Nanoscale FTIR and Mechanical Mapping of Plant Cell Walls for Understanding Biomass Deconstruction
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59 BASIC BIOLOGICAL SCIENCES↗
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Conservation of model degraded pine wood with selected organosilicons studied by XFM and nanoindentation
Previous research found that some organosilicon treatments proved effective in stabilizing waterlogged wood dimensions during drying. The present research aimed to determine the mechanism of wood stabilization by these chemicals to understand their mode of action. Here, the study used chemically (ChP) and biologically degraded (BP) model Scots pine wood treated with Methyltrimethoxysilane (MTMS), (3-Mercaptopropyl) trimethoxysilane (MPTMS), or 1,3-Bis(diethylamino)-3-propoxypropanol)-1,1,3,3-tetramethyldisiloxane (DEAPTMDS). Synchrotron-based X-ray fluorescence microscopy (XFM) was used to investigate the penetration of organosilicons into the wood cellular structure and cell walls, and nanoindentation was used to study the mechanical properties of the treated wood cell walls. All treatments resulted in high volumetric anti-shrink efficiency (ASE V ) values of 74-82%, except for MTMS-treated ChP with an ASE V of 52%. The multiscale XFM results revealed that all applied organosilicons penetrated throughout the whole wooden blocks and deposited in both cell lumina and cell walls. The retention of all applied organosilicons was highest in BP wood, and so was the dimensional stabilization effect. MTMS-treated ChP had the lowest measured cell wall infiltration, which likely contributed to its lower ASE v . DEAPTMDS treatments plasticized the cell walls and resulted in lowered nanoindentation elastic modulus (E s NI ) and hardness (H) for all types of wood. MTMS and MPTMS had modest effects on cell wall mechanical properties, and the effect depended on the type of wood. The final effect of organosilicon treatment on the dimensional wood stabilization and mechanical properties of wood cell walls depended not only on the type of the applied organosilicon but also the type of wood degradation. This means that the treatment cannot be considered universal, and specific approaches are needed for the conservation of individual wooden objects. Although some mechanisms are now better understood, such as the need for organosilicons to infiltrate the cell walls and the plasticizing effect of DEAPTMDS, other aspects will benefit from a more detailed analysis of the molecular interactions between organosilicons and wood polymers.
59 BASIC BIOLOGICAL SCIENCES↗
Xylan Is Critical for Proper Bundling and Alignment of Cellulose Microfibrils in Plant Secondary Cell Walls
Plant biomass represents an abundant and increasingly important natural resource and it mainly consists of a number of cell types that have undergone extensive secondary cell wall (SCW) formation. These cell types are abundant in the stems of Arabidopsis, a well-studied model system for hardwood, the wood of eudicot plants. The main constituents of hardwood include cellulose, lignin, and xylan, the latter in the form of glucuronoxylan (GX). The binding of GX to cellulose in the eudicot SCW represents one of the best-understood molecular interactions within plant cell walls. The evenly spaced acetylation and 4-O-methyl glucuronic acid (MeGlcA) substitutions of the xylan polymer backbone facilitates binding in a linear two-fold screw conformation to the hydrophilic side of cellulose and signifies a high level of molecular specificity. However, the wider implications of GX–cellulose interactions for cellulose network formation and SCW architecture have remained less explored. In this study, we seek to expand our knowledge on this by characterizing the cellulose microfibril organization in three well-characterized GX mutants. The selected mutants display a range of GX deficiency from mild to severe, with findings indicating even the weakest mutant having significant perturbations of the cellulose network, as visualized by both scanning electron microscopy (SEM) and atomic force microscopy (AFM). We show by image analysis that microfibril width is increased by as much as three times in the severe mutants compared to the wild type and that the degree of directional dispersion of the fibrils is approximately doubled in all the three mutants. Further, we find that these changes correlate with both altered nanomechanical properties of the SCW, as observed by AFM, and with increases in enzymatic hydrolysis. Results from this study indicate the critical role that normal GX composition has on cellulose bundle formation and cellulose organization as a whole within the SCWs.
09 BIOMASS FUELS↗
Pectin methylesterase selectively softens the onion epidermal wall yet reduces acid-induced creep
De-esterification of homogalacturonan (HG) is thought to stiffen pectin gels and primary cell walls by increasing calcium cross-linking between HG chains. Contrary to this idea, recent studies found that HG de-esterification correlated with reduced stiffness of living tissues, measured by surface indentation. The physical basis of such apparent wall softening is unclear, but possibly involves complex biological responses to HG modification. To assess the direct physical consequences of HG de-esterification on wall mechanics without such complications, we treated isolated onion (Allium cepa) epidermal walls with pectin methylesterase (PME) and assessed wall biomechanics with indentation and tensile tests. In nanoindentation assays, PME action softened the wall (reduced the indentation modulus). In tensile force/extension assays, PME increased plasticity, but not elasticity. These softening effects are attributed, at least in part, to increased electrostatic repulsion and swelling of the wall after PME treatment. Despite softening and swelling upon HG de-esterification, PME treatment alone failed to induce cell wall creep. Instead, acid-induced creep, mediated by endogenous α-expansin, was reduced. We conclude that HG de-esterification physically softens the onion wall, yet reduces expansin-mediated wall extensibility.
59 BASIC BIOLOGICAL SCIENCES↗
Leaf morphogenesis: The multifaceted roles of mechanics
Plants produce a rich diversity of biological forms, and the diversity of leaves is especially notable. Mechanisms of leaf morphogenesis have been studied in the past two decades, with a growing focus on the interactive roles of mechanics in recent years. Growth of plant organs involves feedback by mechanical stress: growth induces stress, and stress affects growth and morphogenesis. Although much attention has been given to potential stress-sensing mechanisms and cellular responses, the mechanical principles guiding morphogenesis have not been well understood. Here we synthesize the overarching roles of mechanics and mechanical stress in multilevel and multiple stages of leaf morphogenesis, encompassing leaf primordium initiation, phyllotaxis and venation patterning, and the establishment of complex mature leaf shapes. Moreover, the roles of mechanics at multiscale levels, from subcellular cytoskeletal molecules to single cells to tissues at the organ scale, are articulated. In conclusion, by highlighting the role of mechanical buckling in the formation of three-dimensional leaf shapes, this review integrates the perspectives of mechanics and biology to provide broader insights into the mechanobiology of leaf morphogenesis.
59 BASIC BIOLOGICAL SCIENCES↗
Assembly and enlargement of the primary cell wall in plants
Growing plant cells are shaped by an extensible wall that is a complex amalgam of cellulose microfibrils bonded noncovalently to a matrix of hemicelluloses, pectins, and structural proteins. Cellulose is synthesized by complexes in the plasma membrane and is extruded as a self-assembling microfibril, whereas the matrix polymers are secreted by the Golgi apparatus and become integrated into the wall network by poorly understood mechanisms. The growing wall is under high tensile stress from cell turgor and is able to enlarge by a combination of stress relaxation and polymer creep. A pH-dependent mechanism of wall loosening, known as acid growth, is characteristic of growing walls and is mediated by a group of unusual wall proteins called expansins. Expansins appear to disrupt the noncovalent bonding of matrix hemicelluloses to the microfibril, thereby allowing the wall to yield to the mechanical forces generated by cell turgor. Other wall enzymes, such as (1-->4) beta-glucanases and pectinases, may make the wall more responsive to expansin-mediated wall creep whereas pectin methylesterases and peroxidases may alter the wall so as to make it resistant to expansin-mediated creep.
NASA Discipline Plant Biology↗
Nanostructural Analysis of Enzymatic and Non-enzymatic Brown Rot Fungal Deconstruction of the Lignocellulose Cell Wall†
Brown rot (BR) decay mechanisms employ carbohydrate-active enzymes (CAZymes) as well as a unique non-enzymatic chelator-mediated Fenton (CMF) chemistry to deconstruct lignocellulosic materials. Unlike white rot fungi, BR fungi lack peroxidases for lignin deconstruction, and also lack some endoglucanase/cellobiohydrolase activities. The role that the CMF mechanism plays in “opening up” the wood cell wall structure in advance of enzymatic action, and any interaction between CMF constituents and the selective CAZyme suite that BRs possess, is still unclear. Expression patterns for CMF redox metabolites and lytic polysaccharide monooxygenase (LPMO–AA9 family) genes showed that some LPMO isozymes were upregulated with genes associated with CMF at early stages of brown rot by Gloeophyllum trabeum. In the structural studies, wood decayed by the G. trabeum was compared to CMF-treated wood, or CMF-treated wood followed by treatment with either the early-upregulated LPMO or a commercial CAZyme cocktail. Structural modification of decayed/treated wood was characterized using small angle neutron scattering. CMF treatment produced neutron scattering patterns similar to that of the BR decay indicating that both systems enlarged the nanopore structure of wood cell walls to permit enzyme access. Enzymatic deconstruction of cellulose or lignin in raw wood samples was not achieved via CAZyme cocktail or LPMO enzyme action alone. CMF treatment resulted in depolymerization of crystalline cellulose as attack progressed from the outer regions of individual crystallites. Multiple pulses of CMF treatment on raw wood showed a progressive increase in the spacing between the cellulose elementary fibrils (EFs), indicating the CMF eroded the matrix outside the EF bundles, leading to less tightly packed EFs. Peracetic acid delignification treatment enhanced subsequent CMF treatment effects, and allowed both enzyme systems to further increase spacing of the EFs. Moreover, even after a single pulse of CMF treatment, both enzymes were apparently able to penetrate the cell wall to further increase EF spacing. The data suggest the potential for the early-upregulated LPMO enzyme to work in association with CMF chemistry, suggesting that G. trabeum may have adopted mechanisms to integrate non-enzymatic and enzymatic chemistries together during early stages of brown rot decay.
59 BASIC BIOLOGICAL SCIENCES↗
Scintillation detector for carbon-14
Detector consists of plastic, cylindrical double-wall scintillation cell, which is filled with gas to be analyzed. Thin, inner cell wall is isolated optically from outer (guard) scintillator wall by evaporated-aluminum coating. Bonding technique provides mechanical support to cell wall when device is exposed to high temperatures.
Knoll, G. F.↗
Microfibril orientation and compositional heterogeneity in fiber and vessel cell walls of poplar xylem studied by AFM-IR and SFG spectroscopy
Understanding the structural organization of cellulose microfibrils (CMFs) within individual plant cell walls is essential for connecting cell wall architecture to its mechanical and physiological functions. However, due to the complex hierarchical structure and nanoscale heterogeneity of cell walls, it remains technically challenging to resolve detailed compositional and orientational information at subcellular levels of individual cell walls. This study investigates the internal 3D structure, chemical composition, and sublayer organization of fiber and vessel cell walls in the xylem tissue of a two-year-old field-grown hybrid poplar tree (Populus alba × P. glandulosa) using photothermal atomic force microscopy coupled with infrared spectroscopy (AFM-IR) and sum frequency generation (SFG) hyperspectral microscopy. AFM-IR provided nanoscale chemical imaging, revealing localized compositional heterogeneity, including variations between adjacent cell walls and transitional layers beyond the traditional S1, S2, and S3 sublayers. SFG microscopy revealed that CMFs in fiber walls are highly aligned along the stem axis, consistent with their role in mechanical support, while vessel cell walls exhibited slightly tilted CMFs, reflecting their function in hydraulic transport. Together, these results offer new insights into cell-type-specific CMF organization and compositional gradients in hybrid poplar xylem. These findings highlight the structural and chemical complexity of secondary cell walls in woody plants and demonstrate the value of AFM-IR and SFG spectroscopy in elucidating plant cell wall architecture.
60 APPLIED LIFE SCIENCES↗
A Maize GT14 Family Glycosyltransferase Affects Cell Wall Composition and Carbohydrate Export from Source Leaves
Sucrose translocation from photosynthetic leaves to distant parts of a plant, such as seeds and roots, is a critical aspect of plant growth and development and a major determinant of crop yield. To identify genes contributing to this process in maize (Zea mays), we isolated four allelic mutants, carbohydrate partitioning defective7, 48, 49 (cpd7, cpd48, cpd49) and a UniformMu insertion (mu1049954), all of which exhibited reduced growth and fertility and hyperaccumulation of starch and soluble sugars in mature leaves. Consistent with carbohydrate accumulation, cpd7 mutants exhibited reduced sucrose export from mature leaves. Cpd7 encodes a Golgi-resident glucuronosyltransferase belonging to the Glycosyltransferase14 (GT14) family, which is involved in decoration of type II arabinogalactan proteins. No previously described GT14 mutants exhibit reduced sucrose transport or carbohydrate partitioning defects. Additionally, we show that mature leaves of cpd7 mutants have reduced cellulose content and an altered cell wall composition. Further, cpd7 mutants exhibit ectopic phloem lignification likely as a compensatory mechanism for reduced cell wall integrity. Collectively, our data suggest that Cpd7 functions to facilitate cell wall development in the phloem, which is required for efficient sucrose export from mature maize leaves.
59 BASIC BIOLOGICAL SCIENCES↗
Structure and mechanism of biosynthesis of Streptococcus mutans cell wall polysaccharide
Streptococcus mutans, the causative agent of human dental caries, expresses a cell wall attached Serotype c-specific Carbohydrate (SCC) that is critical for cell viability. SCC consists of a polyrhamnose backbone of →3)α-Rha(1 → 2)α-Rha(1→ repeats with glucose (Glc) side-chains and glycerol phosphate (GroP) decorations. This study reveals that SCC has one predominant and two more minor Glc modifications. The predominant Glc modification, α-Glc, attached to position 2 of 3-rhamnose, is installed by SccN and SccM glycosyltransferases and is the site of the GroP addition. The minor Glc modifications are β-Glc linked to position 4 of 3-rhamnose installed by SccP and SccQ glycosyltransferases, and α-Glc attached to position 4 of 2-rhamnose installed by SccN working in tandem with an unknown enzyme. Both the major and the minor β-Glc modifications control bacterial morphology, but only the GroP and major Glc modifications are critical for biofilm formation.
59 BASIC BIOLOGICAL SCIENCES↗
The role of wall calcium in the extension of cell walls of soybean hypocotyls
Calcium crosslinks are load-bearing bonds in soybean (Glycine max (L.) Merr.) hypocotyl cell walls, but they are not the same load-bearing bonds that are broken during acid-mediated cell elongation. This conclusion is reached by studying the relationship between wall calcium, pH and the facilitated creep of frozen-thawed soybean hypocotyl sections. Supporting data include the following observations: 1) 2-[(2-bis-[carboxymethyl]amino-5-methylphenoxy)methyl]-6-methoxy-8-bis[car boxymethyl]aminoquinoline (Quin 2) and ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) caused only limited facilitated creep as compared with acid, despite removal of comparable or larger amounts of wall calcium; 2) the pH-response curves for calcium removal and acid-facilitated creep were different; 3) reversible acid-extension occurred even after removal of almost all wall calcium with Quin 2; and 4) growth of abraded sections did not involve a proportional loss of wall calcium. Removal of wall calcium, however, increased the capacity of the walls to undergo acid-facilitated creep. These data indicate that breakage of calcium crosslinks is not a major mechanism of cell-wall loosening in soybean hypocotyl tissues.
Non-NASA Center↗
Mechanism of rapid suppression of cell expansion in cucumber hypocotyls after blue-light irradiation
Rapid suppression of hypocotyl elongation by blue light in cucumber (Cucumis sativus L.) was studied to examine possible hydraulic and wall changes responsible for diminished growth. Cell-sap osmotic pressure, measured by vapor-pressure osmometry, was not decreased by blue light; turgor pressure, measured by the pressure-probe technique, remained constant during the growth inhibition; and stem hydraulic conductance, measured by dynamic and static methods, was likewise unaffected by blue light. Wall yielding properties were assessed by the pressure-block technique for in-vivo stress relaxation. Blue light reduced the initial rate of relaxation by 77%, but had little effect on the final amount of relaxation. The results demonstrate that blue irradiation acts to decrease the wall yielding coefficient, but not the yield threshold. Stress-strain (Instron) analysis showed that irradiation of the seedlings had little effect on the mechanical extensibilities of the isolated wall. The results indicate that blue light can reduce cell-wall loosening without affecting bulk viscoelastic properties, and indicate a chemorheological mechanism of cell-wall expansion.
NASA Discipline Number 40-30↗
Oxygen Effect on the Low Temperature Tolerance of Facultative Anaerobes from Antarctica, Alaska, and Patagonia
Psychrotolerance as an adaptation to survival in extreme environments is widespread among many of the mesophilic microorganisms. Red-ox potential, pH and salinity could significantly alter the features of ecosystems by providing liquid water at subzero temperatures. Furthermore, organisms can respond to temperature changes by several known mechanisms, including changing the conformation capacities of constitutional proteins or by the synthesis of mucopolysaccharides around the cell wall and membrane. Such protective mechanisms make it possible for cells to not only passively survive low-temperature in a state of anabiosis, but also to be capable of actively metabolizing substrates and reproducing normally. The physiological and biochemical characteristics of species as well as genetics could be remarkably changed due to -on and surviving m extreme environments. The cold shock genes for some of the studied strains of psychrotolerant facultative anaerobes already were published In this paper we present experimental data for psychrotolerant facultative anaerobes isolated from geographically different cold regions of our planet. We show the growth response on the changing of anaerobic conditions to aerobic with cultivation at subzero temperatures.
Pikuta, Elena V.↗
Growth of the Facultative Anaerobes from Antarctica, Alaska, and Patagonia at Low Temperatures
Psychotolerance, as an adaptation for surviving in extreme environments, is widespread among mesophilic microorganisms. Physico-chemical factors such as pressure, red-ox potential, pH and salinity could significantly alter the features of ecosystems by providing liquid water at subzero temperatures. Furthermore, organisms can respond to temperature changes by several known mechanisms, including changing the conformation capacities of constitutional proteins or by the synthesis of mucopolysaccharides around the cell wall and membrane. Such protective mechanisms make it possible for cells to not only passively survive low temperatures in a state of anabiosis, but also to be capable of actively metabolizing substrates and reproducing normally. The physiological and biochemical characteristics of the species, as well as genetics, could be remarkably changed due to adaptation and surviving in extreme environments. The cold shock genes of some of the studied strains of psychotolerant facultative anaerobes were reported previously. In this paper we present experimental data for psychotolerant, non spore-forming, facultative anaerobes isolated from geographically different cold regions of our planet. We show the growth response on changing from anaerobic conditions to aerobic with cultivation at low temperatures.
Pikuta, Elena V.↗
Wall to membrane linkers, stretch activated channels, and the detection of tension, voltage, temperature, auxin, and pH
Introduction. The higher plant is a heterogeneous, mechanically prestressed structure continually subject to shifting forces. When a cell grows in a plant at gravitropic equilibrium, it must create localized maxima of shear in walls of neighboring cells. Such mechanical stress and strain are likely detected in a variety of ways. However, tension-sensitive ion channels are of particular interest because it appears that they are elaborately evolved for sensory function. We hypothesize that 1) the patchy patterns of high shear are focused via wall-to-membrane linkers onto the plasma membrane, where 2) they are translated by mechanosensory cation channels into corresponding patterns of high cytosolic Ca2+, which 3) initiate local enhancement of wall expansion. Further, we hypothesize that the local promotion of enhancement is achieved at least in part by local intensification of auxin transport across the plasma membrane. By implication, when an organ is asymmetrically pressed, rubbed, or bent or when it is displaced in the gravitational field, the net asymmetry of shear stress occurring across the organ would lead to asymmetric redistribution of auxin and corrective asymmetric growth. We shall describe a representative mechanosensitive Ca(2+) -selective cation channel (MCaC) with susceptibilities to xenobiotics implicating it as a force transducer in thigmo- and gravitropism. Then, we shall consider whether a putative wall-to-membrane linker (WML) could be a key feature of the molecular architecture permitting the stress distributed in the wall system to be focused on the channels.
NASA Discipline Number 40-50↗
Archeological wood conservation with selected organosilicon compounds studied by XFM and nanoindentation
Waterlogged wood conservation is a complex and challenging task. Detailed knowledge about the interactions between the applied chemicals and wood is necessary to ensure the effective and safe conservation of wooden artifacts. The present research aims to determine the mechanism of dimensional stabilization of archeological wood by organosilicon compounds using the combination of synchrotron-based X-ray fluorescence microscopy (XFM) and nanoindentation. Archeological oak wood was treated with methyltrimethoxysilane, (3-mercaptopropyl)trimethoxysilane, or 1,3-bis-[(diethylamino)-3-(propoxy)propan-2-ol]-1,1,3,3-tetramethyldisiloxane, which in previous studies were found to be more effective than other organosilicons in stabilizing wood dimensions. The XFM and nanoindentation results showed that all three organosilicons infiltrated wood cell walls and enhanced their mechanical properties. The XFM also showed that part of the chemicals filled some void spaces like cell lumina. Based on the results obtained here and in our previous research, it is determined that the mechanism of archeological wood dimensional stabilization by organosilicon treatment is complex and likely involves both filling cell lumina and infiltration into cell walls where organosilicons interact with wood polymers.
59 BASIC BIOLOGICAL SCIENCES↗
Structural and chemical changes in hardwood cell walls during early stages of flash pyrolysis
Volatile products from thermal decomposition of lignocellulosic biomass have been well characterized, but the solid- and liquid-phase reactions during the early stages of decomposition are largely unknown. Here the initial solid-phase biomass thermal deconstruction reactions were analyzed in situ and with high particle heating rates, delineating how these processes occur. A variety of instrumentation was used to quantify the extent and relative rates of deconstruction, demonstrating that biopolymers resist the thermally energetic conditions to differing degrees, even when ensconced in biomass cell walls. Hemicellulose and the more frangible lignin components decompose and volatilize more readily than cellulose, which temporarily enriches biomass with cellulose. These chemical changes manifest in larger cell wall structural and mechanical property transformations. In all, this investigation concludes that these solid-phase reactions strongly influence the production rates of volatile species and will require additional study before these processes can be modeled precisely to improve yields of desired product.
09 BIOMASS FUELS↗