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This thesis presents an investigation into the development of plasmodesmata (PD), which are specialised pores in plant cell walls through which the cytosol and membranes of neighbouring cells are linked. Modification of PD from their initial single-tube (‘simple’) structures to branched (‘complex’) structures is an important part of tissue maturation as it allows cells to restrict the movement of syplasmically mobile molecules including hormones, RNAs and proteins. Conversion of PD from simple to complex is co-ordinated across large populations of cells to produce symplasmic domains, transport barriers, and preferential transport pathways. The development of PD is therefore intrinsic to the wider development and morphogenesis of cells, tissues, and organs. The aim of this project was to investigate the development of PD from simple to complex, particularly during the predictable, large-scale conversion of PD structure that accompanies the leaf transition from sink state to source. To study this I used transgenic plants expressing a GFP-tagged viral protein which accumulates specifically in complex PD, while leaving simple PD unlabelled. The project follows the development of complex PD from the early stages of leaf development to maturity using a range of microscopy techniques. Structured illumination microscopy was used to view labelled PD at super resolution, which gave new structural details about complex PD using a breakthrough technology. Conventional and high-throughput confocal and electron microscopy were used to localise PD within tissues in a broad survey of PD location in leaves to identify patterns of PD development. An imaging chamber was developed that allowed the development of complex PD to be viewed in real time and identified conditions that can trigger structural conversion of PD. Finally, a high-throughput microscopy study was performed to identify how hormones, sugar availability, environmental stresses, defence responses and inhibitors can affect PD development.

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The C₄ biochemical pathway is the most efficient form of photosynthesis in warm environments and introducing this system into globally significant, but less efficient, C₃ photosynthetic crops could bring major yield increases. The most photosynthetically efficient C₄ grass species have a specialised leaf anatomy characterised by high vein density with two distinct cell types radially arranged around the vascular bundles (Kranz anatomy). Although this anatomy was first described in 1882, the genetic regulators controlling Kranz development are still not known. In recent years, transcriptomic analysis has allowed researchers to identify candidate Kranz regulator genes, and a model for Kranz development has been proposed, however, this model has not been experimentally validated. This study used in situ hybridisation to visualise expression patterns for a set of candidate Kranz regulator genes in maize and the orthologous genes in rice. Further, morphological analysis of rice lines with constitutive expression of the candidate Kranz regulator genes AINTEGUMENTA 1 and DAG-LIKE 1 and 2 were used to characterise protein function during C₃ monocot leaf development. The function of maize AINTEGUMENTA in the C₃ eudicot arabidopsis was also investigated. The results of the in situ hybridisation experiments led to the refinement of the Kranz model and identified potential roles for the candidate regulators during leaf development. The constitutive expression experiments highlighted the regulatory differences between eudicot and monocot leaf development and implicated the regulation of auxin-cytokinin homeostatis as a key factor in Kranz development. Ultimately, this work can be used to guide research into Kranz development and has direct implications for engineering C₄ photosynthesis into rice.

Mikromatriser handlar numera inte bara om att alstra genuttrycksdata i snabb takt, utan det är minst lika viktigt att effektivt ta hand om informationen efteråt. I den här avhandlingen presenteras ett arbetsflöde för att mäta, lagra och analysera genuttrycksdata i asp och poppel (Populus spp.). En Populus} mikromatrisdatabas - UPSC--BASE - tillgänglig för alla intresserade, utvecklades i syfte att samla in och lagra genuttrycksdata. Flertalet analysverktyg gjordes samtidigt tillgängliga, för att möjliggöra ett smidigt arbetsflöde från rådata till biologiska slutsatser. En av de stora utmaningarna i analys av mikromatriser är att kunna särskilja bruset från värdefull biologisk information. Att studera träd som växer utomhus är komplext eftersom de interagerar med omgivningen i mycket större utsträckning än vad som är fallet i växthusets kontrollerade miljö. Det här arbetet visar att det är möjligt, med hjälp av avancerad statistik och god försöksplanering, att följa och jämföra genuttrycket i blad från aspar utomhus under flera år för att dra värdefulla slutsatser om geners reglering. Den lagrade biologiska informationen i UPSC-BASE är avsedd att vara en värdefull tillgång för växtfältet i stort. I databasen finns nästan hundra olika experiment som innefattar alltifrån unga blad till ved, insektsangrepp, kyla och torka samt studier av genmodifierade växter. Informationen kan användas både för jämförande studier inom olika aspförsök, men också för att jämföra med andra växter. För att illustrera möjligheterna, studerades och grupperades gener i blad baserat på hur de uppträder över alla dessa experiment. Dessa grupperingar användes sedan för att definiera gener som är viktiga i bladutvecklingen. Sammanfattningsvis ger arbetet som presenteras i denna avhandling tillgång till verktyg och kunskap för storskaliga studier av genuttryck och den lagrade informationen har bevisats vara en värdefull tillgång för mer ingående studier av geners reglering. / DNA microarray analysis today is not just generation of high-throughput data, much more attention is paid to the subsequent efficient handling of the generated information. In this thesis, a pipeline to generate, store and analyse Populus transcriptional data is presented A public Populus microarray database - UPSC--BASE - was developed to gather and store transcriptomic data. In addition, several tools were provided to facilitate microarray analysis without requirements for expert-level knowledge. The aim has been to streamline the workflow from raw data through to biological interpretation. Differentiating noise from valuable biological information is one of the challenges in DNA microarray analysis. Studying gene regulation in free-growing aspen trees represents a complex analysis scenario as the trees are exposed to, and interacting with, the environment to a much higher extent than under highly controlled conditions in the greenhouse. This work shows that, by using multivariate statistics and experimental planning, it is possible to follow and compare gene expression in leaves from multiple growing seasons, and draw valuable conclusions about gene expression from field-grown samples. The biological information in UPSC-BASE is intended to be a valuable transcriptomic resource also for the wider plant community. The database provides information from almost a hundred different experiments, spanning different developmental stages, tissue types, abiotic and biotic stresses and mutants. The information can potentially be used for both cross-experiment analysis and for comparisons against other plants, such as Arabidopsis or rice. As a demonstration of this, microarray experiments performed on Populus leaves were merged and genes preferentially expressed in leaves were organised in to regulons of co-regulated genes. Those regulons were used to define genes of importance in leaf development in Populus. Taken together, the work presented in this thesis provides tools and knowledge for large-scale transcriptional studies and the stored gene expression information has been proven to be a valuable information resource for in-depth studies about gene regulation.

A semi-herbaceous. pioneer plant Chryzanihtmoidts moniiifera was grown under varying environmental condition; in order to assess whether altering environmental variables would affect its hydraulic conductance and photo synthetic rates The plants were grown under sun and shade conditions, subjected to low and high watering treatments and to two different nutrient regimes. Steady-state gaseous exchange parameters, and whole-plant and leaf hydraulic conductance were measured on plants from each treatment. A key aspect of this study was to investigate how the following leaf components - petiole. major veins, minor veins and extravascular tissue - contributed to die overall resistance to water flow in the leaf (Ricaf). Vein orders were cut in specific sequences to interrupt water flow which then allowed the partitioning of leaf hydraulic resistances. The results showed that die maximum pbotosynthetic rate, under light saturating CO;. (A,^ was significantly affected by both nutrient and light treatments Environmental conditions (light, water and nutrient treatments) did not. however, affect the CO.- compensation point, or dark respiration of the measured A:C, curves for C. moniaftra. In terms of whole-plant hydraulic conductance, the shoot, stem and root were not significantly affected by environmental treatments. When investigating R«„. only the light treatments significantly affected the resistance of the petiole, extra vascular tissue, and minor vans Rf<trfc was found to be positively correlated with and contributed between 34-59 % of the total leaf resistance When considering the resistance of the leaf it was observed that the vascular tissue of the leaf contained up to 90 % of the total leaf resistance. The results from this study show that the hydraulic conductance of C. moniijfera was found to be significantly affected by light treatment; only. Water and nutrient treatments did not have a substantial impact upon the water flow of the plant. Leaf hydraulic resistance was partitioned differently to that of results from ocher studies, in that the petiole and major veins contained the majority of the leaf resistance In retrospect this study would have been more effective if C. monilitfera treatments were more severe, in terms of water and nutrients Further studies should focus on a comparison of leaf hydraulic resistance partitioning of other species, across a range of plant types. / Thesis (M.Sc.)-University of KwaZulu-Natal, Westville, 2008.

The C₄ photosynthetic cycle acts to concentrate CO₂ around the enzyme Rubisco. By doing so, C₄ photosynthesis leads to increased radiation, water and nitrogen use efficiencies. As such, C₄ photosynthesis is the most productive form of photosynthesis known. Because it enables such high levels of productivity there are large international efforts to introduce C₄ photosynthesis into non-C₄ crop species such as rice. Kranz anatomy is a characteristic leaf cellular arrangement of concentric rings of bundle sheath and mesophyll cells around closely spaced veins and is crucial to C₄ photosynthesis in almost all known examples. Despite the fact that Kranz has evolved on over 60 times independently little is known about the genetic regulation of Kranz development, as attempts to elucidate Kranz regulators using conventional mutagenesis screens have provided few insights. However, the advent of next generation DNA sequencing technologies has enabled the interrogation of genetic networks at a previously unprecedented scale. The work in this thesis describes a genome-wide transcriptomic analysis of leaf development in maize, a C₄ species, that develops both Kranz-type and non-Kranz-type leaves. Detailed bioinformatics analyses identified candidate regulators of both Kranz development and additional aspects of maize leaf development. Three of the identified Kranz candidates were functionally characterised in both C₄ and non-C₄ species. Furthermore, expression and phylogenetic analyses of GOLDEN2-LIKE (GLK) genes, a small transcription factor family previously implicated in C₄ development in maize, were extended to determine the generality of GLK function in C₄ evolution.

Two factors which affect leaf ontogeny and ultimate leaf size: (1) the rate and duration of cell expansion, and (2) the rate and duration of cell division, were examined for their role in the slowed early growth rate and smaller ultimate leaf size when plants are exposed to ultraviolet-B (UV-B) radiation. Rumex patientia L. was grown in controlled environment chambers under enhanced UV-B radiation (equivalent to daily solar UV-B irradiation at 40°N latitude in mid-May with an atmospheric ozone concentration of 0.20 atm-cm) and control treatments. The pattern of growth as expressed in changes of mean cell size of two distinct cell types, tissue cell density, and length of the entire blade are consistent with the hypothesis that the radiation primarily affects cell division rather than cell expansion. Furthermore, it appears that the radiation probably alters the rate rather than the duration of cell division. An understanding of the mechanism of radiation damage should facilitate prediction of how this stress may interact with other stresses to which plants are normally subjected.

ABSTRACT: Formation of organ shape is an intriguing yet largely unanswered question in developmental biology. Shapes arise as a result of tightly controlled spatial variation in the rates and directions of tissue expansion over the course of development; therefore, quantifying these growth patterns could provide information about the underlying mechanisms of morphogenesis. Here we present a novel technique and computational tools for quantifying growth and shape changes in developing leaves, with a few unique capabilities. This includes the ability to compute growth from three-dimensional (3D) coordinates, which makes this the first method suitable for studying leaf growth in species or mutants with non-flat leaves, as well as small leaves at early stages of development, and allows us to simultaneously capture 3D shape changes. In the following, we apply these methods to study growth and shape changes in the first rosette leaf of Arabidopsis thaliana. Results reveal clear spatiotemporal patterns in growth rates and directionality, and tissue deformation maps illustrate an intricate balance involved in maintaining a relatively flat leaf surface in wild type leaves. Semi-automated tools presented make a high throughput of data possible with this method, and algorithms for generating mean maps of growth will make it possible to perform standardized comparative analyses of growth patterns between wild type and mutants and/or between species. The methods presented in this thesis will therefore be useful for studying leaf growth and shape, to further investigate the mechanisms of morphogenesis.   RÉSUMÉ: Comment un organe acquiert sa forme particulière au cours du développement est une question intéressante mais largement non résolue. La forme d’un organe résulte de la façon dont les taux et directions de croissance de ses tissues varient dans l’espace et dans le temps. Quantifier les motifs de croissance est donc nécessaire pout élucider les mécanismes sous-jacents de la morphogenèse. Nous présentons ici une nouvelle méthodologie pour quantifier la croissance et les changements de forme dans les feuilles en développement. Cette méthodologie s’appuie sur le développement de nouvelles techniques expérimentales et de programmes informatiques, et présente des avantages uniques : la croissance de la surface des feuilles et le changement de forme peuvent être analysés en trois dimensions (3D), pour une longue période et de large déformations. De plus l’analyse de multiples échantillons permet de générer une cartographie moyenne des motifs de croissance à la surface des feuilles au cours de leur développement, ainsi qu’une description quantitative de la déformation des tissus sous l’effet de leur croissance. Dans cette thèse, nous présentons les résultats de croissance et de changements de forme de la première feuille de rosette d'Arabidopsis thaliana au cours de son développement. Les cartes moyennes de croissance révèlent des motifs spatio-temporels évidents tant pour les taux que pour les directions de croissance. De plus, la description de la déformation des tissus démontre l'équilibre complexe impliqué dans le maintien d'une surface relativement plane dans les feuilles. La méthode proposée et les logiciels associés permettra d’effectuer des analyses comparative de la croissance entre feuilles de type sauvage et feuilles de mutants aux formes altérées, afin d’élucider les mécanismes de la morphogenèse foliaire.