CRISPR-TSKO : a technique for efficient mutagenesis in specific cell types, tissues, or organs in Arabidopsis

Detailed functional analyses of many fundamentally important plant genes via conventional loss-of-function approaches are impeded by the severe pleiotropic phenotypes resulting from these losses. In particular, mutations in genes that are required for basic cellular functions and/or reproduction often interfere with the generation of homozygous mutant plants, precluding further functional studies. To overcome this limitation, we devised a clustered regularly interspaced short palindromic repeats (CRISPR)-based tissue-specific knockout system, CRISPR-TSKO, enabling the generation of somatic mutations in particular plant cell types, tissues, and organs. In Arabidopsis (Arabidopsis thaliana), CRISPR-TSKO mutations in essential genes caused well-defined, localized phenotypes in the root cap, stomatal lineage, or entire lateral roots. The modular cloning system developed in this study allows for the efficient selection, identification, and functional analysis of mutant lines directly in the first transgenic generation. The efficacy of CRISPR-TSKO opens avenues for discovering and analyzing gene functions in the spatial and temporal contexts of plant life while avoiding the pleiotropic effects of system-wide losses of gene function

Study of the role of plant nuclear envelope and lamina-like components in nuclear and chromatin organisation using 3D imaging

The linker of nucleoskeleton and cytoskeleton (LINC) complex is an evolutionarily well-conserved protein bridge connecting the cytoplasmic and nuclear compartments across the nuclear membrane. While recent data supports its function in nuclear morphology and meiosis, its implication for chromatin organisation has been less studied in plants. The fi aim of this work was to develop NucleusJ a simple and user-friendly ImageJ plugin dedicated to the characterisation of nuclear morphol- ogy and chromatin organisation in 3D. NucleusJ quantifies 15 parameters including shape and size of nuclei as well as intra-nuclear objects and their position within the nucleus. A step-by-step documentation is available for self-training, together with data sets of nuclei with diff t nuclear organisation. Several improvements are ongoing to release a new version of this plugin. In a second part of this work, 3D imaging methods have been used to investigate nuclear morphology and chromatin organisation in interphase nuclei of the plant model Arabidopsis thaliana in which heterochromatin domains cluster in conspicuous chromatin regions called chromo- centres. Chromocentres form a repressive chromatin environment contributing to the transcriptional silencing of repeated sequences a general mechanism needed for genome stability. Quantitative measurements of 3D position of chromocentres in the nucleus indicate that most chromocentres are situated in close proximity to the periphery of the nucleus but that this distance can be altered according to nuclear volume or in specific mutants affecting the LINC complex. Finally, the LINC com- plex is proposed to contribute at the proper chromatin organisation and positioning since its alteration is associated with the release of transcriptional silencing as well as decompaction of heterochromatic sequences. The last part of this work takes ad- vantage of available genomic sequences and RNA-seq data to explore the evolution of NE proteins in plants and propose a minimal requirement to built the simplest functional NE. Altogether, work achieved in this thesis associate genetics, molecular biology, bioinformatics and imaging to better understand the contribution of the nuclear envelope in nuclear morphology and chromatin organisation and suggests the functional implication of the LINC complex in these processes

Accurate and versatile 3D segmentation of plant tissues at cellular resolution

Quantitative analysis of plant and animal morphogenesis requires accurate segmentation of individual cells in volumetric images of growing organs. In the last years, deep learning has provided robust automated algorithms that approach human performance, with applications to bio-image analysis now starting to emerge. Here, we present PlantSeg, a pipeline for volumetric segmentation of plant tissues into cells. PlantSeg employs a convolutional neural network to predict cell boundaries and graph partitioning to segment cells based on the neural network predictions. PlantSeg was trained on fixed and live plant organs imaged with confocal and light sheet microscopes. PlantSeg delivers accurate results and generalizes well across different tissues, scales, acquisition settings even on non plant samples. We present results of PlantSeg applications in diverse developmental contexts. PlantSeg is free and open-source, with both a command line and a user-friendly graphical interface

Imaging plant germline differentiation within Arabidopsis flowers by light sheet microscopy

In higher plants, germline differentiation occurs during a relatively short period within developing flowers. Understanding of the mechanisms that govern germline differentiation lags behind other plant developmental processes. This is largely because the germline is restricted to relatively few cells buried deep within floral tissues, which makes them difficult to study. To overcome this limitation, we have developed a methodology for live imaging of the germ cell lineage within floral organs of Arabidopsis using light sheet fluorescence microscopy. We have established reporter lines, cultivation conditions, and imaging protocols for high-resolution microscopy of developing flowers continuously for up to several days. We used multiview imagining to reconstruct a three-dimensional model of a flower at subcellular resolution. We demonstrate the power of this approach by capturing male and female meiosis, asymmetric pollen division, movement of meiotic chromosomes, and unusual restitution mitosis in tapetum cells. This method will enable new avenues of research into plant sexual reproduction.Web of Science9art. no. e5254

ETR1 Integrates Response to Ethylene and Cytokinins into a Single Multistep Phosphorelay Pathway to Control Root Growth

Cytokinins and ethylene control plant development via sensors from the histidine kinase (HK) family. However, downstream signaling pathways for the key phytohormones are distinct. Here we report not only cytokinin but also ethylene is able to control root apical meristem (RAM) size through activation of the multistep phosphorelay (MSP) pathway. We find both cytokinin and ethylene-dependent RAM shortening requires ethylene binding to ETR1 and its HK activity. The receiver domain of ETR1 interacts with MSP signaling intermediates acting downstream of cytokinin receptors, further substantiating the role of ETR1 in MSP signaling. Our studies find both cytokinin and ethylene induce the MSP in similar and distinct cell types with ETR1-mediated ethylene signaling controlling MSP output specifically in the root transition zone. We identified members of the MSP pathway specific and common to both hormones and show that ETR1-regulated ARR3 controls RAM size. ETR1-mediated MSP spatially differs from canonical CTR1/EIN2/EIN3 ethylene signaling and is independent of EIN2, indicating that both pathways can be spatially and functionally separated. Furthermore, we demonstrate that canonical ethylene signaling controls MSP responsiveness to cytokinin specifically in the root transition zone, presumably via regulation of ARR10, one of the positive regulators of MSP signaling in Arabidopsis

Quantitation of Cellular Dynamics in Growing Arabidopsis Roots with Light Sheet Microscopy

To understand dynamic developmental processes, living tissues must be imaged frequently and for extended periods of time. Root development is extensively studied at cellular resolution to understand basic mechanisms underlying pattern formation and maintenance in plants. Unfortunately, ensuring continuous specimen access, while preserving physiological conditions and preventing photo-damage, poses major barriers to measurements of cellular dynamics in indeterminately growing organs such as plant roots. We present a system that integrates optical sectioning through light sheet fluorescence microscopy with hydroponic culture that enables us to image at cellular resolution a vertically growing Arabidopsis root every few minutes and for several consecutive days. We describe novel automated routines to track the root tip as it grows, track cellular nuclei and identify cell divisions. We demonstrate the system's capabilities by collecting data on divisions and nuclear dynamics.Comment: * The first two authors contributed equally to this wor

CRNN: a joint neural network for redundancy detection

This article proposes a novel framework for detecting redundancy in supervised sentence categorisation. Unlike traditional singleton neural network, our model incorporates character-aware convolutional neural network (Char-CNN) with character-aware recurrent neural network (Char-RNN) to form a convolutional recurrent neural network (CRNN). Our model benefits from Char-CNN in that only salient features are selected and fed into the integrated Char-RNN. Char-RNN effectively learns long sequence semantics via sophisticated update mechanism. We compare our framework against the state-of-the-art text classification algorithms on four popular benchmarking corpus. For instance, our model achieves competing precision rate, recall ratio, and F1 score on the Google-news data-set. For twenty-news-groups data stream, our algorithm obtains the optimum on precision rate, recall ratio, and F1 score. For Brown Corpus, our framework obtains the best F1 score and almost equivalent precision rate and recall ratio over the top competitor. For the question classification collection, CRNN produces the optimal recall rate and F1 score and comparable precision rate. We also analyse three different RNN hidden recurrent cells’ impact on performance and their runtime efficiency. We observe that MGU achieves the optimal runtime and comparable performance against GRU and LSTM. For TFIDF based algorithms, we experiment with word2vec, GloVe, and sent2vec embeddings and report their performance differences

Root hydrotropism is controlled via a cortex-specific growth mechanism

Plants can acclimate by using tropisms to link the direction of growth to environmental conditions. Hydrotropism allows roots to forage for water, a process known to depend on abscisic acid (ABA) but whose molecular and cellular basis remains unclear. Here, we show that hydrotropism still occurs in roots after laser ablation removed the meristem and root cap. Additionally, targeted expression studies reveal that hydrotropism depends on the ABA signalling kinase, SnRK2.2, and the hydrotropism-specific MIZ1, both acting specifically in elongation zone cortical cells. Conversely, hydrotropism, but not gravitropism, is inhibited by preventing differential cell-length increases in the cortex, but not in other cell types. We conclude that root tropic responses to gravity and water are driven by distinct tissue-based mechanisms. In addition, unlike its role in root gravitropism, the elongation zone performs a dual function during a hydrotropic response, both sensing a water potential gradient and subsequently undergoing differential growth

Lateral root initiation in Arabidopsis thaliana: a force awakens

Osmotically driven turgor pressure of plant cells can be higher than that of a car tire. It puts tremendous forces onto cell walls and drives cell growth and changes in cell shape. This has given rise to unique mechanisms to control organ formation compared to metazoans. The fascinating interplay between forces and local cellular reorganization is still poorly understood. Growth of lateral roots is a prominent example of a developmental process in which mechanical forces between neighboring cells are generated and must be dealt with. Lateral roots initiate from a single cell layer that resides deep within the primary root. On their way out, lateral roots grow through the overlying endodermal, cortical, and epidermal cell layers. It was recently demonstrated that endodermal cells actively accommodate lateral root formation. Interfering genetically with these accommodating responses in the endodermis completely blocks cell proliferation in the pericycle. The lateral root system provides a unique opportunity to elucidate the molecular and cellular mechanisms whereby mechanical forces and intercellular communication regulate spatial accommodation during plant development