Xyloglucan deficiency leads to a reduction in turgor pressure and changes in cell wall properties, affecting early seedling establishment.

Xyloglucan is believed to play a significant role in cell wall mechanics of dicot plants. Surprisingly, Arabidopsis plants defective in xyloglucan biosynthesis exhibit nearly normal growth and development. We investigated a mutant line, cslc-Δ5, lacking activity in all five Arabidopsis cellulose synthase like-C (CSLC) genes responsible for xyloglucan backbone biosynthesis. We observed that this xyloglucan-deficient line exhibited reduced cellulose crystallinity and increased pectin levels, suggesting the existence of feedback mechanisms that regulate wall composition to compensate for the absence of xyloglucan. These alterations in cell wall composition in the xyloglucan-absent plants were further linked to a decrease in cell wall elastic modulus and rupture stress, as observed through atomic force microscopy (AFM) and extensometer-based techniques. This raised questions about how plants with such modified cell wall properties can maintain normal growth. Our investigation revealed two key factors contributing to this phenomenon. First, measurements of turgor pressure, a primary driver of plant growth, revealed that cslc-Δ5 plants have reduced turgor, preventing the compromised walls from bursting while still allowing growth to occur. Second, we discovered the conservation of elastic asymmetry (ratio of axial to transverse wall elasticity) in the mutant, suggesting an additional mechanism contributing to the maintenance of normal growth. This novel feedback mechanism between cell wall composition and mechanical properties, coupled with turgor pressure regulation, plays a central role in the control of plant growth and is critical for seedling establishment in a mechanically challenging environment by affecting shoot emergence and root penetration.

Macroscopic waves, biological clocks and morphogenesis driven by light in a giant unicellular green alga.

A hallmark of self-organisation in living systems is their capacity to stabilise their own dynamics, often appearing to anticipate and act upon potential outcomes. Caulerpa brachypus is a marine green alga consisting of differentiated organs resembling leaves, stems and roots. While an individual can exceed a metre in size, it is a single multinucleated giant cell. Thus Caulerpa presents the mystery of morphogenesis on macroscopic scales in the absence of cellularization. The experiments reported here reveal self-organised waves of greenness - chloroplasts - that propagate throughout the alga in anticipation of the day-night light cycle. Using dynamical systems analysis we show that these waves are coupled to a self-sustained oscillator, and demonstrate their entrainment to light. Under constant conditions light intensity affects the natural period and drives transition to temporal disorder. Moreover, we find distinct morphologies depending on light temporal patterns, suggesting waves of chlorophyll could link biological oscillators to metabolism and morphogenesis in this giant single-celled organism.

The Cell Tracking Challenge: 10 years of objective benchmarking.

The Cell Tracking Challenge is an ongoing benchmarking initiative that has become a reference in cell segmentation and tracking algorithm development. Here, we present a significant number of improvements introduced in the challenge since our 2017 report. These include the creation of a new segmentation-only benchmark, the enrichment of the dataset repository with new datasets that increase its diversity and complexity, and the creation of a silver standard reference corpus based on the most competitive results, which will be of particular interest for data-hungry deep learning-based strategies. Furthermore, we present the up-to-date cell segmentation and tracking leaderboards, an in-depth analysis of the relationship between the performance of the state-of-the-art methods and the properties of the datasets and annotations, and two novel, insightful studies about the generalizability and the reusability of top-performing methods. These studies provide critical practical conclusions for both developers and users of traditional and machine learning-based cell segmentation and tracking algorithms.

Elliot M. Meyerowitz.

Interview with Elliot Meyerowitz, who studies plant growth and development at Caltech.

Focused Ion Beam Milling and Cryo-electron Tomography Methods to Study the Structure of the Primary Cell Wall in Allium cepa.

Cryo-electron tomography (cryo-ET) is a formidable technique to observe the inner workings of vitrified cells at a nanometric resolution in near-native conditions and in three-dimensions. One consequent drawback of this technique is the sample thickness, for two reasons: i) achieving proper vitrification of the sample gets increasingly difficult with sample thickness, and ii) cryo-ET relies on transmission electron microscopy (TEM), requiring thin samples for proper electron transmittance (<500 nm). For samples exceeding this thickness limit, thinning methods can be used to render the sample amenable for cryo-ET. Cryo-focused ion beam (cryo-FIB) milling is one of them and despite having hugely benefitted the fields of animal cell biology, virology, microbiology, and even crystallography, plant cells are still virtually unexplored by cryo-ET, in particular because they are generally orders of magnitude bigger than bacteria, viruses, or animal cells (at least 10 µm thick) and difficult to process by cryo-FIB milling. Here, we detail a preparation method where abaxial epidermal onion cell wall peels are separated from the epidermal cells and subsequently plunge frozen, cryo-FIB milled, and screened by cryo-ET in order to acquire high resolution tomographic data for analyzing the organization of the cell wall. This protocol was validated in: Curr Biol (2022), DOI: 10.1016/j.cub.2022.04.024.

Tethering of cellulose synthase to microtubules dampens mechano-induced cytoskeletal organization in Arabidopsis pavement cells.

Mechanical forces control development in plants and animals, acting as cues in pattern formation and as the driving force of morphogenesis. In mammalian cells, molecular assemblies residing at the interface of the cell membrane and the extracellular matrix play an important role in perceiving and transmitting external mechanical signals to trigger physiological responses. Similar processes occur in plants, but there is little understanding of the molecular mechanisms and their genetic basis. Here, we show that the number and movement directions of cellulose synthase complexes (CSCs) at the plasma membrane vary during initial stages of development in the cotyledon epidermis of Arabidopsis, closely mirroring the microtubule organization. Uncoupling microtubules and CSCs resulted in enhanced microtubule co-alignment as caused by mechanical stimuli driven either by cell shape or by tissue-scale physical perturbations. Furthermore, micromechanical perturbation resulted in depletion of CSCs from the plasma membrane, suggesting a possible link between cellulose synthase removal from the plasma membrane and microtubule response to mechanical stimuli. Taken together, our results suggest that the interaction of cellulose synthase with cortical microtubules forms a physical continuum between the cell wall, plasma membrane and the cytoskeleton that modulates the mechano-response of the cytoskeleton.

A multiscale analysis of early flower development in Arabidopsis provides an integrated view of molecular regulation and growth control.

We have analyzed the link between the gene regulation and growth during the early stages of flower development in Arabidopsis. Starting from time-lapse images, we generated a 4D atlas of early flower development, including cell lineage, cellular growth rates, and the expression patterns of regulatory genes. This information was introduced in MorphoNet, a web-based platform. Using computational models, we found that the literature-based molecular network only explained a minority of the gene expression patterns. This was substantially improved by adding regulatory hypotheses for individual genes. Correlating growth with the combinatorial expression of multiple regulators led to a set of hypotheses for the action of individual genes in morphogenesis. This identified the central factor LEAFY as a potential regulator of heterogeneous growth, which was supported by quantifying growth patterns in a leafy mutant. By providing an integrated view, this atlas should represent a fundamental step toward mechanistic models of flower development.

Structure of the Bacterial Cellulose Ribbon and Its Assembly-Guiding Cytoskeleton by Electron Cryotomography.

Cellulose is a widespread component of bacterial biofilms, where its properties of exceptional water retention, high tensile strength, and stiffness prevent dehydration and mechanical disruption of the biofilm. Bacteria in the genus Gluconacetobacter secrete crystalline cellulose, with a structure very similar to that found in plant cell walls. How this higher-order structure is produced is poorly understood. We used cryo-electron tomography and focused-ion-beam milling of native bacterial biofilms to image cellulose-synthesizing Gluconacetobacter hansenii and Gluconacetobacter xylinus bacteria in a frozen-hydrated, near-native state. We confirm previous results suggesting that cellulose crystallization occurs serially following its secretion along one side of the cell, leading to a cellulose ribbon that can reach several micrometers in length and combine with ribbons from other cells to form a robust biofilm matrix. We were able to take direct measurements in a near-native state of the cellulose sheets. Our results also reveal a novel cytoskeletal structure, which we have named the cortical belt, adjacent to the inner membrane and underlying the sites where cellulose is seen emerging from the cell. We found that this structure is not present in other cellulose-synthesizing bacterial species, Agrobacterium tumefaciens and Escherichia coli 1094, which do not produce organized cellulose ribbons. We therefore propose that the cortical belt holds the cellulose synthase complexes in a line to form higher-order cellulose structures, such as sheets and ribbons.IMPORTANCE This work's relevance for the microbiology community is twofold. It delivers for the first time high-resolution near-native snapshots of Gluconacetobacter spp. (previously Komagataeibacter spp.) in the process of cellulose ribbon synthesis, in their native biofilm environment. It puts forward a noncharacterized cytoskeleton element associated with the side of the cell where the cellulose synthesis occurs. This represents a step forward in the understanding of the cell-guided process of crystalline cellulose synthesis, studied specifically in the Gluconacetobacter genus and still not fully understood. Additionally, our successful attempt to use cryo-focused-ion-beam milling through biofilms to image the cells in their native environment will drive the community to use this tool for the morphological characterization of other studied biofilms.

The Overlapping and Distinct Roles of HAM Family Genes in Arabidopsis Shoot Meristems.

In Arabidopsis shoot apical meristems (SAMs), a well-characterized regulatory loop between WUSCHEL (WUS) and CLAVATA3 (CLV3) maintains stem cell homeostasis by regulating the balance between cell proliferation and cell differentiation. WUS proteins, translated in deep cell layers, move into the overlaying stem cells to activate CLV3. The secreted peptide CLV3 then regulates WUS levels through a ligand-receptor mediated signaling cascade. CLV3 is specifically expressed in the stem cells and repressed in the deep cell layers despite presence of the WUS activator, forming an apical-basal polarity along the axis of the SAM. Previously, we proposed and validated a hypothesis that the HAIRY MERISTEM (HAM) family genes regulate this polarity, keeping the expression of CLV3 off in interior cells of the SAM. However, the specific role of each individual member of the HAM family in this process remains to be elucidated. Combining live imaging and molecular genetics, we have dissected the conserved and distinct functions of different HAM family members in control of CLV3 patterning in the SAMs and in the de novo shoot stem cell niches as well.

Cytoskeletal organization in isolated plant cells under geometry control.

The cytoskeleton plays a key role in establishing robust cell shape. In animals, it is well established that cell shape can also influence cytoskeletal organization. Cytoskeletal proteins are well conserved between animal and plant kingdoms; nevertheless, because plant cells exhibit major structural differences to animal cells, the question arises whether the plant cytoskeleton also responds to geometrical cues. Recent numerical simulations predicted that a geometry-based rule is sufficient to explain the microtubule (MT) organization observed in cells. Due to their high flexural rigidity and persistence length of the order of a few millimeters, MTs are rigid over cellular dimensions and are thus expected to align along their long axis if constrained in specific geometries. This hypothesis remains to be tested in cellulo Here, we explore the relative contribution of geometry to the final organization of actin and MT cytoskeletons in single plant cells of Arabidopsis thaliana We show that the cytoskeleton aligns with the long axis of the cells. We find that actin organization relies on MTs but not the opposite. We develop a model of self-organizing MTs in three dimensions, which predicts the importance of MT severing, which we confirm experimentally. This work is a first step toward assessing quantitatively how cellular geometry contributes to the control of cytoskeletal organization in living plant cells.