Trans-synaptic Association of Vesicular Zinc Transporter 3 and Shank3 Supports Synapse-Specific Dendritic Spine Structure and Function in the Mouse Auditory Cortex.

Shank3 is a synaptic scaffolding protein that assists in tethering and organizing structural proteins and glutamatergic receptors in the postsynaptic density of excitatory synapses. The localization of Shank3 at excitatory synapses and the formation of stable Shank3 complexes is regulated by the binding of zinc to the C-terminal sterile-alpha-motif (SAM) domain of Shank3. Mutations in the SAM domain of Shank3 result in altered synaptic function and morphology, and disruption of zinc in synapses that express Shank3 leads to a reduction of postsynaptic proteins important for synaptic structure and function. This suggests that zinc supports the localization of postsynaptic proteins via Shank3. Many regions of the brain are highly enriched with free zinc inside glutamatergic vesicles at presynaptic terminals. At these synapses, zinc transporter 3 (ZnT3) moves zinc into vesicles where it is co-released with glutamate. Alterations in ZnT3 are implicated in multiple neurodevelopmental disorders, and ZnT3 knock-out (KO) mice-which lack synaptic zinc-show behavioral deficits associated with autism spectrum disorder and schizophrenia. Here we show that male and female ZnT3 KO mice have smaller dendritic spines and miniature excitatory postsynaptic current amplitudes than wildtype (WT) mice in the auditory cortex. Additionally, spine size deficits in ZnT3 KO mice are restricted to synapses that express Shank3. In WT mice, synapses that express both Shank3 and ZnT3 have larger spines compared to synapses that express Shank3 but not ZnT3. Together these findings suggest a mechanism whereby presynaptic ZnT3-dependent zinc supports postsynaptic structure and function via Shank3 in a synapse-specific manner.

Preparation and Compositional Analysis of Lignocellulosic Plant Biomass as a Precursor for Food Production During Food Crises.

In the event of a sunlight-blocking, temperature-lowering global catastrophe, such as a global nuclear war, super-volcano eruption or large asteroid strike, normal agricultural practices would be severely disrupted with a devastating impact on the global food supply. Despite the improbability of such an occurrence, it is prudent to consider how to sustain the surviving population following a global catastrophe until normal weather and climate patterns resume. Additionally, the ongoing challenges posed by climate change, droughts, flooding, soil salinization, and famine highlight the importance of developing food systems with resilient inputs such as lignocellulosic biomass. With its high proportion of cellulose, the abundant lignocellulosic biomass found across the Earth's land surfaces could be a source of energy and nutrition, but it would first need to be converted into foods. To understand the potential of lignocellulosic biomass to provide energy and nutrition to humans in post-catastrophic and other food crisis scenarios, compositional analyses should be completed to gauge the amount of energy (soluble sugars) and other macronutrients (protein and lipids) that might be available and the level of difficulty in extracting them. Suitable preparation of the lignocellulosic biomass is critical to achieve consistent and comparable results from these analyses. Here we describe a compilation of protocols to prepare lignocellulosic biomass and analyze its composition to understand its potential as a precursor to produce post-catastrophic foods which are those that could be foraged, grown, or produced under the new climate conditions to supplement reduced availability of traditional foods. These foods have sometimes been referred to in the literature as emergency, alternate, or resilient foods. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Convection oven drying (1 to 2 days) Alternate Protocol 1: Air-drying (2 to 3 days) Alternate Protocol 2: Lyophilization (1 to 4 days) Support Protocol 1: Milling plant biomass Support Protocol 2: Measuring moisture content Basic Protocol 2: Cellulose determination Basic Protocol 3: Lignin determination Basic Protocol 4: Crude protein content by total nitrogen Basic Protocol 5: Crude fat determination via soxtec extraction system Basic Protocol 6: Sugars by HPLC Basic Protocol 7: Ash content.

Architecture and functions of stomatal cell walls in eudicots and grasses.

BACKGROUND: Like all plant cells, the guard cells of stomatal complexes are encased in cell walls that are composed of diverse, interacting networks of polysaccharide polymers. The properties of these cell walls underpin the dynamic deformations that occur in guard cells as they expand and contract to drive the opening and closing of the stomatal pore, the regulation of which is crucial for photosynthesis and water transport in plants. SCOPE: Our understanding of how cell wall mechanics are influenced by the nanoscale assembly of cell wall polymers in guard cell walls, how this architecture changes over stomatal development, maturation and ageing and how the cell walls of stomatal guard cells might be tuned to optimize stomatal responses to dynamic environmental stimuli is still in its infancy. CONCLUSION: In this review, we discuss advances in our ability to probe experimentally and to model the structure and dynamics of guard cell walls quantitatively across a range of plant species, highlighting new ideas and exciting opportunities for further research into these actively moving plant cells.

Single-molecule tracking reveals dual front door/back door inhibition of Cel7A cellulase by its product cellobiose.

Degrading cellulose is a key step in the processing of lignocellulosic biomass into bioethanol. Cellobiose, the disaccharide product of cellulose degradation, has been shown to inhibit cellulase activity, but the mechanisms underlying product inhibition are not clear. We combined single-molecule imaging and biochemical investigations with the goal of revealing the mechanism by which cellobiose inhibits the activity of Trichoderma reesei Cel7A, a well-characterized exo-cellulase. We find that cellobiose slows the processive velocity of Cel7A and shortens the distance moved per encounter; effects that can be explained by cellobiose binding to the product release site of the enzyme. Cellobiose also strongly inhibits the binding of Cel7A to immobilized cellulose, with a Ki of 2.1 mM. The isolated catalytic domain (CD) of Cel7A was also inhibited to a similar degree by cellobiose, and binding of an isolated carbohydrate-binding module to cellulose was not inhibited by cellobiose, suggesting that cellobiose acts on the CD alone. Finally, cellopentaose inhibited Cel7A binding at micromolar concentrations without affecting the enzyme's velocity of movement along cellulose. Together, these results suggest that cellobiose inhibits Cel7A activity both by binding to the "back door" product release site to slow activity and to the "front door" substrate-binding tunnel to inhibit interaction with cellulose. These findings point to strategies for engineering cellulases to reduce product inhibition and enhance cellulose degradation, supporting the growth of a sustainable bioeconomy.

Young guard cells function dynamically despite low mechanical anisotropy but gain efficiency during stomatal maturation in Arabidopsis thaliana.

Stomata are pores at the leaf surface that enable gas exchange and transpiration. The signaling pathways that regulate the differentiation of stomatal guard cells and the mechanisms of stomatal pore formation have been characterized in Arabidopsis thaliana. However, the process by which stomatal complexes develop after pore formation into fully mature complexes is poorly understood. We tracked the morphogenesis of young stomatal complexes over time to establish characteristic geometric milestones along the path of stomatal maturation. Using 3D-nanoindentation coupled with finite element modeling of young and mature stomata, we found that despite having thicker cell walls than young guard cells, mature guard cells are more energy efficient with respect to stomatal opening, potentially attributable to the increased mechanical anisotropy of their cell walls and smaller changes in turgor pressure between the closed and open states. Comparing geometric changes in young and mature guard cells of wild-type and cellulose-deficient plants revealed that although cellulose is required for normal stomatal maturation, mechanical anisotropy appears to be achieved by the collective influence of cellulose and additional wall components. Together, these data elucidate the dynamic geometric and biomechanical mechanisms underlying the development process of stomatal maturation.

A core of cell wall proteins functions in wall integrity responses in Arabidopsis thaliana.

Cell walls surround all plant cells, and their composition and structure are tightly regulated to maintain cellular and organismal homeostasis. In response to wall damage, the cell wall integrity (CWI) system is engaged to ameliorate effects on plant growth. Despite the central role CWI plays in plant development, our current understanding of how this system functions at the molecular level is limited. Here, we investigated the transcriptomes of etiolated seedlings of mutants of Arabidopsis thaliana with defects in three major wall polysaccharides, pectin (quasimodo2), cellulose (cellulose synthase3 je5), and xyloglucan (xyloglucan xylosyltransferase1 and 2), to probe whether changes in the expression of cell wall-related genes occur and are similar or different when specific wall components are reduced or missing. Many changes occurred in the transcriptomes of pectin- and cellulose-deficient plants, but fewer changes occurred in the transcriptomes of xyloglucan-deficient plants. We hypothesize that this might be because pectins interact with other wall components and/or integrity sensors, whereas cellulose forms a major load-bearing component of the wall; defects in either appear to trigger the expression of structural proteins to maintain wall cohesion in the absence of a major polysaccharide. This core set of genes functioning in CWI in plants represents an attractive target for future genetic engineering of robust and resilient cell walls.

Altering the substitution and cross-linking of glucuronoarabinoxylans affects cell wall architecture in Brachypodium distachyon.

The Poaceae family of plants provides cereal crops that are critical for human and animal nutrition, and also, they are an important source of biomass. Interacting plant cell wall components give rise to recalcitrance to digestion; thus, understanding the wall molecular architecture is important to improve biomass properties. Xylan is the main hemicellulose in grass cell walls. Recently, we reported structural variation in grass xylans, suggesting functional specialisation and distinct interactions with cellulose and lignin. Here, we investigated the functions of these xylans by perturbing the biosynthesis of specific xylan types. We generated CRISPR/Cas9 knockout mutants in Brachypodium distachyon XAX1 and GUX2 genes involved in xylan substitution. Using carbohydrate gel electrophoresis, we identified biochemical changes in different xylan types. Saccharification, cryo-SEM, subcritical water extraction and ssNMR were used to study wall architecture. BdXAX1A and BdGUX2 enzymes modify different types of grass xylan. Brachypodium mutant walls are likely more porous, suggesting the xylan substitutions directed by both BdXAX1A and GUX2 enzymes influence xylan-xylan and/or xylan-lignin interactions. Since xylan substitutions influence wall architecture and digestibility, our findings open new avenues to improve cereals for food and to use grass biomass for feed and the production of bioenergy and biomaterials.

Lignin impairs Cel7A degradation of in vitro lignified cellulose by impeding enzyme movement and not by acting as a sink.

BACKGROUND: Cellulose degradation by cellulases has been studied for decades due to the potential of using lignocellulosic biomass as a sustainable source of bioethanol. In plant cell walls, cellulose is bonded together and strengthened by the polyphenolic polymer, lignin. Because lignin is tightly linked to cellulose and is not digestible by cellulases, is thought to play a dominant role in limiting the efficient enzymatic degradation of plant biomass. Removal of lignin via pretreatments currently limits the cost-efficient production of ethanol from cellulose, motivating the need for a better understanding of how lignin inhibits cellulase-catalyzed degradation of lignocellulose. Work to date using bulk assays has suggested three possible inhibition mechanisms: lignin blocks access of the enzyme to cellulose, lignin impedes progress of the enzyme along cellulose, or lignin binds cellulases directly and acts as a sink. RESULTS: We used single-molecule fluorescence microscopy to investigate the nanoscale dynamics of Cel7A from Trichoderma reesei, as it binds to and moves along purified bacterial cellulose in vitro. Lignified cellulose was generated by polymerizing coniferyl alcohol onto purified bacterial cellulose, and the degree of lignin incorporation into the cellulose meshwork was analyzed by optical and electron microscopy. We found that Cel7A preferentially bound to regions of cellulose where lignin was absent, and that in regions of high lignin density, Cel7A binding was inhibited. With increasing degrees of lignification, there was a decrease in the fraction of Cel7A that moved along cellulose rather than statically binding. Furthermore, with increasing lignification, the velocity of processive Cel7A movement decreased, as did the distance that individual Cel7A molecules moved during processive runs. CONCLUSIONS: In an in vitro system that mimics lignified cellulose in plant cell walls, lignin did not act as a sink to sequester Cel7A and prevent it from interacting with cellulose. Instead, lignin both blocked access of Cel7A to cellulose and impeded the processive movement of Cel7A along cellulose. This work implies that strategies for improving biofuel production efficiency should target weakening interactions between lignin and cellulose surface, and further suggest that nonspecific adsorption of Cel7A to lignin is likely not a dominant mechanism of inhibition.

Flexible Pectin Nanopatterning Drives Cell Wall Organization in Plants.

Plant cell walls are abundant sources of materials and energy. Nevertheless, cell wall nanostructure, specifically how pectins interact with cellulose and hemicelluloses to construct a robust and flexible biomaterial, is poorly understood. X-ray scattering measurements are minimally invasive and can reveal ultrastructural, compositional, and physical properties of materials. Resonant X-ray scattering takes advantage of compositional differences by tuning the energy of the incident X-ray to absorption edges of specific elements in a material. Using Tender Resonant X-ray Scattering (TReXS) at the calcium K-edge to study hypocotyls of the model plant, Arabidopsis thaliana, we detected distinctive Ca features that we hypothesize correspond to previously unreported Ca-Homogalacturonan (Ca-HG) nanostructures. When Ca-HG structures were perturbed by chemical and enzymatic treatments, cellulose microfibrils were also rearranged. Moreover, Ca-HG nanostructure was altered in mutants with abnormal cellulose, pectin, or hemicellulose content. Our results indicate direct structural interlinks between components of the plant cell wall at the nanoscale and reveal mechanisms that underpin both the structural integrity of these components and the molecular architecture of the plant cell wall.

Stomatal opening efficiency is controlled by cell wall organization in Arabidopsis thaliana.

Stomatal function in plants is regulated by the nanoscale architecture of the cell wall and turgor pressure, which together control stomatal pore size to facilitate gas exchange and photosynthesis. The mechanical properties of the cell wall and cell geometry are critical determinants of stomatal dynamics. However, the specific biomechanical functions of wall constituents, for example, cellulose and pectins, and their impact on the work required to open or close the stomatal pore are unclear. Here, we use nanoindentation in normal and lateral directions, computational modeling, and microscopic imaging of cells from the model plant Arabidopsis thaliana to investigate the precise influences of wall architecture and turgor pressure on stomatal biomechanics. This approach allows us to quantify and compare the unique anisotropic properties of guard cells with normal composition, lower cellulose content, or alterations in pectin molecular weight. Using these data to calculate the work required to open the stomata reveals that the wild type, with a circumferential-to-longitudinal modulus ratio of 3:1, is the most energy-efficient of those studied. In addition, the tested genotypes displayed similar changes in their pore size despite large differences in wall thickness and biomechanical properties. These findings imply that homeostasis in stomatal function is maintained in the face of varying wall compositions and biomechanics by tuning wall thickness.

Measuring calcium content in plants using NEXAFS spectroscopy.

Calcium is important for the growth and development of plants. It serves crucial functions in cell wall and cell membrane structure and serves as a secondary messenger in signaling pathways relevant to nutrient and immunity responses. Thus, measuring calcium levels in plants is important for studies of plant biology and for technology development in food, agriculture, energy, and forest industries. Often, calcium in plants has been measured through techniques such as atomic absorption spectrophotometry (AAS), inductively coupled plasma-mass spectrometry (ICP-MS), and electrophysiology. These techniques, however, require large sample sizes, chemical extraction of samples or have limited spatial resolution. Here, we used near-edge X-ray absorption fine structure (NEXAFS) spectroscopy at the calcium L- and K-edges to measure the calcium to carbon mass ratio with spatial resolution in plant samples without requiring chemical extraction or large sample sizes. We demonstrate that the integrated absorbance at the calcium L-edge and the edge jump in the fluorescence yield at the calcium K-edge can be used to quantify the calcium content as the calcium mass fraction, and validate this approach with onion epidermal peels and ICP-MS. We also used NEXAFS to estimate the calcium mass ratio in hypocotyls of a model plant, Arabidopsis thaliana, which has a cell wall composition that is similar to that of onion epidermal peels. These results show that NEXAFS spectroscopy performed at the calcium edge provides an approach to quantify calcium levels within plants, which is crucial for understanding plant physiology and advancing plant-based materials.

Assessing lignocellulosic biomass as a source of emergency foods.

Catastrophes such as a nuclear war would generate atmospheric soot and reduce sunlight, making it difficult to grow crops. Under such conditions, people might turn to inedible plant biomass for nutrition, but the convertibility and nutritional content of this biomass have not been rigorously analyzed. We found that if plant biomass were converted into food at 30% efficiency, 6.7 kg of biomass per day would yield adequate carbohydrates, but contain potentially toxic or insufficient levels of other nutrients for a family of four. Therefore, exploiting biomass with low mineral content for carbohydrates and consuming other sources of protein, fat, and vitamins such as edible insects/single-cell proteins and vitamin supplements could provide a balanced diet in a global catastrophic environment.

Synaptic zinc potentiates AMPA receptor function in mouse auditory cortex.

Synaptic zinc signaling modulates synaptic activity and is present in specific populations of cortical neurons, suggesting that synaptic zinc contributes to the diversity of intracortical synaptic microcircuits and their functional specificity. To understand the role of zinc signaling in the cortex, we performed whole-cell patch-clamp recordings from intratelencephalic (IT)-type neurons and pyramidal tract (PT)-type neurons in layer 5 of the mouse auditory cortex during optogenetic stimulation of specific classes of presynaptic neurons. Our results show that synaptic zinc potentiates AMPA receptor (AMPAR) function in a synapse-specific manner. We performed in vivo 2-photon calcium imaging of the same classes of neurons in awake mice and found that changes in synaptic zinc can widen or sharpen the sound-frequency tuning bandwidth of IT-type neurons but only widen the tuning bandwidth of PT-type neurons. These results provide evidence for synapse- and cell-type-specific actions of synaptic zinc in the cortex.

Galactosylation of xyloglucan is essential for the stabilization of the actin cytoskeleton and endomembrane system through the proper assembly of cell walls.

Xyloglucan, a major hemicellulose, interacts with cellulose and pectin to assemble primary cell walls in plants. Loss of the xyloglucan galactosyltransferase MURUS3 (MUR3) leads to the deficiency of galactosylated xyloglucan and perturbs plant growth. However, it is unclear whether defects in xyloglucan galactosylation influence the synthesis of other wall polysaccharides, cell wall integrity, cytoskeleton behaviour, and endomembrane homeostasis. Here, we found that in mur3-7 etiolated seedlings cellulose was reduced, CELLULOSE SYNTHASE (CESA) genes were down-regulated, the density and mobility of cellulose synthase complexes (CSCs) were decreased, and cellulose microfibrils become discontinuous. Pectin, rhamnogalacturonan II (RGII), and boron contents were reduced in mur3-7 plants, and B-RGII cross-linking was abnormal. Wall porosity and thickness were significantly increased in mur3-7 seedlings. Endomembrane aggregation was also apparent in the mur3-7 mutant. Furthermore, mutant seedlings and their actin filaments were more sensitive to Latrunculin A (LatA) treatment. However, all defects in mur3-7 mutants were substantially restored by exogenous boric acid application. Our study reveals the importance of MUR3-mediated xyloglucan galactosylation for cell wall structural assembly and homeostasis, which is required for the stabilization of the actin cytoskeleton and the endomembrane system.

A genetically encoded far-red fluorescent indicator for imaging synaptically released Zn2.

Synaptic zinc ion (Zn2+) has emerged as a key neuromodulator in the brain. However, the lack of research tools for directly tracking synaptic Zn2+ in the brain of awake animals hinders our rigorous understanding of the physiological and pathological roles of synaptic Zn2+. In this study, we developed a genetically encoded far-red fluorescent indicator for monitoring synaptic Zn2+ dynamics in the nervous system. Our engineered far-red fluorescent indicator for synaptic Zn2+ (FRISZ) displayed a substantial Zn2+-specific turn-on response and low-micromolar affinity. We genetically anchored FRISZ to the mammalian extracellular membrane via a transmembrane (TM) ⍺ helix and characterized the resultant FRISZ-TM construct at the mammalian cell surface. We used FRISZ-TM to image synaptic Zn2+ in the auditory cortex in acute brain slices and awake mice in response to electric and sound stimuli, respectively. Thus, this study establishes a technology for studying the roles of synaptic Zn2+ in the nervous system.

Automated 3D segmentation of guard cells enables volumetric analysis of stomatal biomechanics.

Automating the three-dimensional (3D) segmentation of stomatal guard cells and other confocal microscopy data is extremely challenging due to hardware limitations, hard-to-localize regions, and limited optical resolution. We present a memory-efficient, attention-based, one-stage segmentation neural network for 3D images of stomatal guard cells. Our model is trained end to end and achieved expert-level accuracy while leveraging only eight human-labeled volume images. As a proof of concept, we applied our model to 3D confocal data from a cell ablation experiment that tests the "polar stiffening" model of stomatal biomechanics. The resulting data allow us to refine this polar stiffening model. This work presents a comprehensive, automated, computer-based volumetric analysis of fluorescent guard cell images. We anticipate that our model will allow biologists to rapidly test cell mechanics and dynamics and help them identify plants that more efficiently use water, a major limiting factor in global agricultural production and an area of critical concern during climate change.

Pectin methyltransferase QUASIMODO2 functions in the formation of seed coat mucilage in Arabidopsis.

Pectin, cellulose, and hemicelluloses are major components of primary cell walls in plants. In addition to cell adhesion and expansion, pectin plays a central role in seed mucilage. Seed mucilage contains abundant pectic rhamnogalacturonan-I (RG-I) and lower amounts of homogalacturonan (HG), cellulose, and hemicelluloses. Previously, accumulated evidence has addressed the role of pectin RG-I in mucilage production and adherence. However, less is known about the function of pectin HG in seed coat mucilage formation. In this study, we analyzed a novel mutant, designated things fall apart2 (tfa2), which contains a mutation in HG methyltransferase QUASIMODO2 (QUA2). Etiolated tfa2 seedlings display short hypocotyls and adhesion defects similar to qua2 and tumorous shoot development2 (tsd2) alleles, and show seed mucilage defects. The diminished uronic acid content and methylesterification degree of HG in mutant seed mucilage indicate the role of HG in the formation of seed mucilage. Cellulosic rays in mutant mucilage are collapsed. The epidermal cells of seed coat in tfa2 and tsd2 display deformed columellae and reduced radial wall thickness. Under polyethylene glycol treatment, seeds from these three mutant alleles exhibit reduced germination rates. Together, these data emphasize the requirement of pectic HG biosynthesis for the synthesis of seed mucilage, and the functions of different pectin domains together with cellulose in regulating its formation, expansion, and release.

Dynamics of pectic homogalacturonan in cellular morphogenesis and adhesion, wall integrity sensing and plant development.

Homogalacturonan (HG) is the most abundant pectin subtype in plant cell walls. Although it is a linear homopolymer, its modification states allow for complex molecular encoding. HG metabolism affects its structure, chemical properties, mobility and binding capacity, allowing it to interact dynamically with other polymers during wall assembly and remodelling and to facilitate anisotropic cell growth, cell adhesion and separation, and organ morphogenesis. HGs have also recently been found to function as signalling molecules that transmit information about wall integrity to the cell. Here we highlight recent advances in our understanding of the dual functions of HG as a dynamic structural component of the cell wall and an initiator of intrinsic and environmental signalling. We also predict how HG might interconnect the cell wall, plasma membrane and intracellular components with transcriptional networks to regulate plant growth and development.

Polygalacturonase activity promotes aberrant cell separation in the quasimodo2 mutant of Arabidopsis thaliana.

In plants, cell adhesion relies on balancing the integrity of the pectin-rich middle lamella with wall loosening during tissue expansion. Mutation of QUASIMODO2 (QUA2), a pectin methyltransferase, causes defective hypocotyl elongation and cell adhesion in Arabidopsis thaliana hypocotyls. However, the molecular function of QUA2 in cell adhesion is obscured by complex genetic and environmental interactions. To dissect the role of QUA2 in cell adhesion, we investigated a qua2 loss-of-function mutant and a suppressor mutant with restored cell adhesion, qua2 esmeralda1, using a combination of imaging and biochemical techniques. We found that qua2 hypocotyls have reductions in middle lamellae integrity, pectin methyl-esterase (PME) activity, pectin content and molecular mass, and immunodetected Ca2+-crosslinking at cell corners, but increased methyl-esterification and polygalacturonase (PG) activity, with qua2 esmd1 having wild type-like or intermediate phenotypes. Our findings suggest that excessive pectin degradation prevents pectin accumulation and the formation of a sufficiently Ca2+-crosslinked network to maintain cell adhesion in qua2 mutants. We propose that PME and PG activities balance tissue-level expansion and cell separation. Together, these data provide insight into the cause of cell adhesion defects in qua2 mutants and highlight the importance of harmonizing pectin modification and degradation during plant growth and development.