Silica Biomineralization with Lignin Involves Si-O-C Bonds That Stabilize Radicals.

Plants undergo substantial biomineralization of silicon, which is deposited primarily in cell walls as amorphous silica. The mineral formation could be moderated by the structure and chemistry of lignin, a polyphenol polymer that is a major constituent of the secondary cell wall. However, the reactions between lignin and silica have not yet been well elucidated. Here, we investigate silica deposition onto a lignin model compound. Polyphenyl propanoid was synthesized from coniferyl alcohol by oxidative coupling with peroxidase in the presence of acidic tetramethyl orthosilicate, a silicic acid precursor. Raman, Fourier transform infrared, and X-ray photoelectron spectroscopies detected changes in lignin formation in the presence of silicic acid. Bonds between the Si-O/Si-OH residues and phenoxyl radicals and lignin functional groups formed during the first 3 h of the reaction, while silica continued to form over 3 days. Thermal gravimetric analysis indicated that lignin yields increased in the presence of silicic acid, possibly via the stabilization of phenolic radicals. This, in turn, resulted in shorter stretches of the lignin polymer. Silica deposition initiated within a lignin matrix via the formation of covalent Si-O-C bonds. The silica nucleants grew into 2-5 nm particles, as observed via scanning transmission electron microscopy and energy-dispersive X-ray spectroscopy. Additional silica precipitated into an extended gel. Collectively, our results demonstrate a reciprocal relation by which lignin polymerization catalyzes the formation of silica, and at the same time silicic acid enhances lignin polymerization and yield.

Deposition of silica in sorghum root endodermis modifies the chemistry of associated lignin.

Silica aggregates at the endodermis of sorghum roots. Aggregation follows a spotted pattern of locally deposited lignin at the inner tangential cell walls. Autofluorescence microscopy suggests that non-silicified (-Si) lignin spots are composed of two distinct concentric regions of varied composition. To highlight variations in lignin chemistry, we used Raman microspectroscopy to map the endodermal cell wall and silica aggregation sites in sorghum roots grown hydroponically with or without Si amendment. In +Si samples, the aggregate center was characterized by typical lignin monomer bands surrounded by lignin with a low level of polymerization. Farther from the spot, polysaccharide concentration increased and soluble silicic acid was detected in addition to silica bands. In -Si samples, the main band at the spot center was assigned to lignin radicals and highly polymerized lignin. Both +Si and -Si loci were enriched by aromatic carbonyls. We propose that at silica aggregation sites, carbonyl rich lignin monomers are locally exported to the apoplast. These monomers are radicalized and polymerized into short lignin polymers. In the presence of silicic acid, bonds typically involved in lignin extension, bind to silanols and nucleate silica aggregates near the monomer extrusion loci. This process inhibits further polymerization of lignin. In -Si samples, the monomers diffuse farther in the wall and crosslink with cell wall polymers, forming a ring of dense lignified cell wall around their export sites.

Raman developmental markers in root cell walls are associated with lodging tendency in tef.

MAIN CONCLUSION: Using Raman micro-spectroscopy on tef roots, we could monitor cell wall maturation in lines with varied genetic lodging tendency. We describe the developing cell wall composition in root endodermis and cylinder tissue. Tef [Eragrostis tef (Zucc.) Trotter] is an important staple crop in Ethiopia and Eritrea, producing gluten-free and protein-rich grains. However, this crop is not adapted to modern farming practices due to high lodging susceptibility, which prevents the application of mechanical harvest. Lodging describes the displacement of roots (root lodging) or fracture of culms (stem lodging), forcing plants to bend or fall from their vertical position, causing significant yield losses. In this study, we aimed to understand the microstructural properties of crown roots, underlining tef tolerance/susceptibility to lodging. We analyzed plants at 5 and 10 weeks after emergence and compared trellised to lodged plants. Root cross sections from different tef genotypes were characterized by scanning electron microscopy, micro-computed tomography, and Raman micro-spectroscopy. Lodging susceptible genotypes exhibited early tissue maturation, including developed aerenchyma, intensive lignification, and lignin with high levels of crosslinks. A comparison between trellised and lodged plants suggested that lodging itself does not affect the histology of root tissue. Furthermore, cell wall composition along plant maturation was typical to each of the tested genotypes independently of trellising. Our results suggest that it is possible to select lines that exhibit slow maturation of crown roots. Such lines are predicted to show reduction in lodging and facilitate mechanical harvest.

Silicon-mediated herbivore defence in a pasture grass under reduced and Anthropocene levels of CO2.

The uptake and accumulation of silicon (Si) in grass plants play a crucial role in alleviating both biotic and abiotic stresses. Si supplementation has been reported to increase activity of defence-related antioxidant enzyme, which helps to reduce oxidative stress caused by reactive oxygen species (ROS) following herbivore attack. Atmospheric CO2 levels are known to affect Si accumulation in grasses; reduced CO2 concentrations increase Si accumulation whereas elevated CO2 concentrations often decrease Si accumulation. This can potentially affect antioxidant enzyme activity and subsequently insect herbivory, but this remains untested. We examined the effects of Si supplementation and herbivory by Helicoverpa armigera on antioxidant enzyme (catalase, CAT; superoxide dismutase, SOD; and ascorbate peroxidase, APX) activity in tall fescue grass (Festuca arundinacea) grown under CO2 concentrations of 200, 410, and 640 ppm representing reduced, ambient, and elevated CO2 levels, respectively. We also quantified foliar Si, carbon (C), and nitrogen (N) concentrations and determined how changes in enzymes and elemental chemistry affected H. armigera relative growth rates and plant consumption. Rising CO2 concentrations increased plant mass and foliar C but decreased foliar N and Si. Si supplementation enhanced APX and SOD activity under the ranging CO2 regimes. Si accumulation and antioxidant enzyme activity were at their highest level under reduced CO2 conditions and their lowest level under future levels of CO2. The latter corresponded with increased herbivore growth rates and plant consumption, suggesting that some grasses could become more susceptible to herbivory under projected CO2 conditions.

Siliplant1 B-domain precipitates silica spheres, aggregates, or gel, depending on Si-precursor to peptide ratios.

Silica is extensively deposited by plants, however, only little is known about the molecular control over this process. Siliplant1 is the only known plant protein to precipitate biosilica. The protein contains seven repeats made of three domains. One of the domains exhibits a conserved sequence, which catalyzes silica precipitation in vitro. Here, silica was synthesized by the activity of a peptide carrying this conserved sequence. Infrared spectroscopy and thermal gravimetric analyses showed that the peptide was bound to the mineral. Scanning electron microscopy showed that silica-peptide particles of 22 ± 4 nm aggregated to spherical structures of 200-300 nm when the ratio of silicic acid to the peptide was below 183:1 molecules. When the ratio was about 183:1, similar particles aggregated into irregular structures, and silica gel formed at higher ratios. Solid-state NMR spectra indicated that the irregular aggregates were richer in Si-O-Si bonds as well as disordered peptide. Our results suggest that the peptide catalyzed the condensation of silicic acid and the formation of ∼20 nm particles, which aggregated into spheres. Excess of the peptide stabilized surface Si-OH groups that prevented spontaneous Si-O-Si bonding between aggregates. Under Si concentrations relevant to plant sap, the peptide and possibly Siliplant1, could catalyze nucleation of silica particles that aggregate into spherical aggregates.

Silica deposition in plants: scaffolding the mineralization.

BACKGROUND: Silicon and aluminium oxides make the bulk of agricultural soils. Plants absorb dissolved silicon as silicic acid into their bodies through their roots. The silicic acid moves with transpiration to target tissues in the plant body, where it polymerizes into biogenic silica. Mostly, the mineral forms on a matrix of cell wall polymers to create a composite material. Historically, silica deposition (silicification) was supposed to occur once water evaporated from the plant surface, leaving behind an increased concentration of silicic acid within plant tissues. However, recent publications indicate that certain cell wall polymers and proteins initiate and control the extent of plant silicification. SCOPE: Here we review recent publications on the polymers that scaffold the formation of biogenic plant silica, and propose a paradigm shift from spontaneous polymerization of silicic acid to dedicated active metabolic processes that control both the location and the extent of the mineralization. CONCLUSION: Protein activity concentrates silicic acid beyond its saturation level. Polymeric structures at the cell wall stabilize the supersaturated silicic acid and allow its flow with the transpiration stream, or bind it and allow its initial condensation. Silica nucleation and further polymerization are enabled on a polymeric scaffold, which is embedded within the mineral. Deposition is terminated once free silicic acid is consumed or the chemical moieties for its binding are saturated.

Outstanding Questions on the Beneficial Role of Silicon in Crop Plants.

Silicon (Si) is widely accepted as a beneficial element for plants. Despite the substantial progress made in understanding Si transport mechanisms and modes of action in plants, several questions remain unanswered. In this review, we discuss such outstanding questions and issues commonly encountered by biologists studying the role of Si in plants in relation to Si bioavailability. In recent years, advances in our understanding of the role of Si-solubilizing bacteria and the efficacy of Si nanoparticles have been made. However, there are many unknown aspects associated with structural and functional features of Si transporters, Si loading into the xylem, and the role of specialized cells like silica cells and compounds preventing Si polymerization in plant tissues. In addition, despite several 1,000 reports showing the positive effects of Si in high as well as low Si-accumulating plant species, the exact roles of Si at the molecular level are yet to be understood. Some evidence suggests that Si regulates hormonal pathways and nutrient uptake, thereby explaining various observed benefits of Si uptake. However, how Si modulates hormonal pathways or improves nutrient uptake remains to be explained. Finally, we summarize the knowledge gaps that will provide a roadmap for further research on plant silicon biology, leading to an exploration of the benefits of Si uptake to enhance crop production.

Hydrogen peroxide modulates silica deposits in sorghum roots.

Hydrated silica (SiO2·nH2O) aggregates in the root endodermis of grasses. Application of soluble silicates (Si) to roots is associated with variations in the balance of reactive oxygen species (ROS), increased tolerance to a broad range of stresses affecting ROS concentrations, and early lignin deposition. In sorghum (Sorghum bicolor L.), silica aggregation is patterned in an active silicification zone (ASZ) by a special type of aromatic material forming a spotted pattern. The deposition has a signature typical of lignin. Since lignin polymerization is mediated by ROS, we studied the formation of root lignin and silica controlled by ROS via modulating hydrogen peroxide (H2O2) concentrations in the growth medium. Sorghum seedlings were grown hydroponically and supplemented with Si, H2O2, and KI, an ionic compound that catalyses H2O2 decomposition. Lignin and silica deposits in the endodermis were studied by histology, scanning electron and Raman microscopies. Cell wall composition was quantified by thermal gravimetric analysis. Endodermal H2O2 concentration correlated to the extent of lignin-like deposition along the root, but did not affect its patterning in spots. Our results show that the ASZ spots were necessary for root silica aggregation, and suggest that silicification is intensified under oxidative stress as a result of increased ASZ lignin-like deposition.

Repetitive hygroscopic snapping movements in awns of wild oats.

Wild oat (Avena sterilis) is a very common annual plant species. Successful seed dispersion support its wide distribution in Africa, Asia and Europe. The seed dispersal units are made of two elongated stiff awns that are attached to a pointy compartment containing two seeds. The awns bend and twist with changes in humidity, pushing the seeds along and into the soil. The present work reveals the material structure of the awns, and models their functionality as two-link robotic arms. Based on nano-to-micro structure analyses the bending and twisting hygroscopic movements are explained. The coordinated movements of two sister awns attached to one dispersal unit were followed. Our work shows that sister awns intersect typically twice every wetting-drying cycle. Once the awns cross each other, epidermal silica hairs are suggested to lock subsequent movements, resulting in stress accumulation. Sudden release of the interlocked awns induces jumps of the dispersal unit and changes in its movement direction. Our findings propose a new role to epidermis silica hairs and a new facet of wild oat seed dispersion. Reversible jumping mechanism in multiple-awn seed dispersal units may serve as a blueprint for reversibly jumping robotic systems. STATEMENT OF SIGNIFICANCE: The seed dispersal unit of wild oats carries two elongated stiff awns covered by unidirectional silica hairs. The awns bend and twist with changes in humidity, pushing the seed capsule along and into the ground. We studied structures constructing the movement mechanism and modeled the awn as a two-link robotic arm. We show that sister awns, attached to the same seed capsule, intersect twice every drying cycle. Once the awns cross each other, the epidermal silica hairs are suggested to lock any subsequent movements, causing stress accumulation. Sudden release of the interlocked awns may cause the dispersal unit to jump and change its direction. Our findings suggest a new role to silica hairs and a new dispersal mechanism in multiple-awn seed dispersal units.

Anti-herbivore silicon defences in a model grass are greatest under Miocene levels of atmospheric CO2.

Silicon (Si) has an important role in mitigating diverse biotic and abiotic stresses in plants, mainly via the silicification of plant tissues. Environmental changes such as atmospheric CO2 concentrations may affect grass Si concentrations which, in turn, can alter herbivore performance. We recently demonstrated that pre-industrial atmospheric CO2 increased Si accumulation in Brachypodium distachyon grass, yet the patterns of Si deposition in leaves and whether this affects insect herbivore performance remains unknown. Moreover, it is unclear whether CO2 -driven changes in Si accumulation are linked to changes in gas exchange (e.g. transpiration rates). We therefore investigated how pre-industrial (reduced; rCO2 , 200 ppm), ambient (aCO2 , 410 ppm) and elevated (eCO2 , 640 ppm) CO2 concentrations, in combination with Si-treatment (Si+ or Si-), affected Si accumulation in B. distachyon and its subsequent effect on the performance of the global insect pest, Helicoverpa armigera. rCO2 increased Si concentrations by 29% and 36% compared to aCO2 and eCO2 respectively. These changes were not related to observed changes in gas exchange under different CO2 regimes, however. The increased Si accumulation under rCO2 decreased herbivore relative growth rate (RGR) by 120% relative to eCO2, whereas rCO2 caused herbivore RGR to decrease by 26% compared to eCO2 . Si supplementation also increased the density of macrohairs, silica and prickle cells, which was associated with reduced herbivore performance. There was a negative correlation among macrohair density, silica cell density, prickle cell density and herbivore RGR under rCO2 suggesting that these changes in leaf surface morphology were linked to reduced performance under this CO2 regime. To our knowledge, this is the first study to demonstrate that increased Si accumulation under pre-industrial CO2 reduces insect herbivore performance. Contrastingly, we found reduced Si accumulation under higher CO2 , which suggests that some grasses may become more susceptible to insect herbivores under projected climate change scenarios.

Protein-driven biomineralization: Comparing silica formation in grass silica cells to other biomineralization processes.

Biomineralization is a common strategy adopted by organisms to support their body structure. Plants practice significant silicon and calcium based biomineralization in which silicon is deposited as silica in cell walls and intracellularly in various cell-types, while calcium is deposited mostly as calcium oxalate in vacuoles of specialized cells. In this review, we compare cellular processes leading to protein-dependent mineralization in plants, diatoms and sponges (phylum Porifera). The mechanisms of biomineralization in these organisms are inherently different. The composite silica structure in diatoms forms inside the cytoplasm in a membrane bound vesicle, which after maturation is exocytosed to the cell surface. In sponges, separate vesicles with the mineral precursor (silicic acid), an inorganic template, and organic molecules, fuse together and are extruded to the extracellular space. In plants, calcium oxalate mineral precipitates in vacuolar crystal chambers containing a protein matrix which is never exocytosed. Silica deposition in grass silica cells takes place outside the cell membrane when the cells secrete the mineralizing protein into the apoplasm rich with silicic acid (the mineral precursor molecules). Our review infers that the organism complexity and precursor reactivity (calcium and oxalate versus silicic acid) are main driving forces for the evolution of varied mineralization mechanisms.

Contrasting effects of Miocene and Anthropocene levels of atmospheric CO2 on silicon accumulation in a model grass.

Grasses are hyper-accumulators of silicon (Si), which they acquire from the soil and deposit in tissues to resist environmental stresses. Given the high metabolic costs of herbivore defensive chemicals and structural constituents (e.g. cellulose), grasses may substitute Si for these components when carbon is limited. Indeed, high Si uptake grasses evolved in the Miocene when atmospheric CO2 concentration was much lower than present levels. It is, however, unknown how pre-industrial CO2 concentrations affect Si accumulation in grasses. Using Brachypodium distachyon, we hydroponically manipulated Si-supply (0.0, 0.5, 1, 1.5, 2 mM) and grew plants under Miocene (200 ppm) and Anthropocene levels of CO2 comprising ambient (410 ppm) and elevated (640 ppm) CO2 concentrations. We showed that regardless of Si treatments, the Miocene CO2 levels increased foliar Si concentrations by 47% and 56% relative to plants grown under ambient and elevated CO2, respectively. This is owing to higher accumulation overall, but also the reallocation of Si from the roots into the shoots. Our results suggest that grasses may accumulate high Si concentrations in foliage when carbon is less available (i.e. pre-industrial CO2 levels) but this is likely to decline under future climate change scenarios, potentially leaving grasses more susceptible to environmental stresses.

FTIR Nanospectroscopy Shows Molecular Structures of Plant Biominerals and Cell Walls.

Plant tissues are complex composite structures of organic and inorganic components whose function relies on molecular heterogeneity at the nanometer scale. Scattering-type near-field optical microscopy (s-SNOM) in the mid-infrared (IR) region is used here to collect IR nanospectra from both fixed and native plant samples. We compared structures of chemically extracted silica bodies (phytoliths) to silicified and nonsilicified cell walls prepared as a flat block of epoxy-embedded awns of wheat (Triticum turgidum), thin sections of native epidermis cells from sorghum (Sorghum bicolor) comprising silica phytoliths, and isolated cells from awns of oats (Avena sterilis). The correlation of the scanning-probe IR images and the mechanical phase image enables a combined probing of mechanical material properties together with the chemical composition and structure of both the cell walls and the phytolith structures. The data reveal a structural heterogeneity of the different silica bodies in situ, as well as different compositions and crystallinities of cell wall components. In conclusion, IR nanospectroscopy is suggested as an ideal tool for studies of native plant materials of varied origins and preparations and could be applied to other inorganic-organic hybrid materials.

Siliplant1 protein precipitates silica in sorghum silica cells.

Silicon is absorbed by plant roots as silicic acid. The acid moves with the transpiration stream to the shoot, and mineralizes as silica. In grasses, leaf epidermal cells called silica cells deposit silica in most of their volume using an unknown biological factor. Using bioinformatics tools, we identified a previously uncharacterized protein in Sorghum bicolor, which we named Siliplant1 (Slp1). Slp1 is a basic protein with seven repeat units rich in proline, lysine, and glutamic acid. We found Slp1 RNA in sorghum immature leaf and immature inflorescence. In leaves, transcription was highest just before the active silicification zone (ASZ). There, Slp1 was localized specifically to developing silica cells, packed inside vesicles and scattered throughout the cytoplasm or near the cell boundary. These vesicles fused with the membrane, releasing their content in the apoplastic space. A short peptide that is repeated five times in Slp1 precipitated silica in vitro at a biologically relevant silicic acid concentration. Transient overexpression of Slp1 in sorghum resulted in ectopic silica deposition in all leaf epidermal cell types. Our results show that Slp1 precipitates silica in sorghum silica cells.

Peptides from diatoms and grasses harness phosphate ion binding to silica to help regulate biomaterial structure.

Many life forms generate intricate submicron biosilica structures with various important biological functions. The formation of such structures, from the silicic acid in the waters and in the soil, is thought to be regulated by unique proteins with high repeats of specific amino acids and unusual sidechain modifications. Some silicifying proteins are characterized by high prevalence of basic amino acids in their primary structures. Lysine-rich domains are found, for instance, in diatom silaffin proteins and in the sorghum grass siliplant1 protein. These domains exhibit catalytic activity in silica chain condensation, owing to molecular interactions of the lysine amine groups with the forming mineral. The use of amine chemistry by two very remote organisms has motivated us to seek other molecular biosilicification processes that may be common to the two life forms. In diatom silaffins, domains rich in phosphoserine residues are thought to assist the assembly of silaffin molecules into an organic supra-structure which serves as a template for the silica to precipitate on. This mold, held by salt bridges between serine phosphates and lysine amines, dictates the shape of the silica particles formed. Yet, silica synthesized with the dephosphorylated silaffin in phosphate buffer showed similar morphology to the one prepared with the native protein, suggesting that a defined spatial arrangement of serine phosphates is not required to generate silica with the desired shape. Concurrently, free phosphates enhanced the activity of siliplant1 in silica formation. It is therefore beneficial to characterize the involvement of these anions as co-factors in regulated silicification by functional peptides from the two proteins and to understand whether they play similar molecular role in the mechanism of mineralization. Here we analyze the molecular interactions of free phosphate ions with silica and the silaffin peptide PL12 and separately with silica and siliplant1 peptide SLP1 in the two biomimetic silica products generated by the two peptides. MAS NMR measurements show that the phosphate ions interact with the peptides and at the same time may be forming bonds with the silica mineral. This bridging capability may add another avenue by which the structure of the silica material is influenced. A model for the molecular/ionic interactions at the bio-inorganic interface is described, which may have bearings for the role of phosphorylated residues beyond the function as intermolecular cross linkers or free phosphate ions as co-factors in regulation of silicification. STATEMENT OF SIGNIFICANCE: The manuscript addresses the question how proteins in diatoms and plants regulate the biosilica materials that are produced for various purposes in organisms. It uses preparation of silica in vitro with functional peptide derivatives from a sorghum grass protein and from a diatom silaffin protein separately to show that phosphate ions are important for the control that is achieved by these proteins on the final shape of the silica material produced. It portrays via magnetic resonance spectroscopic measurements, in atomic detail, the interface between atoms in the peptide, atoms on the surface of the silica formed and the phosphate ions that form chemical bonds with atoms on the silica as part of the mechanism of action of these peptides.

Unique lignin modifications pattern the nucleation of silica in sorghum endodermis.

Silicon dioxide in the form of hydrated silica is a component of plant tissues that can constitute several percent by dry weight in certain taxa. Nonetheless, the mechanism of plant silica formation is mostly unknown. Silicon (Si) is taken up from the soil by roots in the form of monosilicic acid molecules. The silicic acid is carried in the xylem and subsequently polymerizes in target sites to silica. In roots of sorghum (Sorghum bicolor), silica aggregates form in an orderly pattern along the inner tangential cell walls of endodermis cells. Using Raman microspectroscopy, autofluorescence, and scanning electron microscopy, we investigated the structure and composition of developing aggregates in roots of sorghum seedlings. Putative silica aggregation loci were identified in roots grown under Si starvation. These micrometer-scale spots were constructed of tightly packed modified lignin, and nucleated trace concentrations of silicic acid. Substantial variation in cell wall autofluorescence between Si+ and Si- roots demonstrated the impact of Si on cell wall chemistry. We propose that in Si- roots, the modified lignin cross-linked into the cell wall and lost its ability to nucleate silica. In Si+ roots, silica polymerized on the modified lignin and altered its structure. Our work demonstrates a high degree of control over lignin and silica deposition in cell walls.

Formation of root silica aggregates in sorghum is an active process of the endodermis.

Silica deposition in plants is a common phenomenon that correlates with plant tolerance to various stresses. Deposition occurs mostly in cell walls, but its mechanism is unclear. Here we show that metabolic processes control the formation of silica aggregates in roots of sorghum (Sorghum bicolor L.), a model plant for silicification. Silica formation was followed in intact roots and root segments of seedlings. Root segments were treated to enhance or suppress cell wall biosynthesis. The composition of endodermal cell walls was analysed by Raman microspectroscopy, scanning electron microscopy and energy-dispersive X-ray analysis. Our results were compared with in vitro reactions simulating lignin and silica polymerization. Silica aggregates formed only in live endodermal cells that were metabolically active. Silicic acid was deposited in vitro as silica onto freshly polymerized coniferyl alcohol, simulating G-lignin, but not onto coniferyl alcohol or ferulic acid monomers. Our results show that root silica aggregates form under tight regulation by endodermal cells, independently of the transpiration stream. We raise the hypothesis that the location and extent of silicification are primed by the chemistry and structure of polymerizing lignin as it cross-links to the wall.