Browsing by Subject "Arabidopsis"

Development of a single cell into an adult organism is accomplished through an elaborate and complex cascade of spatiotemporal gene expression. While methods exist for capturing spatiotemporal expression patterns---in situ hybridization, reporter constructs, fluorescent tags---these methods have been highly laborious, and results are frequently assessed by subjective qualitative comparisons. To address these issues, methods must be developed for automating the capture of images, as well as for the normalization and quantification of the resulting data. In this thesis, I design computational approaches for high throughput image analysis which can be grouped into three main areas. First, I develop methods for the capture of high resolution images from high throughput platforms. In addition to the informatics aspect of this problem, I also devise a novel multiscale probabilistic model that allows us to identify and segment objects in an automated fashion. Second, high resolution images must be registered and normalized to a common frame of reference for cross image comparisons. To address these issues, I implement approaches for image registration using statistical shape models and non-rigid registration. Lastly, I validate the spatial expression data obtained from microscopy images to other known spatial expression methods, and develop methods for comparing and calculating the significance between spatial expression patterns. I demonstrate these methods on two model developmental organisms: Arabidopsis and Drosophila.

Temperature is an omnipresent environmental factor that shapes the growth, development and survival of plants. However, global warming has been an inevitable process and caused unusual temperature patterns across the world. As a consequence, forestry as well as agricultural plants are reportedly facing challenges from their environment. Several temperature responses in plant have been described, including short-term responses (such as acclimation) that increase tolerance towards sudden temperature stresses; as well as long-term responses (for example vernalization and flowering) that adjust growth and development to cope with seasonal temperature changes. However, the molecular mechanisms of how plants perceive temperature changes remain poorly understood. It has been observed for decades that one earliest response of plants towards low temperature is a transient increase of the cytosolic free Ca2+ concentration ([Ca2+]i). Considering the highly conserved role of [Ca2+]i increases in mediating thermal perception in animals, it has been speculated that [Ca2+]i increases may also play a role in thermal perception in plants. Nevertheless, despite intensive efforts, the molecular components responsible for cold-induced [Ca2+]i increases remain elusive. In this study, we carried out Ca2+-imaging-based forward genetic screen in Arabidopsis thaliana, isolated mutants defective in cold-induced [Ca2+]i increases (coca) and identified corresponding genes responsible for the coca phenotype through physical mapping. One of the mutants, named coca1, is highly specific to low temperature perception versus other stimuli, including osmotic, ionic and oxidative stimuli. coca1 displays compromised cold-induced [Ca2+] increases in both cotyledons and roots, as well as reduced growth fitness under ambient cool temperature. COCA1 encodes the dynamin-related protein 1A (DRP1A) and is localized on the plasma membrane. Our pharmacological studies showed that DRP1A acts upstream of plasma membrane rigidification and may mediate temperature perception by modification of membrane curvature which in turn opens Ca2+ channels. Alternatively, DRP1A may regulate endocytosis and channel activity through endocytosis signaling. Identification of coca1 as the first Arabidopsis mutant defective in cold-induced [Ca2+]i increases and DRP1A as a key player in thermal perception will greatly extend our understanding of plant adaptation to temperature changes, open up new avenues for studying Ca2+ signaling towards other stimuli and provide potential molecular genetic targets for engineering cold-resistant crops.

A cell’s trajectory from stem cell to differentiation, while often portrayed as a linear progression, is best described as a network that produces a mature state through several pathways acting together. There are few examples that describe gene regulatory network changes during the entire trajectory of cell differentiation. The goal of my project was to define the gene regulatory network required for a stem cell to become a differentiated cell in the Arabidopsis thaliana root. The root is a powerful model for identifying basic principles of differentiation. Plant cells do not migrate therefore entire lineages from stem cell to mature progeny are spatially confined. Furthermore, the root displays indeterminate growth, facilitating the study of many different developmental stages at a single time. One cell type of the root, the endodermis, is particularly suitable for study because the molecular components required for its formation and terminal differentiation are established. In order to understand the path from stem cell to differentiated cell in the endodermis, we asked what transcription factors are sufficient to program a non-native cell-type into endodermis. Our results show the transcription factors SHORTROOT and MYB36 have limited ability to reprogram a non-native cell-type (the epidermis) and that this reprogramming is reversible in the absence of additional cues. The stele-derived signaling peptide CIF2 stabilizes SHORTROOT-induced reprogramming. The outcome is a partially impermeable barrier deposited in the sub-epidermal cell layer that has a transcriptional signature similar to endodermis. The induction mechanism depends on MYB36 and CIF2’s receptor, but may be independent of the transcription factor SCARECROW. These results highlight a non cell-autonomous induction mechanism for endodermis that resembles differentiation in many animal systems.