Macro optical projection tomography for large scale 3D imaging of plant structures and gene activity

Optical projection tomography (OPT) is a well-established method for visualising gene activity in plants and animals. However, a limitation of conventional OPT is that the specimen upper size limit precludes its application to larger structures. To address this problem we constructed a macro version called Macro OPT (M-OPT). We apply M-OPT to 3D live imaging of gene activity in growing whole plants and to visualise structural morphology in large optically cleared plant and insect specimens up to 60 mm tall and 45 mm deep. We also show how M-OPT can be used to image gene expression domains in 3D within fixed tissue and to visualise gene activity in 3D in clones of growing young whole Arabidopsis plants. A further application of M-OPT is to visualise plant-insect interactions. Thus M-OPT provides an effective 3D imaging platform that allows the study of gene activity, internal plant structures and plant-insect interactions at a macroscopic scale

Sculpting the surface: Structural patterning of plant epidermis.

Plant epidermis are multifunctional surfaces that directly affect how plants interact with animals or microorganisms and influence their ability to harvest or protect from abiotic factors. To do this, plants rely on minuscule structures that confer remarkable properties to their outer layer. These microscopic features emerge from the hierarchical organization of epidermal cells with various shapes and dimensions combined with different elaborations of the cuticle, a protective film that covers plant surfaces. Understanding the properties and functions of those tridimensional elements as well as disentangling the mechanisms that control their formation and spatial distribution warrant a multidisciplinary approach. Here we show how interdisciplinary efforts of coupling modern tools of experimental biology, physics, and chemistry with advanced computational modeling and state-of-the art microscopy are yielding broad new insights into the seemingly arcane patterning processes that sculpt the outer layer of plants

Structure and development of complex plasmodesmata

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

Imaging as a tool to study leaf development in Arabidopsis thaliana

In contrast to humans and animals, the body plan of a plant is not completely defined within the embryonic stages. Organ formation continues throughout plant development and this iterative and modular process is continuously controlled by environmental cues such as light, gravity, temperature, humidity and chemicals. In most plant species, the above-ground plant body is dominated by leaves, the organs specialized in photosynthesis. This process converts carbon dioxide into organic components utilizing energy from sunlight; making leaves the energy production site and the growth engine of plants. In addition, in many cases the majority of a plant’s biomass consists of leaves, also making them important organs for the production of food, feed and bio-energy. The final leaf size is determined by the total number of cells and the average cell size that result from cell division and cell expansion, respectively. During leaf development of dicotyledonous species, a cell proliferation phase, characterized by actively dividing cells, is followed by a cell expansion phase, characterized by cell growth and differentiation. After expansion, cells mature and the final leaf size is reached. At the proliferation-to-expansion phase transition, cell division ceases along a longitudinal gradient from leaf tip to base. In this thesis, we set out to gain further insight in these developmental processes affecting leaf size, assisted by the use of imaging technology and automated image analysis. For these studies we used the model species Arabidopsis thaliana, focusing primarily on the epidermis of the developing leaves as divisions there are strictly anticlinal. Moreover this layer is thought to be the main tissue layer controlling leaf growth. As a first step, we developed different image analysis tools to allow for a better and more efficient analysis of the leaf developmental process. In the first place we developed an online framework, designated Leaf Image Analysis Interface (LIMANI), in which venation patterns are automatically segmented and measured on dark-field images. Image segmentation may be manually corrected through use of an interactive interface, allowing supervision and rectification steps in the automated image analysis pipeline and ensuring high-fidelity analysis. We subsequently used this framework to study vascular differentiation during leaf development and to analyze the venation pattern in transgenic lines with contrasting cellular and leaf size traits. A major conclusion from this work was that, as vascular differentiation occurs relatively late in development, the influence of a fully functional and differentiated venation pattern on final leaf size is rather limited. Furthermore, we describe a proof-of-concept to automate the kinematic analysis of leaf growth based on DIC pictures, by a sophisticated image processing chain and a data analysis pipeline. Next, we also developed imaging scripts to extract complete seedlings grown on soil and on Petri dishes and integrated those into three phenotyping platforms which monitor plant growth. Finally, we investigated the potential of emerging imaging technologies, particularly X-ray computed tomography, for future applications in plant growth analysis. The newly developed kinematic analysis tools allowed us to show that the transcription factors, SHORT-ROOT (SHR) and SCARECROW (SCR), next to their specific roles in cortex/endodermis differentiation and stem cell maintenance in the root, primarily function as general regulators of cell proliferation in leaves. The analysis of leaf growth revealed how these proteins affect the cellular growth dynamics and formed the basis to unravel the molecular mechanism controlling this. It turned out that they promote leaf growth mainly by the down-regulation of cell cycle inhibitors, known to restrain the activity of the transcription factor, E2Fa, stimulating S-phase progression. Although the dynamics of cell division and cell expansion processes can be analyzed rigorously by the leaf growth kinematics, knowledge of cell cycle duration, cell expansion, and their interaction at the individual cell level is still poorly understood, not only because of technical obstacles to study these phenomena, but also because the processes are intimately intertwined, shown by the fact that a reduced cell proliferation is often compensated by an increase in cell size and vice versa. A mathematical model fitted to detailed cellular measurements retrieved by automated image analysis of microscopic drawings of the leaf epidermis, revealed that average cell cycle duration remains constant throughout leaf development. Surprisingly, no evidence for a maximum cell size threshold for cell division of pavement cells was found in this analysis. We could estimate the division and expansion parameters of pavement and guard cell populations within the growing leaf separately and the model predicted that neighboring cells of different sizes within the epidermis expand at distinctly different relative rates. We could finally verify this by direct observations using live imaging. The mathematical model helped us to gain a better and more detailed insight into the processes that define leaf growth. But the transition from cell proliferation to cell expansion was a developmental time point that was still not characterized in detail. Differences in the timing of this transition strongly affects the number of cells formed and therefore potentially also serves as a control point determining mature leaf size. Several genes have been identified that alter leaf size by affecting the transition from primary to secondary morphogenesis. We characterized the progression of the transition on the morphological and molecular level using transcriptome analysis and imaging algorithms to visualize and quantify the size and shape of pavement cells along the proximal-distal axis of the leaf during transition. Both analyses showed that the transition from cell proliferation to expansion was established and abolished abruptly. Furthermore, the establishment of the cell cycle arrest front occurs simultaneously with the onset of photomorphogenesis. We provide evidence that retrograde signaling from chloroplasts can affect the onset of transition, revealing a previously unknown level of regulatory complexity during the transition from primary to secondary morphogenesis