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Imaging as a tool to study leaf development in Arabidopsis thaliana

Stijn Dhondt UGent (2012)
abstract
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.
Please use this url to cite or link to this publication:
author
promoter
UGent and UGent
organization
year
type
dissertation (monograph)
subject
pages
227 pages
publisher
Ghent University. Faculty of Sciences
place of publication
Ghent, Belgium
defense location
Zwijnaarde : Technologiepark (FSVM building)
defense date
2012-03-02 16:00
language
English
UGent publication?
yes
classification
D1
additional info
dissertation consists of copyrighted material
copyright statement
I have transferred the copyright for this publication to the publisher
id
3005523
handle
http://hdl.handle.net/1854/LU-3005523
date created
2012-10-03 15:50:07
date last changed
2012-10-05 09:23:58
@phdthesis{3005523,
  abstract     = {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{\textquoteright}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.},
  author       = {Dhondt, Stijn},
  language     = {eng},
  pages        = {227},
  publisher    = {Ghent University. Faculty of Sciences},
  school       = {Ghent University},
  title        = {Imaging as a tool to study leaf development in Arabidopsis thaliana},
  year         = {2012},
}

Chicago
Dhondt, Stijn. 2012. “Imaging as a Tool to Study Leaf Development in Arabidopsis Thaliana”. Ghent, Belgium: Ghent University. Faculty of Sciences.
APA
Dhondt, S. (2012). Imaging as a tool to study leaf development in Arabidopsis thaliana. Ghent University. Faculty of Sciences, Ghent, Belgium.
Vancouver
1.
Dhondt S. Imaging as a tool to study leaf development in Arabidopsis thaliana. [Ghent, Belgium]: Ghent University. Faculty of Sciences; 2012.
MLA
Dhondt, Stijn. “Imaging as a Tool to Study Leaf Development in Arabidopsis Thaliana.” 2012 : n. pag. Print.