were used to characterize TFL 1 biochemically. TFL 1 detection by immunoblots in conjunction with mass-spectrometry analysis showed that TFL 1 was not subjected to obvious modifications unlike animal homologues. Moreover, from cellular fractionation experiments TFL 1 was located in the cytosol. To reveal essential downstream functions required for TFL 1 signaling, I characterized a suppressor mutant, called sof1, of plants ectopically expressing TFL 1. I mapped sof1 within a confined region on the bottom of chromosome 3. Physiological analysis of sof1 led to a model of SOT1 action in controlling phase change. TFL 1 mRNA is found in a unique expression domain which comprises a group of cells in the centre of the SAM and yet TFL 1 affects the identity of lateral primordia. By using affinity purified anti-TFL 1 antibodies I showed that TFL 1 protein moves and is distributed throughout the SAM. This might account for the effect of TFL 1 on controlling overall shoot identity and raises important questions on the role of the TFL 1 protein outside its mRNA expression domain.

Maintaining global food security in the presence of a growing food demand and climate change will require an improved understanding of the genetic basis of crop yield components, and their response to environmental stress conditions, such as drought. The reproductive output of plants is further limited by partitioning of finite resources, resulting in trade-offs between traits. Here, I address these issues using Multiparent Advanced Generation Inter-Cross (MAGIC) lines of Arabidopsis thaliana for quantitative trait locus (QTL) analysis, and find that (i) there is no evidence for a genetic basis of the seed size/number trade-off, and (ii) A. thaliana responds to drought predominantly via phenotypic plasticity. I also show that seed size affects the probability of seeds germinating in the field. Moreover, I demonstrate that the inflorescence contributes resources to fitness, which suggests that flowering time and vegetative size do not necessarily impose an upper limit on reproductive output. Lastly, I address the evolution of genomic imprinting, as several imprinted genes have been shown to be vital for seed development. Several hypotheses for the evolution of genomic imprinting are tested simultaneously, and I find evidence to support a link between transposable elements and imprinted genes.

Protein turnover in plants is a key regulatory mechanism for many cellular processes. The ubiquitin (Ub)/ 26S proteasome pathway is probably the most well studied way to attain this goal. An Arabidopsis mutant which has been termed hawaiian skirt {hws) fails to shed its floral organs. The sepals remain fused at their base and fail to abscise. Mutation of HWS leads to overgrowth for both reproductive and vegetative Arabidopsis organs; in contrast over-expression of HWS leads to an opposite effect.I. So HWS protein may function to inhibit cell division or elongation whlch is responsible for the sepal fusion and organ outgrowth phenotype.

Salt, cold, and drought are major abiotic stresses that impact plants and agricultural crops. Many plants and crops are able to increase their ability to survive these and other stresses by altering gene expression. Using the Arabidopsis model plant in studying gene function and regulation is of crucial importance to plant genetics and biotechnology. Our objective was to study plant survival and stress tolerance in Arabidopsis T-DNA lines and, also chemically mutagenized and RNAi lines, for specific genes that are hypothesised or expected to have a role in at least one of the known abiotic stresses. The selection of genes was based on some previous research including QTL analysis. I included in my investigations OSMl, CHXl7, KUPl, bHLH033, CBF4, dreb2a and the NW20 and N163 lines of ERECTA. Other non-mutated genes such as COR 78, DREB2A, CBFl, CBF3, CBF4, and OSMl were used to study the specificity in gene expression in Arabidopsis leaf and epidermal tissues under a combination of cold and osmolytes. The insertional mutant analysis showed that T-DNA can be located in a different site than the one shown in database and sometimes parts of the T-DNA left-border-sequence were absent. Most of the targeted Arabidopsis mutants showed a normal Mendelian pattern of segregation with the one exception of the CBF4 insertional mutant population. RT-PCR transcript detection of bHLH and DREB2A indicated that wild-type Arabidopsis in our growth environment expressed only one splice form of each of these genes rather than the two described by the TAIR and MIPS databases. Stress results indicated that inserting T-DNA within the Arabidopsis genome for a specific gene can lead to negative or positive effects on the gene's expression and plant survival. Investigation of the role of OSMl in cold tolerance indicated that the mutant plants were more tolerant to cold as measured by plant survival and electrolyte leakage. The higher expression of OSMl gene in the mutant plants has driven me to search in the promoter region of OSMl at OSM1::T-DNA junction. The results revealed the presence of different known eis-acting elements which might have affected plant survival. Using RNAi lines for OSMl (gene expression was not tested) is also consistent with the idea that this gene has a role in cold stress. Even though the high transcripts accumulation of bHLH (lCE2) in the mutant line was discovered, there was only slight difference between homozygous and wild type plants in response to cold. DREB2A, which is well known for its dehydration involvement, was tested under cold. Using dreb2a mutant for cold stress revealed that Salt stress tolerance was obtained with osml, ehx17, and kupl mutants as measured by the high average of plant survival. Mineral content was measured in leaves and roots of CHX17 and KUP 1 mutant and wild type plants under salt conditions. The mutant plants accumulated less sodium and higher potassium especially in roots compared to wild type. Also, roots accumulated more calcium and magnesium than the wild type. These results are clearly consistent with the higher survival in kupl mutant and may indicate a correlation with the salt tolerance in ehx17. Also, the high sensitivity to salt stress was strongly linked to some phenotypic and developmental changes, such as leaf wilting, rolling, chlorosis, and desiccation. Mutant plants that had higher survival developed dark leaf colour which probably indicates anthocyanin accumulation. A higher level of expression of genes in some mutant lines than in the wild type lines, linked with higher plant survival indicated there might be some eis-acting elements in the inserted T-DNA that were influencing stress tolerance. Compared to NW20 Landsberg ereeta line, carrying the chemically produced mutant, N163 plants, which carry wild-type ERECT A gene, exhibited more plant survival under salt stress especially at an older growth stage. Abstract IV covering plants during cold acclimation increased plant's sensitivity to cold compared to uncovered plants. This suggests that the role of DREB2A in cold acclimation probably is not through water stress signalling per se but other signalling must be involved. Also, drought stress was studied and was limited to CBF4 gene. The gene expression of CBF4 in the mutant was greatly reduced. However, plant survival under drought was not affected by the mutation. Gene expression-specificity of some cold regulated genes in epidermis and leaves of Arabidopsis wild type was studied under sugar and sorbitol with or without minerals. COR 78, DREB2A, CBF1, Eli-l-o; ACT2, and ACT8 were more highly expressed under cold in epidermis than in leaves. This expression was often several-fold higher when sucrose at 90 mM with mineral salts was supplied. CBF3 also showed a higher level of transcript accumulation by cold in epidermis than in leaves but both tissues exhibited similar and higher expression with sucrose at 40mM than the control and 90mM of sucrose with minerals. It seemed that removing minerals from sugar solution reduced the gene expression but epidermal tissues conserved the preference of most genes to be expressed in higher level (relative to total RNA) than leaves. Sorbitol at 90mM with minerals highly affected the expression of CBFl and CBF3 apart from type of tissue and reduced the expression of DREB2A, CBF4, and ACT8 in both tissues. OSMl had differential gene expression by different treatments. It showed slightly higher expression in epidermis with cold treatment and its expression was completely diminished in leaves at 90mM of sorbitol with minerals. These results indicate the regulatory role of sugar in cold- induced gene expression of several genes in epidermis which reflects its importance in stress tolerance. Abstract v All in all, using T-DNA can affect plant survival and alter gene expression in ways which can be interpreted as a direct or non-direct link in the overall stress tolerance. These investigations have added new facts to understanding stress tolerance in plants but others remain to be more deeply tested. A novel observation was that the T-DNA in the promoter can increase the regulation of gene expression and stress tolerance and this may be due to stress response elements in the T-DNA. Abstract

Continuing root growth is crucial for the ongoing survival of a plant and provides the structure basis for the acquisition of valuable resources such as water and nutrients required for growth and development. The root apical meristem, located at the root apex, contains a stem cell niche which is the source of root cell production and patterning. This project focuses on the influence of phytohormone signalling upon the maintenance of the root apical meristem (RAM) in Arabidopsis thaliana. Through a combination of physiological, genetic and molecular approaches, the project has uncovered evidence of the involvement of three plant hormones, auxin, abscisic acid (ABA) and ethylene, in regulating the differentiation of stem cells and their descendants in Arabidopsis root meristems. Key findings include: 1) exogenous application of auxin, ABA or 1-aminocyclopropane-1-carboxylic acid (ACC), which is an ethylene precursor, rescues the root meristem failure of a1f3-1 seedlings; 2) exogenous application of ABA promotes QC quiescence and suppresses stem cell differentiation in wild type Arabidopsis root meristems; 3) inhibition of ethylene biosynthesis and mutations that cause ethylene insensitivity induce differentiation of stem cells in root meristems and.i.on the other hand, exogenous application of ethylene precursor or mutations causes ethylene over-production suppress stem cell differentiation in Arabidopsis root meristems; 4) ABA and ethylene interact antagonistically with auxin in the regulation of both stem cell differentiation in Arabidopsis root meristem, and the expression of the CLE40 gene, which encodes a negative regulator of stem cell differentiation in root meristems; 5) ABA modulates the abundance of the LM6 arabinan cell wall epitope within at the root meristem; 6) ethylene suppresses the stem cell differentiation in Arabidopsis root meristems induced by nitrogen (N) and phosphorus (P) deficiencies. The results of the project demonstrate that stem cell regulation in Arabidopsis root meristems involves complex interactions of plant hormones and environmental signals.

Increases in global carbon dioxide, temperature and, in some latitudes, humidity are characteristic features of global climatic change. There is overwhelming consensus on the future increase of these environmental factors. How plants respond to these changes has important implications for the performance of both natural and agricultural systems. The aim of this project was to quantify the effects of carbon dioxide, temperature and relative humidity on morphological features and plant fitness of ecotypes of Arabidopsis thaliana, originating from different altitudes and geographical origins. Ecotypes of A. thaliana were grown in controlled environment chambers to assess the extent that genetic constraints influence phenotypic plasticity. The results showed a significant variation among the ecotypes and some of the variables assessed correlated with altitude of origin. Plant branch number and the length of fruits positively correlated with altitude of origin under elevated C02 and elevated humidity respectively. High altitude ecotypes showed decreased leaf area and also matured earlier in increased temperature and relative humidity. High altitude ecotypes also flowered earlier under elevated temperature. Stomatal density and epidermal cell density increased with CO2. Stomatal density showed a positive relationship with stomatal conductance and carbon isotope discrimination under elevated CO2• Both elevated temperature and relative humidity decreased stomatal density and epidermal cell density. The leaf area, floral stem height and inflorescence number increased under both elevated temperature and relative humidity. Elevated temperature also increased plant branch number. Plant fitness and reproductive phenology were significantly promoted under C02 enrichment and affected negatively under sub-ambient CO2. This study shows intra-specific variation between ecotypes of A. thaliana in response to environmental manipulation and therefore the extent of genetic constraints to acclimation processes. Such knowledge of plant responses to more than one variable under predicted environmental change particularly carbon dioxide, temperature and humidity is critical to preservation of vegetation both natural and agricultural.

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