FInCH: FIJI plugin for automated and scalable whole-image analysis of protein expression and cell morphology.

Study of morphogenesis and its regulation requires analytical tools that enable simultaneous assessment of processes operating at cellular level, such as synthesis of transcription factors (TF), with their effects at the tissue scale. Most current studies conduct histological, cellular and immunochemical (IHC) analyses in separate steps, introducing inevitable biases in finding and alignment of areas of interest at vastly distinct scales of organization, as well as image distortion associated with image repositioning or file modifications. These problems are particularly severe for longitudinal analyses of growing structures that change size and shape. Here we introduce a python-based application for automated and complete whole-slide measurement of expression of multiple TFs and associated cellular morphology. The plugin collects data at customizable scale from the cell-level to the entire structure, records each data point with positional information, accounts for ontogenetic transformation of structures and variation in slide positioning with scalable grid, and includes a customizable file manager that outputs collected data in association with full details of image classification (e.g., ontogenetic stage, population, IHC assay). We demonstrate the utility and accuracy of this application by automated measurement of morphology and associated expression of eight TFs for more than six million cells recorded with full positional information in beak tissues across 12 developmental stages and 25 study populations of a wild passerine bird. Our script is freely available as an open-source Fiji plugin and can be applied to IHC slides from any imaging platforms and transcriptional factors.

Adaptation of Bacillus subtilis MreB Filaments to Osmotic Stress Depends on Influx of Potassium Ions.

The circumferential motion of MreB filaments plays a key role in cell shape maintenance in many bacteria. It has recently been shown that filament formation of MreB filaments in Bacillus subtilis is influenced by stress conditions. In response to osmotic upshift, MreB molecules were released from filaments, as seen by an increase in freely diffusive molecules, and the peptidoglycan synthesis pattern became less organized, concomitant with slowed-down cell extension. In this study, biotic and abiotic factors were analysed with respect to a possible function in the adaptation of MreB filaments to stress conditions. We show that parallel to MreB, its interactor RodZ becomes more diffusive following osmotic stress, but the remodeling of MreB filaments is not affected by a lack of RodZ. Conversely, mutant strains that prevent efficient potassium influx into cells following osmotic shock show a failure to disassemble MreB filaments, accompanied by less perturbed cell wall extension than is observed in wild type cells. Because potassium ions are known to negatively affect MreB polymerization in vitro, our data indicate that polymer disassembly is directly mediated by the physical consequences of the osmotic stress response. The lack of an early potassium influx response strongly decreases cell survival following stress application, suggesting that the disassembly of MreB filaments may ensure slowed-down cell wall extension to allow for efficient adaptation to new osmotic conditions.

Establishment of Physalis alkekengi cell suspension culture: time-dependent behavior of genes related to the steroidal compounds, key enzymes, and physalins under static magnetic field.

Cell suspension culture has the potential to be a valuable source for the bioactive compound productions. In this study, an optimized procedure was established for callus and cell suspension culture of Physalis alkekengi for the first time, and the impact of static magnetic field (SMF, 6 mT) was studied on the high-value metabolic compounds through investigation of signaling molecules and gene expressions at the late log-to-stationary phase. Results showed that the growth regulators of 6-benzyl amino purine (BAP, 1.5 mg-1 L) and 1-naphthaleneacetic acid (NAA, 0.4 mg-1 L) induced the highest fresh weight, callus rate, callus index, and total withanolides. Cell suspension culture was established in the liquid MS medium supplied with BAP (1.5 mg-1 L) and NAA (0.1 mg-1 L). SMF application decreased slightly the cell growth and viability and enhanced the number of round-shaped cells. The hydrogen peroxide (H2O2) and nitric oxide (NO) levels increased at an all-time series after SMF exposure, and their maximum contents were observed after 12 h. A significant alteration of malondialdehyde content was also identified after 12 h of SMF exposure. The expression of 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR), 1-deoxyD-xylulose 5-phosphate synthase (DXS), squalene synthase (SQS), sterol Δ7-reductase (DWF5), and C-7,8 sterol isomerase (HYD1) genes was upregulated significantly after 24 and 48 h. An increase in the total withanolides was related to more activity of HMGR and DXS enzymes in SMF-exposed cells and the maximum physalin A (12.8 mg g-1 DW) and physalin B (1.92 mg g-1 DW) obtained after 24 h compared to controls. Findings suggest that SMF can play a supportive factor in inducing steroidal compounds in P. alkekengi through modulating H2O2 and NO levels and the related-gene expressions.

Gears of life: A primer on the simple machines that shape the embryo.

A simple machine is a basic of device that takes mechanical advantage to apply force. Animals and plants self-assemble through the operation of a wide variety of simple machines. Embryos of different species actuate these simple machines to drive the geometric transformations that convert a disordered mass of cells into organized structures with discrete identities and function. These transformations are intrinsically coupled to sequential and overlapping steps of self-organization and self-assembly. The processes of self-organization have been explored through the molecular composition of cells and tissues and their information networks. By contrast, efforts to understand the simple machines underlying self-assembly must integrate molecular composition with the physical principles of mechanics. This primer is concerned with effort to elucidate the operation of these machines, focusing on the "problem" of morphogenesis. Advances in understanding self-assembly will ultimately connect molecular-, subcellular-, cellular- and meso-scale functions of plants and animals and their ability to interact with larger ecologies and environmental influences.

Chondrocyte deformation during the unloading phase of cyclic compression loading.

Cell volume and shape changes play a pivotal role in cellular mechanotransduction, governing cellular responses to external loading. Understanding the dynamics of cell behavior under loading conditions is essential to elucidate cell adaptation mechanisms in physiological and pathological contexts. In this study, we investigated the effects of dynamic cyclic compression loading on cell volume and shape changes, comparing them with static conditions. Using a custom-designed platform which allowed for simultaneous loading and imaging of cartilage tissue, tissues were subjected to 100 cycles of mechanical loading while measuring cell volume and shape alterations during the unloading phase at specific time points. The findings revealed a transient decrease in cell volume (13%) during the early cycles, followed by a gradual recovery to baseline levels after approximately 20 cycles, despite the cartilage tissue not being fully recovered at the unloading phase. This observed pattern indicates a temporal cell volume response that may be associated with cellular adaptation to the mechanical stimulus through mechanisms related to active cell volume regulation. Additionally, this study demonstrated that cell volume and shape responses during dynamic loading were significantly distinct from those observed under static conditions. Such findings suggest that cells in their natural tissue environment perceive and respond differently to dynamic compared to static mechanical cues, highlighting the significance of considering dynamic loading environments in studies related to cellular mechanics. Overall, this research contributes to the broader understanding of cellular behavior under mechanical stimuli, providing valuable insights into their ability to adapt to dynamic mechanical loading.

Involvement of ArlI, ArlJ, and CirA in archaeal type IV pilin-mediated motility regulation.

Many prokaryotes use swimming motility to move toward favorable conditions and escape adverse surroundings. Regulatory mechanisms governing bacterial flagella-driven motility are well-established; however, little is yet known about the regulation underlying swimming motility propelled by the archaeal cell surface structure, the archaella. Previous research showed that the deletion of the adhesion pilins (PilA1-6), subunits of the type IV pili cell surface structure, renders the model archaeon Haloferax volcanii non-motile. In this study, we used ethyl methanesulfonate mutagenesis and a motility assay to identify motile suppressors of the ∆pilA[1-6] strain. Of the eight suppressors identified, six contain missense mutations in archaella biosynthesis genes, arlI and arlJ. In trans expression of arlI and arlJ mutant constructs in the respective multi-deletion strains ∆pilA[1-6]∆arlI and ∆pilA[1-6]∆arlJ confirmed their role in suppressing the ∆pilA[1-6] motility defect. Additionally, three suppressors harbor co-occurring disruptive missense and nonsense mutations in cirA, a gene encoding a proposed regulatory protein. A deletion of cirA resulted in hypermotility, while cirA expression in trans in wild-type cells led to decreased motility. Moreover, quantitative real-time PCR analysis revealed that in wild-type cells, higher expression levels of arlI, arlJ, and the archaellin gene arlA1 were observed in motile early-log phase rod-shaped cells compared to non-motile mid-log phase disk-shaped cells. Conversely, ∆cirA cells, which form rods during both early- and mid-log phases, exhibited similar expression levels of arl genes in both growth phases. Our findings contribute to a deeper understanding of the mechanisms governing archaeal motility, highlighting the involvement of ArlI, ArlJ, and CirA in pilin-mediated motility regulation.IMPORTANCEArchaea are close relatives of eukaryotes and play crucial ecological roles. Certain behaviors, such as swimming motility, are thought to be important for archaeal environmental adaptation. Archaella, the archaeal motility appendages, are evolutionarily distinct from bacterial flagella, and the regulatory mechanisms driving archaeal motility are largely unknown. Previous research has linked the loss of type IV pili subunits to archaeal motility suppression. This study reveals three Haloferax volcanii proteins involved in pilin-mediated motility regulation, offering a deeper understanding of motility regulation in this understudied domain while also paving the way for uncovering novel mechanisms that govern archaeal motility. Understanding archaeal cellular processes will help elucidate the ecological roles of archaea as well as the evolution of these processes across domains.

Expanding science skills: teaching tissue culture, data analysis, and reporting through imaging the actin cytoskeleton.

Within the eukaryotic cell, the actin cytoskeleton is a crucial structural framework that maintains cellular form, regulates cell movement and division, and facilitates the internal transportation of proteins and organelles. External cues induce alterations in the actin cytoskeleton primarily through the activation of Rho GTPases, which then bind to a diverse array of effector proteins to promote the local assembly or disassembly of actin. We have harnessed the extensively studied functions of RhoA in the dynamics of the actin cytoskeleton to craft a practical series for Stage 2 Biology students. This series not only imparts essential tissue culture laboratory skills but also reinforces them through repetition. These activities are presented in a scenario designed for students to explore the function of a hypothetical RhoA family member. Students produce slides from transfected cells, undertake fluorescence microscopy, process the images using ImageJ, and compile their findings in a comprehensive scientific report. The composition of the report requires independent acquisition of new knowledge and synoptic learning. According to student feedback, this early experience greatly aids in solidifying and honing the skills required to report on more extensive and intricate research projects, such as capstone projects.

PRSS3/mesotrypsin as a putative regulator of the biophysical characteristics of epidermal keratinocytes in superficial layers.

Mesotrypsin, encoded by the PRSS3 gene, is a distinctive trypsin isoform renowned for its exceptional resistance to traditional trypsin inhibitors and unique substrate specificity. Within the skin epidermis, this protein primarily expresses in the upper layers of the stratified epidermis and plays a crucial role in processing pro-filaggrin (Pro-FLG). Although prior studies have partially elucidated its functions using primary cultured keratinocytes, challenges persist due to these cells' differentiation-activated cell death program. In the present study, HaCaT keratinocytes, characterized by minimal endogenous mesotrypsin expression and sustained proliferation in differentiated states, were utilized to further scrutinize the function of mesotrypsin. Despite the ready degradation of the intact form of active mesotrypsin in these cells, fusion with Venus, flanked by a peptide linker, enables evasion from the protein elimination machinery, thus facilitating activation of the Pro-FLG processing system. Inducing Venus-mesotrypsin expression in the cells resulted in a flattened phenotype and reduced proliferative capacity. Moreover, these cells displayed altered F-actin assembly, enhanced E-cadherin adhesive activity, and facilitated tight junction formation without overtly influencing epidermal differentiation. These findings underscore mesotrypsin's potentially pivotal role in shaping the characteristic cellular morphology of upper epidermal layers.

Buckling forces and the wavy folds between pleural epithelial cells.

Cell shapes in tissues are affected by the biophysical interaction between cells. Tissue forces can influence specific cell features such as cell geometry and cell surface area. Here, we examined the 2-dimensional shape, size, and perimeter of pleural epithelial cells at various lung volumes. We demonstrated a 1.53-fold increase in 2-dimensional cell surface area and a 1.43-fold increase in cell perimeter at total lung capacity compared to residual lung volume. Consistent with previous results, close inspection of the pleura demonstrated wavy folds between pleural epithelial cells at all lung volumes. To investigate a potential explanation for the wavy folds, we developed a physical simulacrum suggested by D'Arcy Thompson in On Growth and Form. The simulacrum suggested that the wavy folds were the result of redundant cell membranes unable to contract. To test this hypothesis, we developed a numerical simulation to evaluate the impact of an increase in 2-dimensional cell surface area and cell perimeter on the shape of the cell-cell interface. Our simulation demonstrated that an increase in cell perimeter, rather than an increase in 2-dimensional cell surface area, had the most direct impact on the presence of wavy folds. We conclude that wavy folds between pleural epithelial cells reflects buckling forces arising from the excess cell perimeter necessary to accommodate visceral organ expansion.

Epithelial UNC-23 limits mechanical stress to maintain glia-neuron architecture in C. elegans.

For an organ to maintain correct architecture and function, its diverse cellular components must coordinate their size and shape. Although cell-intrinsic mechanisms driving homotypic cell-cell coordination are known, it is unclear how cell shape is regulated across heterotypic cells. We find that epithelial cells maintain the shape of neighboring sense-organ glia-neuron units in adult Caenorhabditis elegans (C. elegans). Hsp co-chaperone UNC-23/BAG2 prevents epithelial cell shape from deforming, and its loss causes head epithelia to stretch aberrantly during animal movement. In the sense-organ glia, amphid sheath (AMsh), this causes progressive fibroblast growth factor receptor (FGFR)-dependent disruption of the glial apical cytoskeleton. Resultant glial cell shape alteration causes concomitant shape change in glia-associated neuron endings. Epithelial UNC-23 maintenance of glia-neuron shape is specific both spatially, within a defined anatomical zone, and temporally, in a developmentally critical period. As all molecular components uncovered are broadly conserved across central and peripheral nervous systems, we posit that epithelia may similarly regulate glia-neuron architecture cross-species.

Discretised Flux Balance Analysis for Reaction-Diffusion Simulation of Single-Cell Metabolism.

Metabolites have to diffuse within the sub-cellular compartments they occupy to specific locations where enzymes are, so reactions could occur. Conventional flux balance analysis (FBA), a method based on linear programming that is commonly used to model metabolism, implicitly assumes that all enzymatic reactions are not diffusion-limited though that may not always be the case. In this work, we have developed a spatial method that implements FBA on a grid-based system, to enable the exploration of diffusion effects on metabolism. Specifically, the method discretises a living cell into a two-dimensional grid, represents the metabolic reactions in each grid element as well as the diffusion of metabolites to and from neighbouring elements, and simulates the system as a single linear programming problem. We varied the number of rows and columns in the grid to simulate different cell shapes, and the method was able to capture diffusion effects at different shapes. We then used the method to simulate heterogeneous enzyme distribution, which suggested a theoretical effect on variability at the population level. We propose the use of this method, and its future extensions, to explore how spatiotemporal organisation of sub-cellular compartments and the molecules within could affect cell behaviour.

Osteopontin Rejuvenates Senescent Adipose-Derived Stem Cells and Restores their Bone Tissue Regenerative Function.

The regenerative function of stem cells is compromised when the proportion of senescent stem cells increases with ageing advance. Therefore, combating stem cell senescence is of great importance for stem cell-based tissue engineering in the elderly, but remains largely unexplored. Osteopontin (OPN), a glycosylated phosphoprotein, is one of the key extracellular matrix molecules in bone tissue. OPN activates various signalling pathways and modulates cellular activities, including cell senescence. However, the role of OPN in stem cell senescence remains largely unknown. This study aims to investigate if OPN modulates cell senescence and bone regenerative function in human adipose-derived mesenchymal stem cells (ASCs), and to determine the underlying mechanisms. We first developed a senescent ASC model using serial passaging until passage 10 (P10), in which senescent cells were characterised by reduced proliferation and osteogenic differentiation capacity compared to P4 ASCs. The conditioned medium from P10 ASCs exhibited a diminished trophic effect on human osteoblasts (HOBs), compared to that from P4 ASCs. P10 ASCs on OPN-coated surface showed rejuvenated phenotype and enhanced osteogenic differentiation. The conditioned medium from P10 ASCs on OPN-coating improved trophic effects on HOBs. OPN regulated the morphology of senescent ASCs, transforming them from a more rounded and flattened cell shape to an elongated shape with a smaller area. These findings demonstrated the effects of OPN in restoring senescent ASCs functions, possibly through a mechanism that involves the modulation of cell morphology, indicating that OPN might hold a great potential for rejuvenating senescent stem cells and could potentially open a new venue for regenerating bone tissue in age-related diseases.

Disease-related non-muscle myosin IIA D1424N rod domain mutation, but not R702C motor domain mutation, disrupts mouse ocular lens fiber cell alignment and hexagonal packing.

The mouse ocular lens is an excellent vertebrate model system for studying hexagonal cell packing and shape changes during tissue morphogenesis and differentiation. The lens is composed of two types of cells, epithelial and fiber cells. During the initiation of fiber cell differentiation, lens epithelial cells transform from randomly packed cells to hexagonally shaped and packed cells to form meridional row cells. The meridional row cells further differentiate and elongate into newly formed fiber cells that maintain hexagonal cell shape and ordered packing. In other tissues, actomyosin contractility regulates cell hexagonal packing geometry during epithelial tissue morphogenesis. Here, we use the mouse lens as a model to study the effect of two human disease-related non-muscle myosin IIA (NMIIA) mutations on lens cellular organization during fiber cell morphogenesis and differentiation. We studied genetic knock-in heterozygous mice with NMIIA-R702C motor domain or NMIIA-D1424N rod domain mutations. We observed that while one allele of NMIIA-R702C has no impact on lens meridional row epithelial cell shape and packing, one allele of the NMIIA-D1424N mutation can cause localized defects in cell hexagonal packing. Similarly, one allele of NMIIA-R702C motor domain mutation does not affect lens fiber cell organization while the NMIIA-D1424N mutant proteins disrupt fiber cell organization and packing. Our work demonstrates that disease-related NMIIA rod domain mutations (D1424N or E1841K) disrupt mouse lens fiber cell morphogenesis and differentiation.

Filament structure and subcellular organization of the bacterial intermediate filament-like protein crescentin.

The protein crescentin is required for the crescent shape of the freshwater bacterium Caulobacter crescentus (vibrioides). Crescentin forms a filamentous structure on the inner, concave side of the curved cells. It shares features with eukaryotic intermediate filament (IF) proteins, including the formation of static filaments based on long and parallel coiled coils, the protein's length, structural roles in cell and organelle shape determination and the presence of a coiled coil discontinuity called the "stutter." Here, we have used electron cryomicroscopy (cryo-EM) to determine the structure of the full-length protein and its filament, exploiting a crescentin-specific nanobody. The filament is formed by two strands, related by twofold symmetry, that each consist of two dimers, resulting in an octameric assembly. Crescentin subunits form longitudinal contacts head-to-head and tail-to-tail, making the entire filament non-polar. Using in vivo site-directed cysteine cross-linking, we demonstrated that contacts observed in the in vitro filament structure exist in cells. Electron cryotomography (cryo-ET) of cells expressing crescentin showed filaments on the concave side of the curved cells, close to the inner membrane, where they form a band. When comparing with current models of IF proteins and their filaments, which are also built from parallel coiled coil dimers and lack overall polarity, it emerges that IF proteins form head-to-tail longitudinal contacts in contrast to crescentin and hence several inter-dimer contacts in IFs have no equivalents in crescentin filaments. Our work supports the idea that intermediate filament-like proteins achieve their shared polymerization and mechanical properties through a variety of filament architectures.

Deep learning classification for macrophage subtypes through cell migratory pattern analysis.

Macrophages can exhibit pro-inflammatory or pro-reparatory functions, contingent upon their specific activation state. This dynamic behavior empowers macrophages to engage in immune reactions and contribute to tissue homeostasis. Understanding the intricate interplay between macrophage motility and activation status provides valuable insights into the complex mechanisms that govern their diverse functions. In a recent study, we developed a classification method based on morphology, which demonstrated that movement characteristics, including speed and displacement, can serve as distinguishing factors for macrophage subtypes. In this study, we develop a deep learning model to explore the potential of classifying macrophage subtypes based solely on raw trajectory patterns. The classification model relies on the time series of x-y coordinates, as well as the distance traveled and net displacement. We begin by investigating the migratory patterns of macrophages to gain a deeper understanding of their behavior. Although this analysis does not directly inform the deep learning model, it serves to highlight the intricate and distinct dynamics exhibited by different macrophage subtypes, which cannot be easily captured by a finite set of motility metrics. Our study uses cell trajectories to classify three macrophage subtypes: M0, M1, and M2. This advancement holds promising implications for the future, as it suggests the possibility of identifying macrophage subtypes without relying on shape analysis. Consequently, it could potentially eliminate the necessity for high-quality imaging techniques and provide more robust methods for analyzing inherently blurry images.

CuticleTrace: A toolkit for capturing cell outlines from leaf cuticle with implications for paleoecology and paleoclimatology.

Premise: Leaf epidermal cell morphology is closely tied to the evolutionary history of plants and their growth environments and is therefore of interest to many plant biologists. However, cell measurement can be time consuming and restrictive with current methods. CuticleTrace is a suite of Fiji and R-based functions that streamlines and automates the segmentation and measurement of epidermal pavement cells across a wide range of cell morphologies and image qualities. Methods and Results: We evaluated CuticleTrace-generated measurements against those from alternate automated methods and expert and undergraduate hand tracings across a taxonomically diverse 50-image data set of variable image qualities. We observed ~93% statistical agreement between CuticleTrace and expert hand-traced measurements, outperforming alternate methods. Conclusions: CuticleTrace is a broadly applicable, modular, and customizable tool that integrates data visualization and cell shape measurement with image segmentation, lowering the barrier to high-throughput studies of epidermal morphology by vastly decreasing the labor investment required to generate high-quality cell shape data sets.

Evolving topological order in the postnatal visceral pleura.

BACKGROUND: Changes in epithelial cell shape reflects optimal cell packing and the minimization of surface free energy, but also cell-cell interactions, cell proliferation, and cytoskeletal rearrangements. RESULTS: Here, we studied the structure of the rat pleura in the first 15 days after birth. After pleural isolation and image segmentation, the analysis demonstrated a progression of epithelial order from postnatal day 1 (P1) to P15. The cells with the largest surface area and greatest shape variability were observed at P1. In contrast, the cells with the smallest surface area and most shape consistency were observed at P15. A comparison of polygonal cell geometries demonstrated progressive optimization with an increase in the number of hexagons (six-sided) as well as five-sided and seven-sided polygons. Analysis of the epithelial organization with Voronoi tessellations and graphlet motif frequencies demonstrated a developmental path strikingly distinct from mathematical and natural reference paths. Graph Theory analysis of cell connectivity demonstrated a progressive decrease in network heterogeneity and clustering coefficient from P1 to P15. CONCLUSIONS: We conclude that the rat pleura undergoes a striking change in pleural structure from P1 to P15. Further, a geometric and network-based approach can provide a quantitative characterization of these developmental changes.

A dynamic bactofilin cytoskeleton cooperates with an M23 endopeptidase to control bacterial morphogenesis.

Bactofilins have emerged as a widespread family of cytoskeletal proteins with important roles in bacterial morphogenesis, but their precise mode of action is still incompletely understood. In this study, we identify the bactofilin cytoskeleton as a key regulator of cell growth in the stalked budding alphaproteobacterium Hyphomonas neptunium. We show that, in this species, bactofilin polymers localize dynamically to the stalk base and the bud neck, with their absence leading to unconstrained growth of the stalk and bud compartments, indicating a central role in the spatial regulation of cell wall biosynthesis. Database searches reveal that bactofilin genes are often clustered with genes for cell wall hydrolases of the M23 peptidase family, suggesting a functional connection between these two types of proteins. In support of this notion, we find that the H. neptunium M23 peptidase homolog LmdC interacts directly with bactofilin in vitro and is required for proper cell shape in vivo. Complementary studies in the spiral-shaped alphaproteobacterium Rhodospirillum rubrum again reveal a close association of its bactofilin and LmdC homologs, which co-localize at the inner curve of the cell, modulating the degree of cell curvature. Collectively, these findings demonstrate that bactofilins and M23 peptidases form a conserved functional module that promotes local changes in the mode of cell wall biosynthesis, thereby driving cell shape determination in morphologically complex bacteria.

The murein endopeptidase MepA regulated by MtrAB and MprAB participate in cell wall homeostasis.

The complete genome of Corynebacterium glutamicum contain a gene encoding murein endopeptidase MepA which maintain cell wall homeostasis by regulating peptidoglycan biosynthesis. In this study, we investigate the physiological function, localization and regulator of MepA. The result shows that mepA overexpression lead to peptidoglycan degradation and the defects in cell division. MepA-EGFP was shown to localizes exclusively at the cell cell septum. In addition, mepA overexpression increased cell permeability and reduced the resistance of cells to isoniazid, an antibiotic used to treat Mycobacterium tuberculosis infection. Furthermore, transcription analysis showed that mepA affected cell division and membrane transport pathways, and was coordinately regulated by the two-component systems MtrAB and MprAB(CgtS/R2).

The mechanism underlying asymmetric bending of lateral petals in Delphinium (Ranunculaceae).

During the process of flower opening, most petals move downward in the direction of the pedicel (i.e., epinastic movement). In most Delphinium flowers, however, their two lateral petals display a very peculiar movement, the mirrored helical rotation, which requires the twist of the petal stalk. However, in some lineages, their lateral petals also exhibit asymmetric bending that increases the degree of mirrored helical rotation, facilitating the formation of a 3D final shape. Notably, petal asymmetric bending is a novel trait that has not been noticed yet, so its morphological nature, developmental process, and molecular mechanisms remain largely unknown. Here, by using D. anthriscifolium as a model, we determined that petal asymmetric bending was caused by the localized expansion of cell width, accompanied by the specialized array of cell wall nano-structure, on the adaxial epidermis. Digital gene analyses, gene expression, and functional studies revealed that a class I homeodomain-leucine zipper family transcription factor gene, DeanLATE MERISTEM IDENTITY1 (DeanLMI1), contributes to petal asymmetric bending; knockdown of it led to the formation of explanate 2D petals. Specifically, DeanLMI1 promotes cell expansion in width and influences the arrangement of cell wall nano-structure on the localized adaxial epidermis. These results not only provide a comprehensive portrait of petal asymmetric bending for the first time but also shed some new insights into the mechanisms of flower opening and helical movement in plants.