Gene expression analysis is essential for understanding the mechanisms involved in plant development. Here, we developed M2WISH, a protocol based on MicroWave treatment for Wholemount mRNA In Situ Hybridization in Arabidopsis. By permeabilizing tissues without damaging cellular organization this protocol results in high and homogeneous hybridization yields that enable systematic analysis of gene expression dynamics. Moreover, when combined with cellular histochemical staining, M2WISH successfully provides a cellular resolution of gene expression. Thus, we demonstrate the robustness of M2WISH with 10 genes on roots, aerial meristems, leaves, and embryos in the seed. We applied M2WISH to study the spatial dynamics of WUSCHEL (WUS) and CLAVATA3 (CLV3) expression during in vitro meristematic conversion of roots into shoot apical meristems. Thus, we showed that shoot apical meristems could arise from two different types of root structures that differed by their CLV3 gene expression patterns. We constructed 3D cellular representations of WUS and CLV3 gene co-expression pattern and stressed the variability inherent to meristem conversion. Thus, this protocol generates a large amount of data on the localization of gene expression, which can be used to model complex systems.

Genome organization within the 3D nuclear volume influences major biological processes but is completely lost during mitosis, which represents a major challenge to maintain cellular identity and cell fate. To restore a functional G1 nucleus for the next cell cycle, it is imperative to reestablish genome organization during post-mitotic nuclear assembly. Importantly, the configuration of linear chromosomes has been shown to directly impact spatial genome architecture. Both centromeres and telomeres are known to associate with nuclear structures, such as the nuclear envelope, and support chromatin distribution. Here, using high-resolution 3D imaging combined with 3D spatial statistics and modeling, we showed that telomeres generally followed a regular distribution compared to what is expected under a random organization. While the preferential localization of telomeres at nuclear periphery was restricted to early G1, we found a strong clustering of centromeres in addition to their predominant peripheral localization at all cell cycle stages. We then conducted a targeted screen using MadID to identify the molecular pathways driving or maintaining telomere anchoring to the nuclear envelope. Among these factors, we could show that LAP2α transiently localizes to telomeres in anaphase, at a stage where LAP2α initiates the reformation of the nuclear envelope. Moreover, co-depletion of LAP proteins and their partner BAF impacted telomere redistribution in the next interphase. There results suggest that in addition to their crucial role in genome protection, telomeres also participate in reshaping functional G1 nuclei after mitosis.

AbstractGene expression analysis is essential for understanding the mechanisms involved in plant development. Here, we developed M2WISH, a protocol based onMicroWave treatment forWholemount mRNA InSituHybridization in Arabidopsis. By permeabilizing tissues without damaging cellular organisation this protocol results in high and homogeneous hybridization yields that enables systematic analysis of gene expression dynamics. Moreover, when combined with cellular histochemical staining, M2WISH provides a cellular resolution of gene expression on roots, aerial meristems, leaves and embryos in the seed. We applied M2WISH to study the spatial dynamics ofWUSCHEL(WUS) andCLAVATA3(CLV3) expression duringin vitromeristematic conversion of roots into shoot apical meristems. Thus, we showed that shoot apical meristems could arise from two different types of root structures that differed by theirCLV3gene expression patterns. We constructed 3D cellular representations ofWUSandCLV3gene co-expression pattern, and stressed the variability inherent to meristem conversion. Thus, this protocol generates a large amount of data on the localization of gene expression, which can be used to model complex systems.

Plant organ morphogenesis spans several orders of magnitude in time and space. Because of limitations in live-imaging, analysing whole organ growth from initiation to mature stages typically rely on static data sampled from different timepoints and individuals. We introduce a new model-based strategy for dating organs and for reconstructing morphogenetic trajectories over unlimited time windows based on static data. Using this approach, we show that Arabidopsis thaliana leaves are initiated at regular 1-day intervals. Despite contrasted adult morphologies, leaves of different ranks exhibited shared growth dynamics, with linear gradations of growth parameters according to leaf rank. At the sub-organ scale, successive serrations from same or different leaves also followed shared growth dynamics, suggesting that global and local leaf growth patterns are decoupled. Analysing mutants leaves with altered morphology highlighted the decorrelation between adult shapes and morphogenetic trajectories, thus stressing the benefits of our approach in identifying determinants and critical timepoints during organ morphogenesis.

Adapting plants to sustainable cropping systems is a major challenge for facing climate change and promoting agroecological transition. Camelina sativa is an emerging oilseed crop species with climate-resilient properties that could be used in double-cropping systems, in particular as a summer catch crop. The availability of early-flowering camelina is essential in such cropping systems to allow full completion of the growth cycle during summer. Targeted induced gene variation (TIGV) was used in camelina on several flowering repressor genes identified in Arabidopsis to obtain early-flowering lines. Multiplex editing of 15 target genes representing FLOWERING LOCUS C, SHORT VEGETATIVE PHASE, LIKE HETEROCHROMATIN PROTEIN 1, TERMINAL FLOWER 1 and EARLY FLOWERING LOCUS 3 induced combinatorial mutations that were screened for early-flowering phenotypes. Certain mutants showing a stable early-flowering trait after five generations also presented additional phenotypes: determinate flowering, shorter stature and/or basal branching. Different combinations of mutations had a positive or negative impact on yield. This work demonstrates that efficient multiplex CRISPR is achievable in hexaploid plants like camelina, providing valuable genetic diversity for better selecting lines adapted to new cropping systems.

Boundary domains delimit and organize organ growth throughout plant development almost relentlessly, building plant architecture and morphogenesis. Boundary domains display reduced growth and orchestrate development of adjacent tissues in a non-cell-autonomous manner. How these two functions are achieved remains elusive despite the identification of several boundary-specific genes. Here, we show using morphometrics at the organ and cellular levels that leaf boundary domain development requires SPINDLY (SPY), an O-fucosyltransferase, to act as cell growth repressor. Furthermore, we show that SPY acts redundantly with the CUP-SHAPED COTYLEDON transcription factors (CUC2 and CUC3), which are major determinants of boundaries development. Accordingly, at the molecular level CUC2 and SPY repress a common set of genes involved in cell wall loosening, providing a molecular framework for the growth repression associated with boundary domains. Atomic force microscopy confirmed that young leaf boundary domain cells have stiffer cell walls than marginal outgrowth. This differential cell wall stiffness was reduced in spy mutant plants. Taken together, our data reveal a concealed CUC2 cell wall-associated gene network linking tissue patterning with cell growth and mechanics.

During organ development, morphological and topological changes jointly occur at the cellular and tissue levels. Hence, the systematic and integrative quantification of cellular parameters during growth is essential to better understand organogenesis. We developed an atlasing strategy to quantitatively map cellular parameters during organ growth. Our approach is based on the computation of prototypical shapes, which are average shapes of individual organs at successive developmental stages, whereupon statistical descriptors of cellular parameters measuredfrom individual organs are projected. We describe here the algorithmic pipeline we developed for 3D organ shape registration, based on the establishment of an organ-centered coordinate system and on the automatic parameterization of organ surface. Using our framework, dynamic developmental trajectories can be readily reconstructed using point-to-point interpolation between parameterized organ surfaces at different time points. We illustrate and validate our pipeline using 3D confocal images of developing plant leaves.

Analyzing gene expression patterns over the tissue of an organ during growth is a means to decipher the biological mechanisms that control its shaping. For this, the organ is often sampled and imaged in different individuals at different developmental stages. However, estimating a continuous developmental process from a discrete dataset is an arduous task, especially if both size and shape evolve. We propose a strategy to: 1) integrate individual data to produce a comprehensive representation of the dynamic of expression patterns in a growing tissue, and 2) superimpose and compare patterns coming from different experimental groups. This opens the way to the integrative atlasing of expression patterns in an organ during its development. To illustrate our approach, we use a particularly challenging organ that can encompass huge shape changes during growth, the plant leaf.

Morphogenesis is a complex process that integrates several mechanisms from the molecular to the organ scales. In plants, division and growth are the two fundamental cellular mechanisms that drive morphogenesis. However, little is known about how these mechanisms are coordinated to establish functional tissue structure. A fundamental bottleneck is the current lack of techniques to systematically quantify the spatio-temporal evolution of 3D cell morphology during organ growth. Using leaf development as a relevant and challenging model to study morphogenesis, we developed a computational framework for cell analysis and quantification from 3D images and for the generation of 3D cell shape atlas. A remarkable feature of leaf morphogenesis being the formation of a laminar-like structure, we propose to automatically separate the cells corresponding to the leaf sides in the segmented leaves, by applying a clustering algorithm. The performance of the proposed pipeline was experimentally assessed on a dataset of 46 leaves in an early developmental state.

The localization of proteins in specific domains or compartments in the 3D cellular space is essential for many fundamental processes in eukaryotic cells. Deciphering spatial organization principles within cells is a challenging task, in particular because of the large morphological variations between individual cells. We present here an approach for normalizing variations in cell morphology and for statistically analyzing spatial distributions of intracellular compartments from collections of 3D images. The method relies on the processing and analysis of 3D geometrical models that are generated from image stacks and that are used to build representations at progressively increasing levels of integration, ultimately revealing statistical significant traits of spatial distributions. To make this methodology widely available to end-users, we implemented our algorithmic pipeline into a user-friendly, multi-platform, and freely available software. To validate our approach, we generated 3D statistical maps of endomembrane compartments at subcellular resolution within an average epidermal root cell from collections of image stacks. This revealed unsuspected polar distribution patterns of organelles that were not detectable in individual images. By reversing the classical 'measure-then-average' paradigm, one major benefit of the proposed strategy is the production and display of statistical 3D representations of spatial organizations, thus fully preserving the spatial dimension of image data and at the same time allowing their integration over individual observations. The approach and software are generic and should be of general interest for experimental and modeling studies of spatial organizations at multiple scales (subcellular, cellular, tissular) in biological systems.

A major challenge in morphometrics is to analyse complex biological shapes formed by structures at different scales. Leaves exemplify this challenge as they combine differences in their overall shape with smaller shape variations at their margin, leading to lobes or teeth. Current methods based on contour or on landmark analysis are successful in quantifying either overall leaf shape or leaf margin dissection, but fail in combining the two. Here, we present a comprehensive strategy and its associated freely available platform for the quantitative, multiscale analysis of the morphology of leaves with different architectures. For this, biologically relevant landmarks are automatically extracted and hierarchised, and used to guide the reconstruction of accurate average contours that properly represent both global and local features. Using this method, we establish a quantitative framework of the developmental trajectory of Arabidopsis leaves of different ranks and retrace the origin of leaf heteroblasty. When applied to different mutant forms, our method can contribute to a better understanding of gene function, as we show here for the role of CUC2 during Arabidopsis leaf serration. Finally, we illustrate the wider applicability of our tool by analysing hand morphometrics.

Plant growth and organ formation depend on the oriented deposition of load-bearing cellulose microfibrils in the cell wall. Cellulose is synthesized by a large relative molecular weight cellulose synthase complex (CSC), which comprises at least three distinct cellulose synthases. Cellulose synthesis in plants or bacteria also requires the activity of an endo-1,4-β-d-glucanase, the exact function of which in the synthesis process is not known. Here, we show, to our knowledge for the first time, that a leaky mutation in the Arabidopsis (Arabidopsis thaliana) membrane-bound endo-1,4-β-d-glucanase KORRIGAN1 (KOR1) not only caused reduced CSC movement in the plasma membrane but also a reduced cellulose synthesis inhibitor-induced accumulation of CSCs in intracellular compartments. This suggests a role for KOR1 both in the synthesis of cellulose microfibrils and in the intracellular trafficking of CSCs. Next, we used a multidisciplinary approach, including live cell imaging, gel filtration chromatography analysis, split ubiquitin assays in yeast (Saccharomyces cerevisiae NMY51), and bimolecular fluorescence complementation, to show that, in contrast to previous observations, KOR1 is an integral part of the primary cell wall CSC in the plasma membrane.

During cytokinesis a new crosswall is rapidly laid down. This process involves the formation at the cell equator of a tubulo-vesicular membrane network (TVN). This TVN evolves into a tubular network (TN) and a planar fenestrated sheet, which extends at its periphery before fusing to the mother cell wall. The role of cell wall polymers in cell plate assembly is poorly understood. We used specific stains and GFP-labelled cellulose synthases (CESAs) to show that cellulose, as well as three distinct CESAs, accumulated in the cell plate already at the TVN stage. This early presence suggests that cellulose is extruded into the tubular membrane structures of the TVN. Co-localisation studies using GFP-CESAs suggest the delivery of cellulose synthase complexes (CSCs) to the cell plate via phragmoplast-associated vesicles. In the more mature TN part of the cell plate, we observed delivery of GFP-CESA from doughnut-shaped organelles, presumably Golgi bodies. During the conversion of the TN into a planar fenestrated sheet, the GFP-CESA density diminished, whereas GFP-CESA levels remained high in the TVN zone at the periphery of the expanding cell plate. We observed retrieval of GFP-CESA in clathrin-containing structures from the central zone of the cell plate and from the plasma membrane of the mother cell, which may contribute to the recycling of CESAs to the peripheral growth zone of the cell plate. These observations, together with mutant phenotypes of cellulose-deficient mutants and pharmacological experiments, suggest a key role for cellulose synthesis already at early stages of cell plate assembly.

In eukaryotes, the interphase nucleus is organized in morphologically and/or functionally distinct nuclear "compartments". Numerous studies highlight functional relationships between the spatial organization of the nucleus and gene regulation. This raises the question of whether nuclear organization principles exist and, if so, whether they are identical in the animal and plant kingdoms. We addressed this issue through the investigation of the three-dimensional distribution of the centromeres and chromocenters. We investigated five very diverse populations of interphase nuclei at different differentiation stages in their physiological environment, belonging to rabbit embryos at the 8-cell and blastocyst stages, differentiated rabbit mammary epithelial cells during lactation, and differentiated cells of Arabidopsis thaliana plantlets. We developed new tools based on the processing of confocal images and a new statistical approach based on G- and F- distance functions used in spatial statistics. Our original computational scheme takes into account both size and shape variability by comparing, for each nucleus, the observed distribution against a reference distribution estimated by Monte-Carlo sampling over the same nucleus. This implicit normalization allowed similar data processing and extraction of rules in the five differentiated nuclei populations of the three studied biological systems, despite differences in chromosome number, genome organization and heterochromatin content. We showed that centromeres/chromocenters form significantly more regularly spaced patterns than expected under a completely random situation, suggesting that repulsive constraints or spatial inhomogeneities underlay the spatial organization of heterochromatic compartments. The proposed technique should be useful for identifying further spatial features in a wide range of cell types.

Background: Fluorescent hybridization techniques are widely used to study the functional organization of different compartments within the mammalian nucleus. However, few examples of such studies are known in the plant kingdom. Indeed, preservation of nuclei 3D structure, which is required for nuclear organization studies, is difficult to fulfill.Results: We report a rapid protocol for fluorescent in situ hybridization (FISH) performed on 3D isolated nuclei and thin cryosectioned leaves of Arabidopsis thaliana. The use of direct labeling minimized treatment steps, shortening the overall procedure. Using image analysis, we measured different parameters related to nucleus morphology and overall 3D structure.Conclusion: Our work describes a 3D-FISH protocol that preserves the 3D structure of Arabidopsis interphase nuclei. Moreover, we report for the first time FISH using cryosections of Arabidopsis leaves. This protocol is a valuable tool to investigate nuclear architecture and chromatin organization.

In confocal cellular imaging, fluorescent markers are used to label target structures. These elements of interest frequently appear as spots within a noisy background. Several algorithms have been proposed to extract spots in such conditions. However, specific methods are required when other structures are also labelled. In this paper, a new spot extraction filter is proposed to discriminate between spots and other structures using the shape of the local spatial intensity distribution. Preliminary results in the analysis of the intra-cellular distribution of the KORRIGAN1 protein in plant cells are presented. Very low detection error rates are reported.