Since their discovery long non-coding RNAs (lncRNAs) have in turn been described as essential genomic regulators or as transcriptional noise. Examples of lncRNAs with experimentally-validated function are limited, with poor nucleotide sequence conservation calling apparent functionality into question. COOLAIR lncRNAs are transcribed in the antisense direction at the Arabidopsis thaliana floral repressor gene, Flowering Locus C (FLC). Previous work has revealed a role for COOLAIR antisense RNAs in regulation of the FLC protein-coding sense transcript and, consequently, flowering time. FLC homologues are wide-spread in flowering plants, but nucleotide sequence conservation of COOLAIR-specific regions is low. COOLAIR has a complex secondary structure, and covariant base-pair mutations predict strong conservation of this secondary structure across flowering plants. Syntenic transcription of COOLAIR was confirmed in vivo for several species within the family Brassicaceae, including three commercially important Brassica crops: B. rapa, B. oleracea and B. napus. COOLAIR transcription was detected from at least three of four ancient FLC clades within the latter three polyploid and paleopolyploid species. Each FLC homologue has distinct nucleotide sequence, expression patterns, and COOLAIR isoforms. Further variation in COOLAIR was identified between winter and spring cultivars. It was hypothesised that this could affect cis-regulation of FLC. Correlation between COOLAIR modifications and flowering time was tested by introducing antisense splice site mutations into Brassica FLC transgenes in A. thaliana. These experiments suggested that a shorter COOLAIR isoform with a disrupted structural motif was a weaker negative regulator of FLC. This work supports conservation of COOLAIR expression, structure and function in Brassica crop plants, and a role for RNA structure in COOLAIR function. We propose an evolutionarily conserved lncRNA that is neither essential regulator nor transcriptional noise, but rather adapts with the environment to fine-tune the transition to flowering.

Despite the sizes of organs and organisms being a key defining feature, very little is known about how the final sizes of organs is determined. Recent progress has highlighted the important role of the DA1 peptidase as a negative regulator of organ size in Arabidopsis thaliana. Previous studies have proposed that DA1, and the E3 ligase BIG BROTHER (a protein known to regulate DA1 activity), work synergistically to regulate the duration of cell proliferation. In the prulresent study, we take a multidisciplinary approach to further our understanding of the biological activities of DA1 and BB. Protoplast transient expression analyses were used to explore potential new substrates for DA1 peptidase activity, and to work towards identifying a conserved target site for DA1mediated cleavage. Using confocal microscopy and bespoke segmentation software, I embarked on a global analysis of leaf cellular phenotypes in the da1-1, bb, and da1-1bb mutants throughout early development. This allowed a holistic comparison to wild type of parameters such as total cell number, and cell area, density, and circularity. In addition, scanning electron microscopy was used to examine cells in mature leaves of wild type, and da1-1, bb, and da1-1bb mutants, revealing novel insights into the control of final organ size in these mutants relative to wild type. Finally, innovative live cell imaging has, for the first time, allowed cell divisions to be observed in plants carrying the da1-1, bb, and da1-1bb mutations. My observations and interpretation establish new insights into how DA1 and BB control growth by controlling the arrest of cell proliferation, and the population-level rate of cell proliferation. The approaches I have developed show the promise of quantitative cell imaging for understanding organ growth, and establish a framework for precisely comparing the effects of different mutations on organ growth.

Spores suspensions of Colletotrichum lindemuthianum or Botrytis cinerea incubated for 24 hr. in pod cavities of French bean became inhibitory to germination and germ-tube growth of spores in bio-sasays. This was apparently due to the formation of a phytoalexin. Spores of the two fungi did not respond in the same way to the phytoalexin. The phytoalexin was extractable into diethyl ether but not petroleum ether and gave maximum u.v. absorption between 280 and 290 mg, It could be separated by paper chromatography and appeared to be phenolic. Its properties fitted most closely a substance described by Pierre and not phaseollin. Phytoalexin production was accompanied by the formation of substances stimulatory to spore germination, including sugars and amino-acids, and by an increase in pH. Varying the concentration of the phytoalexin had a much greater effect on spore germination than varying the concentration of stimulants. The phytoalexin appeared after 8 hr. in some cases and did not much increase after 24 hr. Higher concentrations were produced by young pods. The phytoalexin was destroyed by sunlight, Its formation could be induced by cell-free solutions. Various combinations of bean variety and race of Colletotrichum were used for phytoalexin production and no consistent difference was found after 24 hr. between resistant and susceptible combinations. Resistance and susceptibility could not be distinguished visually until six days after inoculation of pod cavities. The phytoalexin could be detected in extracts from infected endocarp but not with certainty in extracts from infected leaves and hypocotyls. Technical difficulties made it impossible to use tissue extracts for quantitative comparisons of phytoalexin production, Prior inoculation of leaves with spore suspension of a race of Colletotrichum to which the leaves were resistant gave a degree of local immunity against a race to which they were susceptible.

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Plants are fundamental to life on Earth, especially for the role they play in the food chain. With the world human population predicted to exceed 9 billion by 2050 the pressure on our agricultural system is set to increase. Not only does agriculture have to expand in order to feed this ever growing population it also needs to effectively deal with the threat that plant pathogens pose to food security. The relationship between plants and their pathogens is immensely complex, with subtle differences at the molecular level defining whether a host becomes infected or remains healthy. These molecular differences are exemplified by pathogen effectors that manipulate processes inside the host cell to benefit colonisation. Knowledge of how these effectors and their targets function is crucial to develop strategies to enhance crop resistance. This project focuses on one such effector, Pi17316, which is secreted by the potato late blight pathogen, Phytophthora infestans, to target a host MAP3K, VIK. Inside the plant cell Pi17316 localises to the plasma membrane, where it interacts with VIK in order to suppress the INF1 and R2/Avr2 cell death pathways and to enhance P. infestans colonisation. VIK itself was shown to be a susceptibility factor, required for full P. infestans colonisation. Overexpression of StVIK results in the suppression of INF1 and R2/Avr2 responses and enhances P. infestans ability to colonise the host. Furthermore, silencing of NbVIK attenuates infection and negates any positive effect on colonisation that Pi17316 provides, demonstrating that VIK activity is required for disease. Here it is shown that Pi17316 requires plasma membrane localisation and the presence of VIK in the host in order to suppress the INF1 and R2/Avr2 pathways to promote P. infestans colonisation.

The evolution of plant roots transformed the Earth's surface, engineering new ecosystems and environments, and providing the nutrient and water uptake, as well as anchorage necessary to support the first trees. The lycophytes (clubmosses) were the first group of plants to evolve roots and the study of their morphology and evolution has been a major goal for evolutionary scientists working on both extant and extinct lycopsids. The aim of the research described in this thesis is to increase our understanding of both the morphology and evolution of lycopsid roots. This is achieved by presenting three papers on the theme of root morphology and evolution. First, I report the discovery of root hairs on extinct stigmarian rootlets, highlighting the conserved morphology of all Isoetalean rootlets. Second, in my discovery of the oldest fossilized root meristem, I illustrate how the interpretation of exceptionally well preserved fossils can change the way we think about the evolution of development of living plant roots. Third, I identify that the rootlets of Isoetes echinospora and roots of Selaginella moellendorffii have similar gene expression profiles. The new results reported in this thesis taken together with a review of the literature of extant and extinct lycopsid rooting structures, enabled me to identify two contrasting evolutionary patterns: conservatism of lycopsid roots, and huge disparity in the structures to which roots are attached. The highly conserved nature of lycopsid roots, supported by the new data presented in this thesis, is consistent with the hypothesis that all lycopsid roots are homologous (described as the lycopsid root hypothesis). In recognising the homology of lycopsid roots, and the two contrasting patterns of rooting structure evolution, the research presented in this thesis makes a significant contribution to our understanding of the morphology and evolution of lycopsid roots.

A fundamental challenge in biology is to explain how different organisms develop the intricate anatomical forms observed in nature. During morphogenesis, biochemical, mechanical, and geometric information has to be integrated across multiple organisational scales, from multicellular organs over individual cells down to subcellular components. In multicellular plants, individual cells are fixed in their relative position in the tissue by their surrounding rigid cell wall. Therefore, plant organogenesis requires control over division plane orientation and anisotropic cell wall growth, which each require spatial patterning of cells. Polyhedral plant cells can display complex patterning in which individual faces are established as biochemically distinct domains by endomembrane trafficking. This thesis shows that the specification of an additional cellular spatial domain by the endomembrane system, a cell's geometric edges, is crucial for lateral root organogenesis. Membrane vesicles lying immediately beneath the plasma membrane at cell edges were revealed through localisation of RAB-A5c, a plant GTPase of the Rab family of membrane-trafficking regulators. RAB-A5c compartments were enriched at growing edges, and associated with the cortical microtubule array. Specific inhibition of RAB-A5c activity grossly perturbed cell geometry and organ growth in developing lateral organs. Quantitative 4D cell growth analysis revealed RAB-A5c inhibition indedependently caused perturbations of cell growth anisotropy and cytokinesis without disrupting default membrane trafficking. To investigate the underlying mechanism of RAB-A5c-mediated growth regulation, hypotheses for RAB-A5c function in modulating cortical microtubule array orientation and cell wall mechanical properties were tested. Based on a combination of quantitative image analysis, pharmacological and genetic approaches, and mechanical modelling, RAB-A5c is proposed to locally modify cell wall properties during growth, and two possible models for RAB-A5c function and their implications for cell-, tissue- and organ-level growth control are discussed.

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