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Sessile plants evolve diverse structures in response to complex environmental cues. These factors, in essence, involve mechanical stimuli, which must be sensed and coordinated properly by the plants to ensure effective growth and development. While we have accumulated substantial knowledge on plant mechanobiology, how plants translate mechanical information into three-dimensional structures is still an open question. In this review, we summarize our current understanding of plant mechanosensing at different levels, particularly using Arabidopsis as a model plant system. We also attempt to abstract the mechanosensing process and link the gaps from mechanical cues to the generation of complex plant structures. Here we review the recent advancements on mechanical response and transduction in plant morphogenesis, and we also raise several questions that interest us in different sections.

Growth and morphogenesis in plants depend on cell wall mechanics and on turgor pressure. Nanoindentation methods, such as atomic force microscopy (AFM), enable measurements of mechanical properties of a tissue at subcellular resolution, while confocal microscopy of tissues expressing fluorescent reporters indicates cell identity. Associating mechanical data with specific cells is essential to reveal the links between cell identity and cell mechanics. Here we describe an image analysis protocol that allows us to segment AFM scans containing information on tissue topography and/or mechanics, to stitch several scans in order to reconstitute an entire region of the tissue investigated, to segment the scans and label cells, and to associate labeled cells to the projection of confocal images. Thus all mechanical data can be mapped to the corresponding cells and to their identity. This protocol is implemented using NanoIndentation, a plugin that we are developing in the Fiji distribution of ImageJ.

Growth and morphogenesis in plants depend on cell wall mechanics and on turgor pressure. Nanoindentation methods, such as atomic force microscopy (AFM), enable measurements of mechanical properties of a tissue at subcellular resolution, while confocal microscopy of tissues expressing fluorescent reporters indicates cell identity. Associating mechanical data with specific cells is essential to reveal the links between cell identity and cell mechanics. Here we describe an image analysis protocol that allows us to segment AFM scans containing information on tissue topography and/or mechanics, to stitch several scans in order to reconstitute an entire region of the tissue investigated, to segment the scans and label cells, and to associate labeled cells to the projection of confocal images. Thus all mechanical data can be mapped to the corresponding cells and to their identity. This protocol is implemented using NanoIndentation, a plugin that we are developing in the Fiji distribution of ImageJ.

International audience; The formation of spatial and temporal patterns is an essential component of organismal development. Patterns can be observed on every level from subcellular to organismal and may emerge from local rules that correspond to the interactions between molecules, cells, or tissues. The emergence of robust patterns may seem in contradiction with the prominent heterogeneity at subcellular and cellular scales, however it has become increasingly clear that heterogeneity can be instrumental for pattern formation. Here we review recent examples in plant development, involving genetic regulation, cell arrangement, growth and signal gradient. We discuss how patterns emerge from local rules, whether heterogeneity is stochastic or can be patterned, and whether stochastic noise is amplified or requires filtering for robust patterns to be achieved. We also stress the importance of modelling in investigating such questions.

Protein-protein interactions (PPI) are essential for a plethora of biological processes. These interactions can be visualized and quantified with spatial resolution using Förster resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM) technology. Currently, FRET-FLIM is routinely used in cell biology, and it has become a powerful tool to map protein interactions in native environments. However, implementing this technology in living multicellular organism remains challenging, especially when dealing with developing plant embryos where tissues are confined in multiple cell layers preventing direct imaging. In this chapter, we describe a step-by-step protocol for studying PPI using FRET-FLIM of the two transcription factors SCARECROW and SHORTROOT in Arabidopsis embryos. We provide a detailed description from embryo isolation to data analysis and representation.

Protein-protein interactions (PPI) are essential for a plethora of biological processes. These interactions can be visualized and quantified with spatial resolution using Forster resonance energy transfer (FRET) measured by fluorescence lifetime imaging microscopy (FLIM) technology. Currently, FRET-FLIM is routinely used in cell biology, and it has become a powerful tool to map protein interactions in native environments. However, implementing this technology in living multicellular organism remains challenging, especially when dealing with developing plant embryos where tissues are confined in multiple cell layers preventing direct imaging. In this chapter, we describe a step-by-step protocol for studying PPI using FRET-FLIM of the two transcription factors SCARECROW and SHORTROOT in Arabidopsis embryos. We provide a detailed description from embryo isolation to data analysis and representation.

The shoot apical meristem (SAM) gives rise to all aerial organs of the plant. The cell walls are supposed to play a central role in this process, translating molecular regulation into dynamic changes of growth rates and directions, although their precise role in morphogenesis during organ formation remains not well understood. Here we investigate the role of xyloglucans (XyGs), which form a major, yet functionally poorly characterized, wall component in the SAM. Using immunolabeling, biochemical analysis, genetic approaches, micro-indentation, laser ablations and live imaging, we show that XyGs are important for meristem shape and phyllotaxis, although no difference in cell wall stiffness could be observed when XyGs are perturbed. Mutations in enzymes required for XyG synthesis also affect other cell wall components such as cellulose content and the pectin methylation status. Interestingly, we show that the control of cortical microtubules dynamics by the severing enzyme KATANIN becomes vital when XyGs are perturbed or absent. This suggests an active role of the cytoskeleton in compensating for altered wall composition.

The continuous solvent extraction operations for the removal of molybdenum from ammonium tungstate solution was conducted using a solvent extraction system consisting of 10% (v/v) N263 as extractant and 30% (v/v) isooctanol as modifier in sulfonated kerosene for 63 h under optimized conditions in mixer-settlers. The concentration of Mo in raffinate was less than 10 mg/L with mass ratio of Mo/WO3 less than 6.0 × 10−5 and the concentration of Mo in strip liquor reached 30 g/L with mass ratio of WO3/Mo less than 1.0 × 10−2, which was consistent with the results of batch tests. The loaded organic solution and the oxidized organic solution were characterized by infrared spectroscopy and the results demonstrated that molybdenum exists in the form of [Mo2O11(H2O)2]2− in oxidized organic solution. The theoretical calculation of temperature change of the organic solution in oxidation process was conducted. The oxidant of 7 wt% H2O2 was recommended to be used during industrial production and a cooling water system has to be installed around the oxidation tank at the same time. The new process exhibited significant advantages of low overall costs, large value increase due to molybdenum producibility of MSA and environmental friendliness without wastewater and residue generation.

Cell-to-cell heterogeneity is observed in many biological phenomena like gene expression, signalling, cell size regulation and growth. Notably, heterogeneity in cell size and growth rate prevails in many systems and impacts tissue patterning and macroscopic growth robustness. From physical perspective, cell volume change is driven by osmosis and the subsequent intracellular hydrostatic pressure, which sustains cellular osmotic potential and is confined by peripheral constraints (plasma membrane, cytoskeletal cortex, extracellular matrix or cell wall) in plant, animal, tumorous and microbial cells. Despite numerous studies in unicellular systems, the spatial variation of hydrostatic pressure in multicellular tissues, and its relation with cell-to-cell growth variability, remain elusive. Here, using atomic force microscopy, we demonstrate that hydrostatic pressure is highly heterogeneous between adjacent cells in the epidermis of Arabidopsis shoot apical meristem, and it unexpectedly correlates either positively or negatively with cellular growth rate depending on growth conditions. Combining experimental arguments and physical modelling of cell wall mechanics and osmosis within multicellular tissues, we show that heterogeneities in pressure and growth are not random, and they spontaneously emerge from cell size and tissue topology. Together, we propose that cellular pressure build-up, a physical phenomenon, and growth rate, a biological property, are innately heterogeneous and modulate cell size homeostasis in any compact tissue with inhomogeneous topology.

In plant biology, transient expression systems have become valuable approaches used routinely to rapidly study protein expression, subcellular localization, protein-protein interactions, and transcriptional activity prior to in vivo studies. When studying transcriptional regulation, luciferase reporter assays offer a sensitive readout for assaying promoter behavior in response to different regulators or environmental contexts and to confirm and assess the functional relevance of predicted binding sites in target promoters. This chapter aims to provide detailed methods for using luciferase reporter system as a rapid, efficient, and versatile assay to analyze transcriptional regulation of target genes by transcriptional regulators. We describe a series of optimized transient expression systems consisting of Arabidopsis thaliana protoplasts, infiltrated Nicotiana benthamiana leaves, and human HeLa cells to study the transcriptional regulations of two well-characterized transcriptional regulators SCARECROW (SCR) and SHORT-ROOT (SHR) on one of their targets, CYCLIN D6 (CYCD6).Here, we illustrate similarities and differences in outcomes when using different systems. The plant-based systems revealed that the SCR-SHR complex enhances CYCD6 transcription, while analysis in HeLa cells showed that the complex is not sufficient to strongly induce CYCD6 transcription, suggesting that additional, plant-specific regulators are required for full activation. These results highlight the importance of the system and suggest that including heterologous systems, such as HeLa cells, can provide a more comprehensive analysis of a complex gene regulatory network.

During multicellular development, specification of distinct cell fates is often regulated by the same transcription factors operating differently in distinct cis-regulatory modules, either through different protein complexes, conformational modification of protein complexes, or combinations of both. Direct visualization of different transcription factor complex states guiding specific gene expression programs has been challenging. Here we use in vivo FRET-FLIM (Förster resonance energy transfer measured by fluorescence lifetime microscopy) to reveal spatial partitioning of protein interactions in relation to specification of cell fate. We show that, in Arabidopsis roots, three fully functional fluorescently tagged cell fate regulators establish cell-type-specific interactions at endogenous expression levels and can form higher order complexes. We reveal that cell-type-specific in vivo FRET-FLIM distributions reflect conformational changes of these complexes to differentially regulate target genes and specify distinct cell fates., This work was supported by an NWO VIDI grant to I.B. and Y.L. Y.L. was further supported by ERC Advanced Grant SysArc and NWO Spinoza Grant to B.S. M.P. was supported by an NWO ALW-VIDI grant 864.09.015 and S.W.P. was supported by DFG-project WE 5343/1-1.

The Arabidopsis (Arabidopsis thaliana) CLAVATA2 (CLV2) gene encodes a leucine-rich repeat receptor-like protein (RLP) that is involved in controlling the stem cell population size in the shoot apical meristem. Our previous genome-wide functional analysis of 57 AtRLP genes revealed only a few phenotypes for mutant alleles, despite screening a wide range of growth and developmental stages and assaying sensitivity to various stress responses, including susceptibility toward pathogens. To gain further insight into the biological role of AtRLPs, in particular CLV2-related AtRLP genes, we tested their ability to complement the clv2 mutant phenotype. We found that out of four close CLV2 homologs tested, AtRLP2 and AtRLP12 could functionally complement the clv2 mutant when expressed under the control of the CLV2 promoter. This indicates that the functional specificity of these three genes is determined at the level of their transcriptional regulation. Single and double mutant combinations with impaired AtRLP2 and/or AtRLP12 did not show an aberrant phenotype, suggesting that other genes are redundant with these CLV2-like genes. To understand which protein domains are essential for CLV2 function and which parts are interchangeable between related CLV2-like proteins, we performed domain-deletion and domain-swap experiments. These experiments revealed that CLV2 remains functional without the island domain, whereas the C1 and C3 regions of the leucine-rich repeat domain are essential for functionality. Analysis of domain-swap constructs showed that the C3-G region of CLV2 can be replaced by that of AtRLP38, although it could not complement the clv2 mutant under control of the CLV2 promoter. This suggests that the C3-G region is conserved among related AtRLP members, whereas the C1 domain may determine the functional specificity of CLV2.