Top five unanswered questions in plant cell surface research.

Plant cell wall researchers were asked their view on what the major unanswered questions are in their field. This article summarises the feedback that was received from them in five questions. In this issue you can find equivalent syntheses for researchers working on bacterial, unicellular parasite and fungal systems.

Mind the mechanical strength: tailoring a 3D matrix to encapsulate isolated human preantral follicles.

STUDY QUESTION: Would a hydrogel with similar mechanical properties to the human ovarian cortex support preantral follicle development? SUMMARY ANSWER: Yes, our tailored PEGylated fibrin hydrogel was shown to significantly improve follicle growth in vitro. WHAT IS KNOWN ALREADY: One of the main challenges in developing an engineered ovary is to provide a 3D matrix that supports the follicle architecture and the interaction between granulosa cells and the oocyte as they are essential for folliculogenesis. Thanks to its biocompatibility and bioactivity, fibrin has been employed to fabricate a 3D matrix to encapsulate ovarian follicles. However, follicles lose their physical support within a few days owing to rapid fibrin degradation. Therefore, different strategies, including physical and chemical modifications, have been developed to enhance the stability of fibrin. STUDY DESIGN SIZE DURATION: By developing a matrix made of a synthetic (polyethylene glycol: PEG) and natural polymer (fibrin), we aimed to overcome fibrin degradation by the chemical reaction of PEGylation and tailor a PEGylated fibrin hydrogel formulation with mechanical strength similar to the ovarian cortex in women of reproductive age. To this end, response surface methodology was employed to obtain a tailored formulation of PEGylated fibrin. This hydrogel was then tested to encapsulate and support isolated human preantral follicles in vitro. PARTICIPANTS/MATERIALS SETTING METHODS: A PEGylated fibrin formulation was tailored using mathematical modeling software to mimic the mechanical properties of human ovarian tissue at reproductive age. Human preantral follicles were isolated from 11 patients of reproductive age and encapsulated in the tailored hydrogels, which were cultured in vitro for 4 or 7 days. Follicle survival and diameter were assessed on Days 1 and 7. Furthermore, the follicles were subjected to confocal microscopy to evaluate their growth (Ki67 staining) on Day 7 and analyze cell-cell communication (connexin 43 and transzonal projection staining) on Day 4. MAIN RESULTS AND THE ROLE OF CHANCE: In this study, mathematical modeling was applied to achieve the biomechanically tailored PEGylated fibrin formulation by targeting the specific goal of 3178 ± 245 Pascal, Young's modulus of ovarian cortical tissue in reproductive-age women. Our results demonstrated that the PEGylated fibrin hydrogel consisting of 39.06 mg/ml of PEGylated fibrinogen and 50.36 IU/ml of thrombin was the optimum condition with the desirability of 97.5%. This tailored hydrogel yielded a high follicle survival rate (83%) after 7 days of in vitro culture and supported its development up to the secondary stage. Follicle growth was confirmed by the presence of Ki67-positive granulosa cells on Day 7. Additionally, connexin 43 and Phalloidin staining indicated the retention of connections between granulosa cells and the oocyte. LARGE SCALE DATA: N/A. LIMITATIONS REASONS FOR CAUTION: In this study, our tailored hydrogel was only tested in vitro, which is not the same as the physiological environment. It is crucial to conduct a study assessing the follicles following their encapsulation in the tailored hydrogel and transplantation, which will be the next step of our investigation. WIDER IMPLICATIONS OF THE FINDINGS: The findings from this study introduced a suitable biomaterial similar to the ovarian cortex in reproductive-age women in terms of biomechanical properties for encapsulating human preantral follicles. This biomaterial allowed the radial growth of follicles and preserved their viability. Furthermore, PEGylation improved the stability of fibrin and the physical support of follicles. STUDY FUNDING/COMPETING INTERESTS: This study was supported by grants from the Fondation Louvain (PhD scholarship awarded to S.M., as part of a legacy from Mr Frans Heyes, and PhD scholarship awarded to A.D. as part of a legacy from Mrs Ilse Schirmer). The authors declare no competing interests.

Hormone-regulated expansins: Expression, localization, and cell wall biomechanics in Arabidopsis root growth.

Expansins facilitate cell expansion by mediating pH-dependent cell wall (CW) loosening. However, the role of expansins in controlling CW biomechanical properties in specific tissues and organs remains elusive. We monitored hormonal responsiveness and spatial specificity of expression and localization of expansins predicted to be the direct targets of cytokinin signaling in Arabidopsis (Arabidopsis thaliana). We found EXPANSIN1 (EXPA1) homogenously distributed throughout the CW of columella/lateral root cap, while EXPA10 and EXPA14 localized predominantly at 3-cell boundaries in the epidermis/cortex in various root zones. EXPA15 revealed cell-type-specific combination of homogenous vs. 3-cell boundaries localization. By comparing Brillouin frequency shift and AFM-measured Young's modulus, we demonstrated Brillouin light scattering (BLS) as a tool suitable for non-invasive in vivo quantitative assessment of CW viscoelasticity. Using both BLS and AFM, we showed that EXPA1 overexpression upregulated CW stiffness in the root transition zone (TZ). The dexamethasone-controlled EXPA1 overexpression induced fast changes in the transcription of numerous CW-associated genes, including several EXPAs and XYLOGLUCAN:XYLOGLUCOSYL TRANSFERASEs (XTHs), and associated with rapid pectin methylesterification determined by in situ Fourier-transform infrared spectroscopy in the root TZ. The EXPA1-induced CW remodeling is associated with the shortening of the root apical meristem, leading to root growth arrest. Based on our results, we propose that expansins control root growth by a delicate orchestration of CW biomechanical properties, possibly regulating both CW loosening and CW remodeling.

Contrasting Cd accumulation of Arabidopsis halleri populations: a role for (1→4)-ß-galactan in pectin.

Cadmium (Cd) accumulation is highly variable among Arabidopsis halleri populations. To identify cell wall (CW) components that contribute to the contrasting Cd accumulation between PL22-H (Cd-hyperaccumulator) and I16-E (Cd-excluder), Cd absorption capacity of CW polysaccharides, CW mono- and poly- saccharides contents and CW glycan profiles were compared between these two populations. PL22-H pectin contained 3-fold higher Cd concentration than I16-E pectin in roots, and (1→4)-ß-galactan pectic epitope showed the biggest difference between PL22-H and I16-E. CW-related differentially expressed genes (DEGs) between PL22-H and I16-E were identified and corresponding A. thaliana mutants were phenotyped for Cd tolerance and accumulation. A higher Cd translocation was observed in GALACTAN SYNTHASE1 A. thaliana knockout and overexpressor mutants, which both showed a lengthening of the RG-I sidechains after Cd treatment, contrary to the wild-type. Overall, our results support an indirect role for (1→4)-ß-galactan in Cd translocation, possibly by a joint effect of regulating the length of RG-I sidechains, the pectin structure and interactions between polysaccharides in the CW. The characterization of other CW-related DEGs between I16-E and PL22-H selected allowed to identify a possible role in Zn translocation for BIIDXI and LEUNIG-HOMOLOG genes, which are both involved in pectin modification.

Biochemical characterization of Pectin Methylesterase Inhibitor 3 from Arabidopsis thaliana.

The de-methylesterification of the pectic polysaccharide homogalacturonan (HG) by pectin methylesterases (PMEs) is a critical step in the control of plant cell expansion and morphogenesis. Plants have large gene families encoding PMEs but also PME inhibitors (PMEIs) with differ in their biochemical properties. The Arabidopsis thaliana PECTIN METHYLESTERASE INHIBITOR 3 (PMEI3) gene is frequently used as a tool to manipulate pectin methylesterase activity in studies assessing its role in the control of morphogenesis. One limitation of these studies is that the exact biochemical activity of this protein has not yet been determined. In this manuscript we produced the protein in Pichia pastoris and characterized its activity in vitro. Like other PMEIs, PMEI3 inhibits PME activity at acidic pH in a variety of cell wall extracts and in purified PME preparations, but does not affect the much stronger PME activity at neutral pH. The protein is remarkable heat stable and shows higher activity against PME3 than against PME2, illustrating how different members of the large PMEI family can differ in their specificities towards PME targets. Finally, growing Arabidopsis thaliana seedlings in the presence of purified PMEI3 caused a dose-dependent inhibition of root growth associated with the overall inhibition of HG de-methylesterification of the root surface. This suggests an essential in vivo role for PME activity at acidic pH in HG de-methylesterification and growth control. These results show that purified recombinant PMEI3 is a powerful tool to study the connection between pectin de-methylesterification and cell expansion.

A cell wall-associated gene network shapes leaf boundary domains.

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.

Proteome-wide and matrisome-specific atlas of the human ovary computes fertility biomarker candidates and open the way for precision oncofertility.

Our modern era is witnessing an increasing infertility rate worldwide. Although some of the causes can be attributed to our modern lifestyle (e.g., persistent organic pollutants, late pregnancy), our knowledge of the human ovarian tissue has remained limited and insufficient to reverse the infertility statistics. Indeed, all efforts have been focused on the endocrine and cellular function in support of the cell theory that dates back to the 18th century, while the human ovarian matrisome is still under-described. Hereby, we unveil the extracellular side of the story during different periods of the ovary life, demonstrating that follicle survival and development, and ultimately fertility, would not be possible without its involvement. We examined the human ovarian matrisome and described its remodeling from prepuberty until menopause, creating the first ovarian proteomic codex. Here, we confidently identified and quantified 98 matrisome proteins present in the three ovary groups. Among them, 26 were expressed differently among age groups, delineating a peculiar matrisomal fingerprint at each stage. Such proteins could be potential biomarkers phenotyping ovarian ECM at each age phase of female reproductive life. Beyond proteomics, our study presents a unique approach to understanding the data and depicting the spatiotemporal ECM-intracellular signaling networks and remodeling with age through imaging, advanced text-mining based on natural language processing technology, machine learning, and data sonification. Our findings provide essential context for healthy ovarian physiology, identifying and characterizing disease states, and recapitulating physiological tissues or development in vitro. This comprehensive proteomics analysis represents the ovarian proteomic codex and contributes to an improved understanding of the critical roles that ECM plays throughout the ovarian life span.

Second-order division in sectors as a prepattern for sensory organs in vertebrate development.

We present in vivo observations of chicken embryo development which show that the early chicken embryo presents a principal structure made out of concentric rings and a secondary structure composed of radial sectors. During development, physical forces deform the main rings into axially directed, antero-posterior tubes, while the sectors roll up to form cylinders that are perpendicular to the antero-posterior axis. As a consequence, the basic structure of the chicken embryo is a series of encased antero-posterior tubes (gut, neural tube, body envelope, amnion, chorion) decorated with smaller orifices (ear duct, eye stalk, nasal duct, gills, mouth) forming at right angles to the main body axis. We argue that the second-order divisions reflect the early pattern of cell cleavage, and that the transformation of radial and orthoradial lines into a body with sensory organs is a generic biophysical mechanism more general than the chicken embryo.

A blueprint of the topology and mechanics of the human ovary for next-generation bioengineering and diagnosis.

Although the first dissection of the human ovary dates back to the 17th century, the biophysical characteristics of the ovarian cell microenvironment are still poorly understood. However, this information is vital to deciphering cellular processes such as proliferation, morphology and differentiation, as well as pathologies like tumor progression, as demonstrated in other biological tissues. Here, we provide the first readout of human ovarian fiber morphology, interstitial and perifollicular fiber orientation, pore geometry, topography and surface roughness, and elastic and viscoelastic properties. By determining differences between healthy prepubertal, reproductive-age, and menopausal ovarian tissue, we unravel and elucidate a unique biophysical phenotype of reproductive-age tissue, bridging biophysics and female fertility. While these data enable to design of more biomimetic scaffolds for the tissue-engineered ovary, our analysis pipeline is applicable for the characterization of other organs in physiological or pathological states to reveal their biophysical markers or design their bioinspired analogs.

Protocol for multicolor three-dimensional dSTORM data analysis using MATLAB-based script package Grafeo.

This protocol describes the step-by-step analysis of the multicolor (one-, two-, or three-color) two- and three-dimensional dSTORM (direct STochastic Optical Reconstruction Microscopy) data using MATLAB-based script package Grafeo. Grafeo primarily uses pointillist data visualization and analysis frameworks, including the nearest neighbors, approach, Voronoi tessellation, Delaunay triangulation, Ripley's (K, L) and two-point correlation functions, and graph-based clustering. For complete details on the use and execution of this protocol, please refer to Peaucelle et al. (2020), Haas et al., 2018, Haas et al. (2020b).

The role of pectin phase separation in plant cell wall assembly and growth.

A rapidly increasing body of literature suggests that many biological processes are driven by phase separation within polymer mixtures. Liquid-liquid phase separation can lead to the formation of membrane-less organelles, which are thought to play a wide variety of roles in cell metabolism, gene regulation or signaling. One of the characteristics of these systems is that they are poised at phase transition boundaries, which makes them perfectly suited to elicit robust cellular responses to often very small changes in the cell's "environment". Recent observations suggest that, also in the semi-solid environment of plant cell walls, phase separation not only plays a role in wall patterning, hydration and stress relaxation during growth, but also may provide a driving force for cell wall expansion. In this context, pectins, the major polyanionic polysaccharides in the walls of growing cells, appear to play a critical role. Here, we will discuss (i) our current understanding of the structure-function relationship of pectins, (ii) in vivo evidence that pectin modification can drive critical phase transitions in the cell wall, (iii) how such phase transitions may drive cell wall expansion in addition to turgor pressure and (iv) the periodic cellular processes that may control phase transitions underlying cell wall assembly and expansion.

Multicolor 3D-dSTORM Reveals Native-State Ultrastructure of Polysaccharides' Network during Plant Cell Wall Assembly.

The plant cell wall, a form of the extracellular matrix, is a complex and dynamic network of polymers mediating a plethora of physiological functions. How polysaccharides assemble into a coherent and heterogeneous matrix remains mostly undefined. Further progress requires improved molecular-level visualization methods that would gain a deeper understanding of the cell wall nanoarchitecture. dSTORM, a type of super-resolution microscopy, permits quantitative nanoimaging of the cell wall. However, due to the lack of single-cell model systems and the requirement of tissue-level imaging, its use in plant science is almost absent. Here we overcome these limitations; we compare two methods to achieve three-dimensional dSTORM and identify optimal photoswitching dyes for tissue-level multicolor nanoscopy. Combining dSTORM with spatial statistics, we reveal and characterize the ultrastructure of three major polysaccharides, callose, mannan, and cellulose, in the plant cell wall precursor and provide evidence for cellulose structural re-organization related to callose content.

The hook shape of growing leaves results from an active regulatory process.

The rachis of most growing compound leaves observed in nature exhibits a stereotypical hook shape. In this study, we focus on the canonical case of Averrhoa carambola. Combining kinematics and mechanical investigation, we characterize this hook shape and shed light on its establishment and maintenance. We show quantitatively that the hook shape is a conserved bent zone propagating at constant velocity and constant distance from the apex throughout development. A simple mechanical test reveals non-zero intrinsic curvature profiles for the rachis during its growth, indicating that the hook shape is actively regulated. We show a robust spatial organization of growth, curvature, rigidity, and lignification, and their interplay. Regulatory processes appear to be specifically localized: in particular, differential growth occurs where the elongation rate drops. Finally, impairing the graviception of the leaf on a clinostat led to reduced hook curvature but not to its loss. Altogether, our results suggest a role for proprioception in the regulation of the leaf hook shape, likely mediated via mechanical strain.

Pectin homogalacturonan nanofilament expansion drives morphogenesis in plant epidermal cells.

The process by which plant cells expand and gain shape has presented a challenge for researchers. Current models propose that these processes are driven by turgor pressure acting on the cell wall. Using nanoimaging, we show that the cell wall contains pectin nanofilaments that possess an intrinsic expansion capacity. Additionally, we use growth models containing such structures to show that a complex plant cell shape can derive from chemically induced local and polarized expansion of the pectin nanofilaments without turgor-driven growth. Thus, the plant cell wall, outside of the cell itself, is an active participant in shaping plant cells. Extracellular matrix function may similarly guide cell shape in other kingdoms, including Animalia.

ELIMÄKI Locus Is Required for Vertical Proprioceptive Response in Birch Trees.

Tree architecture has evolved to support a top-heavy above-ground biomass, but this integral feature poses a weight-induced challenge to trunk stability. Maintaining an upright stem is expected to require vertical proprioception through feedback between sensing stem weight and responding with radial growth. Despite its apparent importance, the principle by which plant stems respond to vertical loading forces remains largely unknown. Here, by manipulating the stem weight of downy birch (Betula pubescens) trees, we show that cambial development is modulated systemically along the stem. We carried out a genetic study on the underlying regulation by combining an accelerated birch flowering program with a recessive mutation at the ELIMÄKI locus (EKI), which causes a mechanically defective response to weight stimulus resulting in stem collapse after just 3 months. We observed delayed wood morphogenesis in eki compared with WT, along with a more mechanically elastic cambial zone and radial compression of xylem cell size, indicating that rapid tissue differentiation is critical for cambial growth under mechanical stress. Furthermore, the touch-induced mechanosensory pathway was transcriptionally misregulated in eki, indicating that the ELIMÄKI locus is required to integrate the weight-growth feedback regulation. By studying this birch mutant, we were able to dissect vertical proprioception from the gravitropic response associated with reaction wood formation. Our study provides evidence for both local and systemic responses to mechanical stimuli during secondary plant development.

Multitarget Immunohistochemistry for Confocal and Super-resolution Imaging of Plant Cell Wall Polysaccharides.

The plant cell wall (PCW) is a pecto-cellulosic extracellular matrix that envelopes the plant cell. By integrating extra-and intra-cellular cues, PCW mediates a plethora of essential physiological functions. Notably, it permits controlled and oriented tissue growth by tuning its local mechano-chemical properties. To refine our knowledge of these essential properties of PCW, we need an appropriate tool for the accurate observation of the native (in muro) structure of the cell wall components. The label-free techniques, such as AFM, EM, FTIR, and Raman microscopy, are used; however, they either do not have the chemical or spatial resolution. Immunolabeling with electron microscopy allows observation of the cell wall nanostructure, however, it is mostly limited to single and, less frequently, multiple labeling. Immunohistochemistry (IHC) is a versatile tool to analyze the distribution and localization of multiple biomolecules in the tissue. The subcellular resolution of chemical changes in the cell wall component can be observed with standard diffraction-limited optical microscopy. Furthermore, novel chemical imaging tools such as multicolor 3D dSTORM (Three-dimensional, direct Stochastic Optical Reconstruction Microscopy) nanoscopy makes it possible to resolve the native structure of the cell wall polymers with nanometer precision and in three dimensions. Here we present a protocol for preparing multi-target immunostaining of the PCW components taking as example Arabidopsis thaliana, Star fruit (Averrhoa carambola), and Maize thin tissue sections. This protocol is compatible with the standard confocal microscope, dSTORM nanoscope, and can also be implemented for other optical nanoscopy such as STED (Stimulated Emission Depletion Microscopy). The protocol can be adapted for any other subcellular compartments, plasma membrane, cytoplasmic, and intracellular organelles.

Primary wall cellulose synthase regulates shoot apical meristem mechanics and growth.

How organisms attain their specific shapes and modify their growth patterns in response to environmental and chemical signals has been the subject of many investigations. Plant cells are at high turgor pressure and are surrounded by a rigid yet flexible cell wall, which is the primary determinant of plant growth and morphogenesis. Cellulose microfibrils, synthesized by plasma membrane-localized cellulose synthase complexes, are major tension-bearing components of the cell wall that mediate directional growth. Despite advances in understanding the genetic and biophysical regulation of morphogenesis, direct studies of cellulose biosynthesis and its impact on morphogenesis of different cell and tissue types are largely lacking. In this study, we took advantage of mutants of three primary cellulose synthase (CESA) genes that are involved in primary wall cellulose synthesis. Using field emission scanning electron microscopy, live cell imaging and biophysical measurements, we aimed to understand how the primary wall CESA complex acts during shoot apical meristem development. Our results indicate that cellulose biosynthesis impacts the mechanics and growth of the shoot apical meristem.

The FERONIA Receptor Kinase Maintains Cell-Wall Integrity during Salt Stress through Ca2+ Signaling.

Cells maintain integrity despite changes in their mechanical properties elicited during growth and environmental stress. How cells sense their physical state and compensate for cell-wall damage is poorly understood, particularly in plants. Here we report that FERONIA (FER), a plasma-membrane-localized receptor kinase from Arabidopsis, is necessary for the recovery of root growth after exposure to high salinity, a widespread soil stress. The extracellular domain of FER displays tandem regions of homology with malectin, an animal protein known to bind di-glucose in vitro and important for protein quality control in the endoplasmic reticulum. The presence of malectin-like domains in FER and related receptor kinases has led to widespread speculation that they interact with cell-wall polysaccharides and can potentially serve a wall-sensing function. Results reported here show that salinity causes softening of the cell wall and that FER is necessary to sense these defects. When this function is disrupted in the fer mutant, root cells explode dramatically during growth recovery. Similar defects are observed in the mur1 mutant, which disrupts pectin cross-linking. Furthermore, fer cell-wall integrity defects can be rescued by treatment with calcium and borate, which also facilitate pectin cross-linking. Sensing of these salinity-induced wall defects might therefore be a direct consequence of physical interaction between the extracellular domain of FER and pectin. FER-dependent signaling elicits cell-specific calcium transients that maintain cell-wall integrity during salt stress. These results reveal a novel extracellular toxicity of salinity, and identify FER as a sensor of damage to the pectin-associated wall.

Non-invasive Protocol for Kinematic Monitoring of Root Growth under Infrared Light.

Phenotyping the dynamics of root responses to environmental cues is necessary to understand plant acclimation to their environment. Continuous monitoring of root growth is challenging because roots normally grow belowground and are very sensitive to their growth environment. This protocol combines infrared imaging with hydroponic cultivation for kinematic analyses. It allows continuous imaging at fine spatiotemporal resolution and disturbs roots minimally. Examples are provided of how the procedure and materials can be adapted for 3D monitoring and of how environmental stress may be manipulated for experimental purposes.