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Title
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A systems biology approach to understand leaf growth regulation in **Arabidopsis thaliana**
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Author
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Abstract
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| Adaptation of growth of individual organs in the context of an entire organism is crucial for plants, and influenced by various genetic and environmental factors. Besides its direct function as a food source, the leaf is the main photosynthetic organ that perceives light and converts solar energy into organic carbon. Moreover, from an evolutionary point of view, all floral organs (e.g. flowers and fruits) are modified leaves. Therefore, understanding the regulation of leaf growth is important and has two main objectives. The first one is to comprehend the molecular/biological processes that control leaf growth and development, and determine the final shape and size in response to the surrounding environment. The second aim is to improve crop yield in the context of changing climatic conditions, such as increase in temperature, salinity and drought by means of genetic engineering approaches. The development of leaves is an intriguing process, which is the result of a complex interplay of different (yet often overlapping) pathways. At the cellular level, leaf growth is strictly determined by spatial and temporal regulation of (only) two processes: cell proliferation which involves cell growth (i.e. enlargement of cell volume by increasing cytoplasmic volume) in combination with cell division (the process of dividing cellular content over two daughter cells and placing a new cell wall) and cell expansion (mostly vacuolar enlargement and turgor driven cell wall extension in absence of cell division), which are regulated at the molecular level by chemical concentrations (metabolites such as sugars, hormones and signaling peptides) and physical interactions (particularly turgor, wall stresses). This means that the plethora of molecular pathways, that have been demonstrated to be involved in leaf development, must be linked to these two processes. Therefore, it is essential to use an interdisciplinary approach to unravel the complex networks involved in leaf growth and development, instead of studying these regulatory processes separately. To comprehend leaf growth regulation in more detail, we performed a first analysis of the dynamics of cell division and expansion in all three dimensions throughout the development of Arabidopsis leaf. To achieve this, we extended the existing kinematic analysis from a single tissue layer to the full three dimensional structure of the leaf. The analysis showed that the leaf growth is highly anisotropic, where expansion rates in the lateral direction are higher than in the longitudinal direction. While, expansion rates in the anticlinal direction are an order of magnitude lower throughout leaf development. Moreover, petiole elongation rates are higher than longitudinal expansion rates in the blade. Detailed analysis of tissue layers showed that anticlinal expansion rates differ between cell types, with mesophyll tissues having higher expansion rates than epidermal cells. We subsequently demonstrated that low light affects cell division and expansion at different developmental stages of the leaf. Low light reduces leaf expansion rates, which are partly compensated by an increased duration of expansion. We found that the reduced leaf thickness caused by low light is associated with a reduced number of palisade cell layers, established prior to emergence of the leaf. We also studied the role of the phytohormone auxin in the response of leaf development to osmotic stress. Our work provides the first evidence of crosstalk between auxin signaling and osmotic stress response. Kinematic analysis showed the dual nature of auxin where it promotes leaf growth under optimal concentration but increases sensitivity under osmotic stress. Interestingly, transcript profiling of proliferating leaves shows the upregulation of auxin biosynthesis genes under stress while downregulation in response to exogenous auxin. In contrast, increased expression of transcripts related to deconjugation and degradation suggest low levels of conjugated auxin under osmotic stress, which was confirmed by detailed hormone measurements. Moreover, increased growth by exogenous auxin is also reflected by increased expression of transcripts related to cell division and expansion, while their reduced expression under stress is consistent with the decreased leaf growth under osmotic stress. One of the key findings from our work is that cell division plays a central role in leaf growth responses to environmental conditions. Therefore, to increase the understanding of the regulators controlling cell cycle, we studied the role of negative regulators of the cell cycle, Kip Related Proteins (KRPs) during leaf development. As KRPs is a family of seven genes having redundant function, single and multiple mutants were studied. It is demonstrated that single mutants cause only minor or no significant change in leaf growth, while double and triple mutants display increased leaf growth as a result of enhanced cell proliferation. Remarkably, the enlarged leaf of these mutants is a consequence of an increased embryo size. Furthermore, transcriptome analysis revealed the upregulation of the DNA synthesis machinery during leaf growth, which was also reflected in increased endoreduplication. In addition, we developed a mathematical model of the cell cycle which provides a theoretical explanation for the two-fold increase in endoreplication by inhibition of KRPs. In addition to KRPs, we also studied the effect of mild overexpression of positive cell cycle regulator CDKB2, which also results in enlarged leaves due to increased cell proliferation while cell expansion is not affected. In addition, embryo development is also found to be responsible for part of the increased leaf growth in the CDKB2 overexpression line. |
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Language
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English
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Publication
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Antwerpen
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Universiteit Antwerpen, Faculteit Wetenschappen, Departement Biologie
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2014
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ISBN
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978-90-5728-467-0
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Volume/pages
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277 p.
: ill.
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Full text (open access)
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